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Volume 18,Issue 4,2011 Table of Contents
2011, 18(4):351-354.
DOI: 10.3872/j.issn.1007-385X.2011.4.002
Abstract:
Cancer immunotherapy has been widely accepted in clinic and has been applied for treatment of multiple malignancies in recent years. Different from chemotherapy, most patients can well tolerate immunotherapy and benefit from the improvement of life quality after immunotherapy; furthermore, immunotherapy shows lower risk of severe toxic side effects. Regrettably, disappointing results always occur when the conventional response evaluation criteria are used to assess the clinical efficiency of cancer immunotherapy, which might be responsible for the failure of quite a number of phase Ⅲ clinical trials of cancer immunotherapy. Since it is very difficult to use the existing WHO and RECIST criteria for accurate elucidation and evaluation of the clinical responses to cancer immunotherapy, Dr. Wolchok of New York Memorial Sloan-Kettering Cancer Center and his colleagues published a paper in Clinical Cancer Research in 2009, entitled "Guidelines for the evaluation of immune therapy activity in solid tumors: Immune-related response criteria", in which a novel response evaluation criterion system was proposed for cancer immunotherapy, and the clinical application of this system was discussed thoroughly. This paper briefly introduces the hot issue in onclology- "new evaluation criteria for efficiency of cancer immunotherapy".
Cancer immunotherapy has been widely accepted in clinic and has been applied for treatment of multiple malignancies in recent years. Different from chemotherapy, most patients can well tolerate immunotherapy and benefit from the improvement of life quality after immunotherapy; furthermore, immunotherapy shows lower risk of severe toxic side effects. Regrettably, disappointing results always occur when the conventional response evaluation criteria are used to assess the clinical efficiency of cancer immunotherapy, which might be responsible for the failure of quite a number of phase Ⅲ clinical trials of cancer immunotherapy. Since it is very difficult to use the existing WHO and RECIST criteria for accurate elucidation and evaluation of the clinical responses to cancer immunotherapy, Dr. Wolchok of New York Memorial Sloan-Kettering Cancer Center and his colleagues published a paper in Clinical Cancer Research in 2009, entitled "Guidelines for the evaluation of immune therapy activity in solid tumors: Immune-related response criteria", in which a novel response evaluation criterion system was proposed for cancer immunotherapy, and the clinical application of this system was discussed thoroughly. This paper briefly introduces the hot issue in onclology- "new evaluation criteria for efficiency of cancer immunotherapy".
2011, 18(4):355-361.
DOI: 10.3872/j.issn.1007-385X.2011.4.003
Abstract:
Objective: To primarily identify the antigenic epitopes of agonist type anti-CD40 monoclonal antibody, 5C11, which was constructed in our previous research, by means of computer modeling and site-directed mutation experiments. Methods:The structures of antigen and antibody were modeled by Insight Ⅱ software and the immune complex was constructed. The antigenic epitope of 5C11 antibody was calculated and speculated. The full length of wide type human CD40 (wtCD40) gene and two site-directed mutant CD40 gene (70muCD40 and 114muCD40) were amplified by RT-PCR; pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 recombinant vectors were constructed. These vectors were transfected into HEK293 cells by Lipofect method, and HEK293 cells stably transfected with pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 vectors (named HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells, respectively) were screened. The binding abilities of HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells with 5C11 were examined by flow cytometry and Western blotting analysis. Results: The recombinant eukaryotic expression vectors pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 were successfully constructed and the corresponding stably transfected HEK293 cells were obtained. The binding ability between 5C11 antibody and HEK293/70muCD40 and HEK293/114muCD40 cells were lower than that with HEK293/wtCD40 cells. Western blotting results showed that 5C11 antibody only recognized HEK293/wtCD40 cells but not HEK293/70muCD40 and HEK293/114muCD40 cells. Conclusion: The 70th threonine and 114th glutamic acids in human CD40 amino acid sequence are the antigenic epitopes of 5C11 monoclonal antibody, which has potential clinical significance for humanized CD40 antibody research.
Objective: To primarily identify the antigenic epitopes of agonist type anti-CD40 monoclonal antibody, 5C11, which was constructed in our previous research, by means of computer modeling and site-directed mutation experiments. Methods:The structures of antigen and antibody were modeled by Insight Ⅱ software and the immune complex was constructed. The antigenic epitope of 5C11 antibody was calculated and speculated. The full length of wide type human CD40 (wtCD40) gene and two site-directed mutant CD40 gene (70muCD40 and 114muCD40) were amplified by RT-PCR; pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 recombinant vectors were constructed. These vectors were transfected into HEK293 cells by Lipofect method, and HEK293 cells stably transfected with pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 vectors (named HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells, respectively) were screened. The binding abilities of HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells with 5C11 were examined by flow cytometry and Western blotting analysis. Results: The recombinant eukaryotic expression vectors pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 were successfully constructed and the corresponding stably transfected HEK293 cells were obtained. The binding ability between 5C11 antibody and HEK293/70muCD40 and HEK293/114muCD40 cells were lower than that with HEK293/wtCD40 cells. Western blotting results showed that 5C11 antibody only recognized HEK293/wtCD40 cells but not HEK293/70muCD40 and HEK293/114muCD40 cells. Conclusion: The 70th threonine and 114th glutamic acids in human CD40 amino acid sequence are the antigenic epitopes of 5C11 monoclonal antibody, which has potential clinical significance for humanized CD40 antibody research.
ZHU Lin
,
ZHENG Hu-yong
,
LIU Xiao
,
ZOU Li-min
,
DU Chao-hao
,
ZHAO Xiao-xi
,
LI Zhi-gang
,
WU Hui-lan
,
BAO Shi-lai
2011, 18(4):362-367.
DOI: 10.3872/j.issn.1007-385X.2011.4.004
Abstract:
Objective:To investigate the expression of transcription factor genes BCL6, KLF5 and nuclear protein gene NCL in the bone marrow cells of pediatric patients with acute lymphoblastic leukemia (ALL) in different ALL stages. Methods: A total of 100 pediatric ALL samples were selected from patients treated in Beijing Children's Hospital from January, 2004 to December, 2005. Five non-ALL patients were used as control. The aberrantly expressed genes in the bone marrow cells were examined by microarray assay; the mRNA levels of BCL6, KLF5, and NCL genes were detected in paired bone marrow samples during preliminary diagnosis and after complete remission by GeXP multiple gene expression analysis system. Results: The microarray results suggested that BCL6 and KLF5 mRNA levels were down-regulated and NCL mRNA level was up-regulated in all the 100 ALL samples, which including various subtypes. The mRNA levels of BCL6 and KLF5 in 10 pediatric ALL were low during preliminary diagnosis and elevated after complete remission (0.380±0.16 vs 0.850±0.10, 0.074±0.021 vs 0.228±0.049, both P<0.01); the mRNA level of NCL was high during preliminary diagnosis and decreased after complete remission (0.234±0.054 vs 0.151±0.055, P<0.01). The expression patterns of the three genes were similar between TEL-AML1 and E2A-PBX1 positive ALL patients during preliminary diagnosis and after complete remission. Conclusion: BCL6 and KLF5 are down-regulated in the bone marrow cells of pediatric ALL during preliminary diagnosis and are elevated after complete remission; the expression pattern of NCL shows an opposite trend to those of BCL6 and KLF5. The three genes have a potential to serve as predictive biomarkers for evaluating the therapeutic effect of leukemia.
Objective:To investigate the expression of transcription factor genes BCL6, KLF5 and nuclear protein gene NCL in the bone marrow cells of pediatric patients with acute lymphoblastic leukemia (ALL) in different ALL stages. Methods: A total of 100 pediatric ALL samples were selected from patients treated in Beijing Children's Hospital from January, 2004 to December, 2005. Five non-ALL patients were used as control. The aberrantly expressed genes in the bone marrow cells were examined by microarray assay; the mRNA levels of BCL6, KLF5, and NCL genes were detected in paired bone marrow samples during preliminary diagnosis and after complete remission by GeXP multiple gene expression analysis system. Results: The microarray results suggested that BCL6 and KLF5 mRNA levels were down-regulated and NCL mRNA level was up-regulated in all the 100 ALL samples, which including various subtypes. The mRNA levels of BCL6 and KLF5 in 10 pediatric ALL were low during preliminary diagnosis and elevated after complete remission (0.380±0.16 vs 0.850±0.10, 0.074±0.021 vs 0.228±0.049, both P<0.01); the mRNA level of NCL was high during preliminary diagnosis and decreased after complete remission (0.234±0.054 vs 0.151±0.055, P<0.01). The expression patterns of the three genes were similar between TEL-AML1 and E2A-PBX1 positive ALL patients during preliminary diagnosis and after complete remission. Conclusion: BCL6 and KLF5 are down-regulated in the bone marrow cells of pediatric ALL during preliminary diagnosis and are elevated after complete remission; the expression pattern of NCL shows an opposite trend to those of BCL6 and KLF5. The three genes have a potential to serve as predictive biomarkers for evaluating the therapeutic effect of leukemia.
2011, 18(4):368-372.
DOI: 10.3872/j.issn.1007-385X.2011.4.005
Abstract:
Objective: To explore the effect of Krüppel-like factors 4 (KLF4) on self-renewal and proliferation potential of tumor stem cells （T3A-A3）. Methods: A lentiviral vector carrying shRNA targeting KLF4 (pLVTHM-shKLF4) was constructed. Tumor stem cells (T3A-A3 cells) were isolated from a human hepatocarcinoma and were identified in our previous study. The expression of KLF4 mRNA and protein in T3A-A3 cells was analyzed by RT-PCR and Western blotting analysis after pLVTHM-shKLF4 infection. Self-renewal ability of T3A-A3 cells was evaluated by tumor sphere formation assay after pLVTHM-shKLF4 infection; clonogenic assay was used to determine the clonogenic ability of T3A-A3 cells; and cell cycle phase distribution was analyzed by flow cytometry. Influence of KLF4 knockdown on the growth of T3A-A3-transplanted tumors was examined in xenograft model of nude mice. Results: T3A-A3 expressed higher level of KLF4 than human hepatocarcinoma cell line Bel-7402 and HepG2. pLVTHM-shKLF4 infection significantly decreased the expression of KLF4 mRNA and protein in T3A-A3 cells. The formed tumor spheres of T3A-A3 cells were significantly smaller in pLVTHM-shKLF4 infection group compared with that in the pLVTHM-shNC control group (\[104.33±16.28\] μm vs \[186.67±28.15\] μm, P<0.01). pLVTHM-shKLF4 infection significantly inhibited the number of T3A-A3 cell-colonies compared with control group (83.5±7.78 vs 125±9.19, P<0.01). Flow cytometry analysis showed that pLVTHM-shKLF4 infection significantly increased G1 population when compared with the control vector (\[39.65±403\]% vs \[29.35±1.00\]%, P<0.01). Furthermore, the growth of T3A-A3-transplanted tumors in pLVTHM-shKLF4 infection group was significantly slower than that in the control group (33 days after cell inoculation, \[46.14±1294\] vs \[228.12±94.86\] mm3, P<0.01). Conclusion: KLF4 knockdown can inhibit the self-renewal of tumor stem cells (T3A-A3 cells), and inhibit the proliferation potential of T3A-A3 both in vitro and in vivo.
Objective: To explore the effect of Krüppel-like factors 4 (KLF4) on self-renewal and proliferation potential of tumor stem cells （T3A-A3）. Methods: A lentiviral vector carrying shRNA targeting KLF4 (pLVTHM-shKLF4) was constructed. Tumor stem cells (T3A-A3 cells) were isolated from a human hepatocarcinoma and were identified in our previous study. The expression of KLF4 mRNA and protein in T3A-A3 cells was analyzed by RT-PCR and Western blotting analysis after pLVTHM-shKLF4 infection. Self-renewal ability of T3A-A3 cells was evaluated by tumor sphere formation assay after pLVTHM-shKLF4 infection; clonogenic assay was used to determine the clonogenic ability of T3A-A3 cells; and cell cycle phase distribution was analyzed by flow cytometry. Influence of KLF4 knockdown on the growth of T3A-A3-transplanted tumors was examined in xenograft model of nude mice. Results: T3A-A3 expressed higher level of KLF4 than human hepatocarcinoma cell line Bel-7402 and HepG2. pLVTHM-shKLF4 infection significantly decreased the expression of KLF4 mRNA and protein in T3A-A3 cells. The formed tumor spheres of T3A-A3 cells were significantly smaller in pLVTHM-shKLF4 infection group compared with that in the pLVTHM-shNC control group (\[104.33±16.28\] μm vs \[186.67±28.15\] μm, P<0.01). pLVTHM-shKLF4 infection significantly inhibited the number of T3A-A3 cell-colonies compared with control group (83.5±7.78 vs 125±9.19, P<0.01). Flow cytometry analysis showed that pLVTHM-shKLF4 infection significantly increased G1 population when compared with the control vector (\[39.65±403\]% vs \[29.35±1.00\]%, P<0.01). Furthermore, the growth of T3A-A3-transplanted tumors in pLVTHM-shKLF4 infection group was significantly slower than that in the control group (33 days after cell inoculation, \[46.14±1294\] vs \[228.12±94.86\] mm3, P<0.01). Conclusion: KLF4 knockdown can inhibit the self-renewal of tumor stem cells (T3A-A3 cells), and inhibit the proliferation potential of T3A-A3 both in vitro and in vivo.
2011, 18(4):373-377.
DOI: 10.3872/j.issn.1007-385X.2011.4.006
Abstract:
Objective : To investigate the specific cytotoxicity effect of 5/35 chimeric oncolytic adenovirus SG635 on hepatocellular carcinoma HepG2 and SMMC-7721 cells. Methods: The knob and shaft domains of type 5 adenovirus (Ad5) in SG600 plasmid were replaced by the domains of type 35 adenovirus (Ad35), and chimeric oncolytic adenovirus Ad5/35 was established. Flow cytometry was used to examine the infection efficiency of chimeric adenovirus Ad5/35 (Ad5/35-EGFP) in HepG2 and SMMC-7721 cells; replication assay was used to evaluate the replication of oncolytic adenovirus SG635; Western blotting analysis was used to examine the expression of E1A in cells after SG635 infection; and Kit-8 assay was used to assess the cytotoxicity of SG635 and SG600 on HepG2 and SMMC-7721 cells. Results: The infection efficiency of Ad5/35-EGFP in HepG2 and SMMC-7721 cells was obviously enhanced compared with Ad5-EGFP. The replication activity of SG635 in HepG2 and SMMC-7721 cells was higher than that of SG600 72 h after infection (15 848.93, 6 309.57 vs 6 309.57, 5 011.87, P<0.01), but SG635 did not replicate in normal BJ cells. Moreover, SG635 induced a higher expression of E1A protein in HepG2 and SMMC-7721 cells than SG600, but did not induce E1A expression in normal BJ cells. At a certain MOI, SG635 showed increasing cytotoxicity on HepG2 (MOI=1, 90% vs 60%) and SMMC-7721 (MOI=10, 90% vs 50%) cells, and the cytotoxicity was stronger than SG600, without causing significant cytotoxicity on normal BJ cells. Conclusion: The 5/35 chimeric oncolytic adenovirus SG635 can effectively infect and specifically kill hepatocarcinoma cells with satisfactory safety and specificity.
Objective : To investigate the specific cytotoxicity effect of 5/35 chimeric oncolytic adenovirus SG635 on hepatocellular carcinoma HepG2 and SMMC-7721 cells. Methods: The knob and shaft domains of type 5 adenovirus (Ad5) in SG600 plasmid were replaced by the domains of type 35 adenovirus (Ad35), and chimeric oncolytic adenovirus Ad5/35 was established. Flow cytometry was used to examine the infection efficiency of chimeric adenovirus Ad5/35 (Ad5/35-EGFP) in HepG2 and SMMC-7721 cells; replication assay was used to evaluate the replication of oncolytic adenovirus SG635; Western blotting analysis was used to examine the expression of E1A in cells after SG635 infection; and Kit-8 assay was used to assess the cytotoxicity of SG635 and SG600 on HepG2 and SMMC-7721 cells. Results: The infection efficiency of Ad5/35-EGFP in HepG2 and SMMC-7721 cells was obviously enhanced compared with Ad5-EGFP. The replication activity of SG635 in HepG2 and SMMC-7721 cells was higher than that of SG600 72 h after infection (15 848.93, 6 309.57 vs 6 309.57, 5 011.87, P<0.01), but SG635 did not replicate in normal BJ cells. Moreover, SG635 induced a higher expression of E1A protein in HepG2 and SMMC-7721 cells than SG600, but did not induce E1A expression in normal BJ cells. At a certain MOI, SG635 showed increasing cytotoxicity on HepG2 (MOI=1, 90% vs 60%) and SMMC-7721 (MOI=10, 90% vs 50%) cells, and the cytotoxicity was stronger than SG600, without causing significant cytotoxicity on normal BJ cells. Conclusion: The 5/35 chimeric oncolytic adenovirus SG635 can effectively infect and specifically kill hepatocarcinoma cells with satisfactory safety and specificity.
2011, 18(4):374-350.
DOI: 10.3872/j.issn.1007-385X.2011.4.001
Abstract:
第98届美国免疫学年会于2011年5月13－17日在美国旧金山市成功召开，年会在抗体、B细胞免疫、疫苗、肿瘤免疫学、免疫细胞代谢学等方面进行了重点研讨。聚焦年会上肿瘤免疫学相关的报道，其热点集中在肿瘤免疫抑制、肿瘤免疫治疗新的理念及exosome在肿瘤免疫中的两面性等方面。综观年会众多热门话题，免疫细胞代谢学极有可能成为今后免疫学发展的重要方向；与之相呼应，肿瘤细胞的代谢改变被认为是肿瘤的第 8大特征，可能是未来肿瘤免疫学研究的潜在方向。
第98届美国免疫学年会于2011年5月13－17日在美国旧金山市成功召开，年会在抗体、B细胞免疫、疫苗、肿瘤免疫学、免疫细胞代谢学等方面进行了重点研讨。聚焦年会上肿瘤免疫学相关的报道，其热点集中在肿瘤免疫抑制、肿瘤免疫治疗新的理念及exosome在肿瘤免疫中的两面性等方面。综观年会众多热门话题，免疫细胞代谢学极有可能成为今后免疫学发展的重要方向；与之相呼应，肿瘤细胞的代谢改变被认为是肿瘤的第 8大特征，可能是未来肿瘤免疫学研究的潜在方向。
2011, 18(4):378-382.
DOI: 10.3872/j.issn.1007-385X.2011.4.007
Abstract:
Objective : To study the cytotoxicity of oncolytic adenovirus SG7605-11R-P53 containing cell-penetrating peptide (11R) on hepatocellular carcinoma cells Hep3B and Huh7 in vitro. Methods: Western blotting analysis was used to detect the expression levels of P53 and 11R-P53 in hepatocellular carcinoma cell lines HepG2, SMMC-7721, Hep3B and Huh7, and normal cell line BJ after SG7605-11R-P53 and SG7605-P53 infection. TCID50 assay was used to evaluate the replication ability of SG7605-11R-P53 and SG7605-P53 in hepatocellular carcinoma cell lines; the cytotoxicity of SG7605-11R-P53 on hepatocellular carcinoma cell lines and normal cell line was evaluated by MTT assay. Results: P53 and 11R-P53 proteins were highly expressed in both SG7605-11R-P53 and SG7605-P53 infected hepatocellular carcinoma cell lines. SG7605-11R-P53 replicated in HepG2, SMMC-7721, Hep3B and Huh7 cells but could hardly replicate in normal BJ cells, and SG7605-11R-P53 had a 10-100 times higher replication than SG7605-P53. When MOI=1, the cytotoxicity rate of SG7605-11R-P53 against Hep3B cells was 90%, and when MOI increased to 50, SG7605-11R-P53 only had a weak inhibitory effect against normal BJ cells. The cytotoxicity effect of SG7605-11R-P53 against Hep3B, HepG2, Huh7 and SMMC-7721 cells decreased gradually. Conclusion: Adenovirus SG7605-11R-P53 containing cell-penetrating peptide (11R) can efficiently kill the 4 hepatocellular carcinoma cell lines in vitro, with the strongest effect seen on Hep3B cells. This study lays a foundation for further investigating the effect of SG7605-11R-P53 against liver cancer in vivo.
Objective : To study the cytotoxicity of oncolytic adenovirus SG7605-11R-P53 containing cell-penetrating peptide (11R) on hepatocellular carcinoma cells Hep3B and Huh7 in vitro. Methods: Western blotting analysis was used to detect the expression levels of P53 and 11R-P53 in hepatocellular carcinoma cell lines HepG2, SMMC-7721, Hep3B and Huh7, and normal cell line BJ after SG7605-11R-P53 and SG7605-P53 infection. TCID50 assay was used to evaluate the replication ability of SG7605-11R-P53 and SG7605-P53 in hepatocellular carcinoma cell lines; the cytotoxicity of SG7605-11R-P53 on hepatocellular carcinoma cell lines and normal cell line was evaluated by MTT assay. Results: P53 and 11R-P53 proteins were highly expressed in both SG7605-11R-P53 and SG7605-P53 infected hepatocellular carcinoma cell lines. SG7605-11R-P53 replicated in HepG2, SMMC-7721, Hep3B and Huh7 cells but could hardly replicate in normal BJ cells, and SG7605-11R-P53 had a 10-100 times higher replication than SG7605-P53. When MOI=1, the cytotoxicity rate of SG7605-11R-P53 against Hep3B cells was 90%, and when MOI increased to 50, SG7605-11R-P53 only had a weak inhibitory effect against normal BJ cells. The cytotoxicity effect of SG7605-11R-P53 against Hep3B, HepG2, Huh7 and SMMC-7721 cells decreased gradually. Conclusion: Adenovirus SG7605-11R-P53 containing cell-penetrating peptide (11R) can efficiently kill the 4 hepatocellular carcinoma cell lines in vitro, with the strongest effect seen on Hep3B cells. This study lays a foundation for further investigating the effect of SG7605-11R-P53 against liver cancer in vivo.
GENG Ji-wei
,
DONG Jian
,
LIU Wei-qing
,
GAO Chang-e
,
XIONG Qiu-xia
,
MIAO Yan-dong
,
ZHOU Hua-hua
,
LI Qiu-tian
,
LI Chen
2011, 18(4):383-388.
DOI: 10.3872/j.issn.1007-385X.2011.4.008
Abstract:
Objective : To investigate the targeted killing effect of breast cancer specific peptide PI-mediated HSV-TK/GCV system against human breast cancer MDA-MB-231 cells in vitro. Methods: PI-TK gene was amplified from pORF-HSV-TK plasmid by PCR, and was re-inserted into the prokaryotic expression plasmid pET-28a (+); the pET-28a (+)-PI-TK plasmid was constructed and transfected into the host bacteria. After induction with IPTG, PI-TK fusion protein was purified by His-Tag and further identified by SDS-PAGE and Western blotting analysis. MDA-MB-231 cells were cultured with different dosages of PI-TK fusion protein; after further treatment with ganciclovier (GCV), the morphology changes of MDA-MB-231 cells were observed under inverted microscope, and the cell proliferation was evaluated by CCK-8 assay. Results: Recombinant prokaryotic expression plasmid pET-28a (+)-PI-TK was successfully constructed. The purified PI-TK fusion protein was obtained and confirmed by the SDS-PAGE and Western blotting analysis. PI-TK fusion protein alone failed to affect the morphology and proliferation of MDA-MB-231 cells, but when combined with GCV, PI-TK fusion protein specifically inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner, with inhibitory rate of 200 μg/ml PI-TK + 10 mg/L GCV being (68.9±7.57)%, and IC50 being 152.64 μg/ml, but it had no effects on MDA-MB-435 cells (P<0.05). Conclusion: The breast cancer specific peptides PI-mediated HSV-TK anti-tumor system can specifically kill MDA-MB-231 cells.
Objective : To investigate the targeted killing effect of breast cancer specific peptide PI-mediated HSV-TK/GCV system against human breast cancer MDA-MB-231 cells in vitro. Methods: PI-TK gene was amplified from pORF-HSV-TK plasmid by PCR, and was re-inserted into the prokaryotic expression plasmid pET-28a (+); the pET-28a (+)-PI-TK plasmid was constructed and transfected into the host bacteria. After induction with IPTG, PI-TK fusion protein was purified by His-Tag and further identified by SDS-PAGE and Western blotting analysis. MDA-MB-231 cells were cultured with different dosages of PI-TK fusion protein; after further treatment with ganciclovier (GCV), the morphology changes of MDA-MB-231 cells were observed under inverted microscope, and the cell proliferation was evaluated by CCK-8 assay. Results: Recombinant prokaryotic expression plasmid pET-28a (+)-PI-TK was successfully constructed. The purified PI-TK fusion protein was obtained and confirmed by the SDS-PAGE and Western blotting analysis. PI-TK fusion protein alone failed to affect the morphology and proliferation of MDA-MB-231 cells, but when combined with GCV, PI-TK fusion protein specifically inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner, with inhibitory rate of 200 μg/ml PI-TK + 10 mg/L GCV being (68.9±7.57)%, and IC50 being 152.64 μg/ml, but it had no effects on MDA-MB-435 cells (P<0.05). Conclusion: The breast cancer specific peptides PI-mediated HSV-TK anti-tumor system can specifically kill MDA-MB-231 cells.
MENG Xi-ting
,
LIN Chen
,
MEI Jia
,
WANG Hai-juan
,
MA Fei
,
ZHANG Jin-long
,
ZHANG Ying
,
QIAN Hai-li
2011, 18(4):389-393.
DOI: 10.3872/j.issn.1007-385X.2011.4.009
Abstract:
Objective : To study the expression of luciferase in mice delivered by replication-deficient adenovirus carrying luciferase (Ad-luciferase, Ad-Luci) through different injection routes, so at to provide information for adenovirus-mediated tumor gene therapy. Methods: Nude mouse model bearing human pancreatic cancer PANC-1 cells was established and Ad-Luci was administered into transplanted tumors with single or multiple intratumoral injections. Ad-Luci was also administered into normal mice through intramuscular, intravenous and intraperitoneal injections. The distribution and expression of luciferase in mice were monitored by in vivo IVIS Lumina imaging system and the toxicity of Ad-Luci was also studied. Results: Single intratumoral injection of Ad-Luci resulted in continuous luciferase expression for at least 15 d, while multiple injections resulted in even longer expression. Intramuscular injection of Ad-Luci gave a weaker and shorter duration of luciferase expression for no longer than 48 h. The majority of Ad-Luci injected through tail-vein homed to liver and expressed luciferase for at least 18 d. Expression of luciferase by intraperitoneal injection decreased with time-lapse and lasted for about 7 d. No significant toxic reactions were observed in all groups during the study. Conclusion: Intratumoral injection of adenovirus carrying exogenous gene gives a continuous expression of gene for a long duration, while multiple injections result in even longer expression; liver is the main target organ after tail intravenous administration and provides long expression duration.
Objective : To study the expression of luciferase in mice delivered by replication-deficient adenovirus carrying luciferase (Ad-luciferase, Ad-Luci) through different injection routes, so at to provide information for adenovirus-mediated tumor gene therapy. Methods: Nude mouse model bearing human pancreatic cancer PANC-1 cells was established and Ad-Luci was administered into transplanted tumors with single or multiple intratumoral injections. Ad-Luci was also administered into normal mice through intramuscular, intravenous and intraperitoneal injections. The distribution and expression of luciferase in mice were monitored by in vivo IVIS Lumina imaging system and the toxicity of Ad-Luci was also studied. Results: Single intratumoral injection of Ad-Luci resulted in continuous luciferase expression for at least 15 d, while multiple injections resulted in even longer expression. Intramuscular injection of Ad-Luci gave a weaker and shorter duration of luciferase expression for no longer than 48 h. The majority of Ad-Luci injected through tail-vein homed to liver and expressed luciferase for at least 18 d. Expression of luciferase by intraperitoneal injection decreased with time-lapse and lasted for about 7 d. No significant toxic reactions were observed in all groups during the study. Conclusion: Intratumoral injection of adenovirus carrying exogenous gene gives a continuous expression of gene for a long duration, while multiple injections result in even longer expression; liver is the main target organ after tail intravenous administration and provides long expression duration.
2011, 18(4):394-399.
DOI: 10.3872/j.issn.1007-385X.2011.4.010
Abstract:
Objective : To construct a dual function expressional plasmid pCDNA3.1-ENDO-VEGI151/survivin-shRNA (pEV/si-survivin), and study its effect on the proliferation and apoptosis of breast cancer MDA-MB-231 cells and vascular endothelial cells (HUEVCs), so as to evaluate its feasibility for gene therapy of cancer. Methods: The efficient siRNA sequences targeting survivin was screened in MDA-MB-231 cells; the pEV/si-survivin expression vector was constructed and transfected into MDA-MB-231 and HUEVC cells, and the expression levels of ENDO-VEGI151 and survivin were detected by the real-time PCR and Western blotting analysis; MTT assay was used to detect the proliferation inhibition in the cells of the two groups after transfection; and flow cytometry was used to detect the changes of cell cycles and apoptosis. Results: The dual function recombinant plasmid pEV/si-survivin was successfully constructed and it was correctly expressed in both MDA-MB-231 and HUEVC cells. The plasmid significantly inhibited the expression of survivin and the cell proliferation (inhibition rate being \[39.36±4.16\]% at 48 h and \[48.43±3.49)% at 72 h); it also significantly promoted cell apoptosis (\[18.33±1.48\]% vs \[4.80±1.01\]%, P<001) and induced cell cycles arrest (P<0.05) in MDA-MB-231 cells. The plasmid also significantly inhibited cell proliferation (inhibition rate being \[38.16±3.37\]%) at 48 h and \[53.75±453\]% at 72 h), promoted apoptosis, and arrested the cell cycles (P<0.05) in HUEVC cells. Conclusion: The dual function expressional plasmid pEV/si-survivin possess both angiogenesis inhibition and apoptosis promotion functions, and is expected to exert synergistic effect in vivo to improve the therapeutic outcome for patients with cancer.
Objective : To construct a dual function expressional plasmid pCDNA3.1-ENDO-VEGI151/survivin-shRNA (pEV/si-survivin), and study its effect on the proliferation and apoptosis of breast cancer MDA-MB-231 cells and vascular endothelial cells (HUEVCs), so as to evaluate its feasibility for gene therapy of cancer. Methods: The efficient siRNA sequences targeting survivin was screened in MDA-MB-231 cells; the pEV/si-survivin expression vector was constructed and transfected into MDA-MB-231 and HUEVC cells, and the expression levels of ENDO-VEGI151 and survivin were detected by the real-time PCR and Western blotting analysis; MTT assay was used to detect the proliferation inhibition in the cells of the two groups after transfection; and flow cytometry was used to detect the changes of cell cycles and apoptosis. Results: The dual function recombinant plasmid pEV/si-survivin was successfully constructed and it was correctly expressed in both MDA-MB-231 and HUEVC cells. The plasmid significantly inhibited the expression of survivin and the cell proliferation (inhibition rate being \[39.36±4.16\]% at 48 h and \[48.43±3.49)% at 72 h); it also significantly promoted cell apoptosis (\[18.33±1.48\]% vs \[4.80±1.01\]%, P<001) and induced cell cycles arrest (P<0.05) in MDA-MB-231 cells. The plasmid also significantly inhibited cell proliferation (inhibition rate being \[38.16±3.37\]%) at 48 h and \[53.75±453\]% at 72 h), promoted apoptosis, and arrested the cell cycles (P<0.05) in HUEVC cells. Conclusion: The dual function expressional plasmid pEV/si-survivin possess both angiogenesis inhibition and apoptosis promotion functions, and is expected to exert synergistic effect in vivo to improve the therapeutic outcome for patients with cancer.
2011, 18(4):400-403.
DOI: 10.3872/j.issn.1007-385X.2011.4.011
Abstract:
Objective : To study the effects of heparanase antisense oligodeoxynucleotide (HPSE-ASODN) on adhesion, invasion and apoptosis of human lung cancer A549 cells. Methods: HPSE specific ASODN was designed, synthesized, and transfected into A549 cells by lipofectin assay. HPSE protein expression was detected by Western blotting analysis in A549 cells, and the effects of HPSE-ASODN on adhesion and invasion of A549 cells were measured by adhesion and invasion assays, respectively. Hoechst 3222 staining was used to detect the effect of HPSE-ASODN on apoptosis of A549 cells. Results: Compared with control and lipofectin, HPSE-ASODN transfection inhibited HPSE protein expression, adhesion and invasion of A549 cells (P<0.01). HPSE-ASODN transfection induced apoptosis of A549 cells, with the apoptotic rate being significantly higher than those in the control and lipofectin groups (\[44.7±18.9\]% vs \[1.2±33\]%, \[5.8±20.1\]%, P<0.01). Conclusion: HPSE-ASODN can inhibit HPSE protein expression, adhesion and invasion in lung cancer A549 cells; it can also induce the apoptosis of lung cancer A549 cells.
Objective : To study the effects of heparanase antisense oligodeoxynucleotide (HPSE-ASODN) on adhesion, invasion and apoptosis of human lung cancer A549 cells. Methods: HPSE specific ASODN was designed, synthesized, and transfected into A549 cells by lipofectin assay. HPSE protein expression was detected by Western blotting analysis in A549 cells, and the effects of HPSE-ASODN on adhesion and invasion of A549 cells were measured by adhesion and invasion assays, respectively. Hoechst 3222 staining was used to detect the effect of HPSE-ASODN on apoptosis of A549 cells. Results: Compared with control and lipofectin, HPSE-ASODN transfection inhibited HPSE protein expression, adhesion and invasion of A549 cells (P<0.01). HPSE-ASODN transfection induced apoptosis of A549 cells, with the apoptotic rate being significantly higher than those in the control and lipofectin groups (\[44.7±18.9\]% vs \[1.2±33\]%, \[5.8±20.1\]%, P<0.01). Conclusion: HPSE-ASODN can inhibit HPSE protein expression, adhesion and invasion in lung cancer A549 cells; it can also induce the apoptosis of lung cancer A549 cells.
2011, 18(4):404-408.
DOI: 10.3872/j.issn.1007-385X.2011.4.012
Abstract:
Objective : To investigate the cytotoxicity effect of homologous cytokine-induced killer cells (CIKs), which was co-cultured with dendritic cells (DCs), against leukemic stem cells (LSCs) of acute myelogenous leukemic KG-1a cells. Methods: The peripheral blood mononuclear cells were isolated from healthy donors, and the adherent cells were induced to differentiate into DCs with GM-CSF and IL-4; the suspension cells were induced to differentiate into CIK cells with IL-1, IL-2, IFN-γ and CD3 mAb. KG-1a cells were frozen-thawed and the lysate antigen was obtained; the DCs pulsed with or without lysate antigen were co-cultured with CIK (Ag-DC-CIK, DC-CIK groups), and the ratios of CD34+CD38-CD123+LSCs were measured by flow cytometry in different groups; and CIK cultured alone served as blank control. DC-CIK or Ag-DC-CIK was further co-cultured with KG-1a cells, and the apoptotic rates of KG-1a and CD34+CD38-CD123+ cells were also examined by flow cytometry in different groups. Results: DCs were successfully induced from the peripheral blood mononuclear cells. The ratios of CD3+CD56+ cells in CIK, DC-CIK and Ag-DC-CIK groups were sequentially increased to (17.36±4.44)%, (28.22±3.66)%, and (36.16±5.88)%, respectively (P<001). Compared with the control group, the ratio of CD34+CD38-CD123+ in DC-CIK and Ag-DC-CIK groups were significantly decreased (\[3.95±0.53\]%, \[3.03±0.62\]% vs \[8.78±0.62\]%, P<0.01). DC-CIK induced apoptosis of KG-1a cells, with the apoptotic rate increased from (2.34±0.74)% to (12.27±1.01)%, but it showed no evident effect on apoptosis of CD34+CD38-CD123+ cells. Conclusion: DCs co-cultured with CIK can effectively kill acute myelogenous leukemic stem cells, but have no evident proapoptosis effect.
Objective : To investigate the cytotoxicity effect of homologous cytokine-induced killer cells (CIKs), which was co-cultured with dendritic cells (DCs), against leukemic stem cells (LSCs) of acute myelogenous leukemic KG-1a cells. Methods: The peripheral blood mononuclear cells were isolated from healthy donors, and the adherent cells were induced to differentiate into DCs with GM-CSF and IL-4; the suspension cells were induced to differentiate into CIK cells with IL-1, IL-2, IFN-γ and CD3 mAb. KG-1a cells were frozen-thawed and the lysate antigen was obtained; the DCs pulsed with or without lysate antigen were co-cultured with CIK (Ag-DC-CIK, DC-CIK groups), and the ratios of CD34+CD38-CD123+LSCs were measured by flow cytometry in different groups; and CIK cultured alone served as blank control. DC-CIK or Ag-DC-CIK was further co-cultured with KG-1a cells, and the apoptotic rates of KG-1a and CD34+CD38-CD123+ cells were also examined by flow cytometry in different groups. Results: DCs were successfully induced from the peripheral blood mononuclear cells. The ratios of CD3+CD56+ cells in CIK, DC-CIK and Ag-DC-CIK groups were sequentially increased to (17.36±4.44)%, (28.22±3.66)%, and (36.16±5.88)%, respectively (P<001). Compared with the control group, the ratio of CD34+CD38-CD123+ in DC-CIK and Ag-DC-CIK groups were significantly decreased (\[3.95±0.53\]%, \[3.03±0.62\]% vs \[8.78±0.62\]%, P<0.01). DC-CIK induced apoptosis of KG-1a cells, with the apoptotic rate increased from (2.34±0.74)% to (12.27±1.01)%, but it showed no evident effect on apoptosis of CD34+CD38-CD123+ cells. Conclusion: DCs co-cultured with CIK can effectively kill acute myelogenous leukemic stem cells, but have no evident proapoptosis effect.
MA Jie
,
ZHAO Wei-dong
,
MA Juan
,
JIN Xia
,
ZHANG Li-na
,
DING Yu-lan
,
ZHOU Hu
,
XUAN Heng-hua
,
GUI Yun
2011, 18(4):409-413.
DOI: 10.3872/j.issn.1007-385X.2011.4.013
Abstract:
Objective : To explore the cytotoxic activity of IL-15 gene modified-NK cells (NK-ustc cells) against primary ovarian cancer cells in vitro and in vivo. Methods: Primary ovarian cancer cells were isolated from ascites of patients. NK-ustc cells were irradiated with different dosages of gamma ray (0, 1, 2, 4, 8, 16 Gy), and the proliferation of irradiated NK-ustc cells were detected by 3H-TdR incorporation assay. Cytotoxic activities of NK-ustc cells against K562 and primary ovarian cancer cells were measured by 51Cr release assay. The tumor-bearing mouse model was established using primary ovarian cancer cells and randomly divided into NK-ustc treatment group (intraperitoneal injection of 8 Gy irradiated NK-ustc cells) and medium control group; moreover, blank control group (8 Gy irradiated NK-ustc cells were injected into nude mice) was also included in the present study. The body weight, abdomen circumference and survival time of nude mice were monitored. Results: After 1, 2, 4, 8 and 16 Gy irradiation, the proliferation rates of NK-ustc cells were (62.1±98)%, (41.3±8.7)%, (14.6±4.1)%, (0.1±0.03)% and (0.2±0.04)%, respectively. The cytotoxic rates of 0 and 8 Gy irradiated-NK-ustc cells against K562 cells were (45.4±8.9)% and (43.1±6.4)% when the effector to target ratio was 10 ∶1, and those against ovarian cancer cells were (54.6±6.4)% and (48.3±5.8)%, respectively. Thus, irradiation had no influence on cytotoxicity of NK-ustc cells (P>0.05). The median survival time of irradiated-NK-ustc cells treated mice was 75 d, and that of control group was 39 d (P<0.05). All the mice in the blank control group survived. Conclusion: Gamma ray irradiation can effectively inhibit proliferation of NK-ustc cells, but retain their cytotoxic activities against primary ovarian cancer cells.
Objective : To explore the cytotoxic activity of IL-15 gene modified-NK cells (NK-ustc cells) against primary ovarian cancer cells in vitro and in vivo. Methods: Primary ovarian cancer cells were isolated from ascites of patients. NK-ustc cells were irradiated with different dosages of gamma ray (0, 1, 2, 4, 8, 16 Gy), and the proliferation of irradiated NK-ustc cells were detected by 3H-TdR incorporation assay. Cytotoxic activities of NK-ustc cells against K562 and primary ovarian cancer cells were measured by 51Cr release assay. The tumor-bearing mouse model was established using primary ovarian cancer cells and randomly divided into NK-ustc treatment group (intraperitoneal injection of 8 Gy irradiated NK-ustc cells) and medium control group; moreover, blank control group (8 Gy irradiated NK-ustc cells were injected into nude mice) was also included in the present study. The body weight, abdomen circumference and survival time of nude mice were monitored. Results: After 1, 2, 4, 8 and 16 Gy irradiation, the proliferation rates of NK-ustc cells were (62.1±98)%, (41.3±8.7)%, (14.6±4.1)%, (0.1±0.03)% and (0.2±0.04)%, respectively. The cytotoxic rates of 0 and 8 Gy irradiated-NK-ustc cells against K562 cells were (45.4±8.9)% and (43.1±6.4)% when the effector to target ratio was 10 ∶1, and those against ovarian cancer cells were (54.6±6.4)% and (48.3±5.8)%, respectively. Thus, irradiation had no influence on cytotoxicity of NK-ustc cells (P>0.05). The median survival time of irradiated-NK-ustc cells treated mice was 75 d, and that of control group was 39 d (P<0.05). All the mice in the blank control group survived. Conclusion: Gamma ray irradiation can effectively inhibit proliferation of NK-ustc cells, but retain their cytotoxic activities against primary ovarian cancer cells.
2011, 18(4):414-418.
DOI: 10.3872/j.issn.1007-385X.2011.4.014
Abstract:
Objective : To study the effect of siRNA interference of ornithine decarboxylase antizyme inhibitor-1 (OAZI-1) expression on the proliferation of mouse melanoma B16-F1 cells. Methods: siRNA expression plasmid psilencer21-U6/OAZI-1 targeting OAZI-1 gene and control psilencer2.1-U6/scrambled plasmid were constructed and transfected into B16-F1 cells by Lipofect assay. Then the OAZI-1 expression in B16-F1 cells was examined by Western blotting analysis and real-time PCR. The effects of psilencer2.1-U6/OAZI-1 on proliferation and cell cycle of B16-F1 cells were detected by MTT and FCM, respectively. The sensitivity of B16-F1 cells to antitumor drug docetaxel was measured by MTT method after interfering OAZI-1 expression. Results: B16-F1 cells stably transfected with psilencer2.1-U6/OAZI-1 plasmid (B16/OAZI-1) were successfully obtained, and the expressions of OAZI-1 mRNA and protein in B16/OAZI-1 were 25% and 18.9% of those in control B16/scrambled cells, respectively. Interference of OAZI-1 expression inhibited the proliferation of B16-F1 cells, significantly increased the cells in G0/G1 phase (\[57.0±0.8\]% vs \[63.5±0.7\]%, P<0.01) and significantly decreased cells in S and G2/M phases (\[31.5±0.7\]% vs \[27.5±0.3\]%, P<0.05; \[11.5±0.3\]% vs \[9.1±0.6\]%, P<0.01). Interference of OAZI-1 expression also decreased the sensitivity of B16-F1 cells to the antitumor drug docetaxel. Conclusion: Interfering OAZI-1 expression can suppress the proliferation of mouse melanoma B16-F1 cells and decrease their sensitivity to docetaxel.
Objective : To study the effect of siRNA interference of ornithine decarboxylase antizyme inhibitor-1 (OAZI-1) expression on the proliferation of mouse melanoma B16-F1 cells. Methods: siRNA expression plasmid psilencer21-U6/OAZI-1 targeting OAZI-1 gene and control psilencer2.1-U6/scrambled plasmid were constructed and transfected into B16-F1 cells by Lipofect assay. Then the OAZI-1 expression in B16-F1 cells was examined by Western blotting analysis and real-time PCR. The effects of psilencer2.1-U6/OAZI-1 on proliferation and cell cycle of B16-F1 cells were detected by MTT and FCM, respectively. The sensitivity of B16-F1 cells to antitumor drug docetaxel was measured by MTT method after interfering OAZI-1 expression. Results: B16-F1 cells stably transfected with psilencer2.1-U6/OAZI-1 plasmid (B16/OAZI-1) were successfully obtained, and the expressions of OAZI-1 mRNA and protein in B16/OAZI-1 were 25% and 18.9% of those in control B16/scrambled cells, respectively. Interference of OAZI-1 expression inhibited the proliferation of B16-F1 cells, significantly increased the cells in G0/G1 phase (\[57.0±0.8\]% vs \[63.5±0.7\]%, P<0.01) and significantly decreased cells in S and G2/M phases (\[31.5±0.7\]% vs \[27.5±0.3\]%, P<0.05; \[11.5±0.3\]% vs \[9.1±0.6\]%, P<0.01). Interference of OAZI-1 expression also decreased the sensitivity of B16-F1 cells to the antitumor drug docetaxel. Conclusion: Interfering OAZI-1 expression can suppress the proliferation of mouse melanoma B16-F1 cells and decrease their sensitivity to docetaxel.
JU Xiao-ping
,
XU Xin-yan
,
ZHANG Xiao-qing
,
LIU Yong-ming
,
YU Chun-shan
,
CAO Yang-sen
,
LU Ming-zhi
,
CHEN Ying
2011, 18(4):419-423.
DOI: 10.3872/j.issn.1007-385X.2011.4.015
Abstract:
Objective : To prepare the fusion protein of SDF-1 and rhGM-CSF (SDF-1γ/rhGM-CSF) by genetic engineering technology, and investigate its hematopoietic and immune promotion functions in tumor patients. Methods: The expression vector for SDF-1γ/rhGM-CSF fusion protein, pPIC9k-SDF1-rhGM-CSF1, was constructed and the protein expression was induced by yeast transfection. SDF-1γ/rhGM-CSF fusion protein was further identified by Western blotting analysis. Colony-formation assay and chemoattract assay were used to study the roles of the prepared fusion protein in stimulating bone marrow cell colony-formation and in chemoattracting immature dendritic cells. Results: SDF-1γ/rhGM-CSF fusion gene vector, pPIC9k-SDF1-rhGM-CSF1, was successfully constructed and expressed high level of SDF-1γ/rhGM-CSF fusion gene. The molecular weight of the expressed protein was about 25 000 and was recognized by GM-CSF specific antibody. The fusion protein had a stronger effect in stimulating bone marrow cell colony-formation than GM-CSF (P<005) and in chemoattracting immature dendritic cells than SDF-1 (P<0.05). Conclusion: SDF-1γ/rhGM-CSF fusion protein can promote bone marrow cell colony-formation and chemoattraction of immature dendritic cells, which might be used for promoting hematopoiesis and immune function of tumor patients after chemotherapy.
Objective : To prepare the fusion protein of SDF-1 and rhGM-CSF (SDF-1γ/rhGM-CSF) by genetic engineering technology, and investigate its hematopoietic and immune promotion functions in tumor patients. Methods: The expression vector for SDF-1γ/rhGM-CSF fusion protein, pPIC9k-SDF1-rhGM-CSF1, was constructed and the protein expression was induced by yeast transfection. SDF-1γ/rhGM-CSF fusion protein was further identified by Western blotting analysis. Colony-formation assay and chemoattract assay were used to study the roles of the prepared fusion protein in stimulating bone marrow cell colony-formation and in chemoattracting immature dendritic cells. Results: SDF-1γ/rhGM-CSF fusion gene vector, pPIC9k-SDF1-rhGM-CSF1, was successfully constructed and expressed high level of SDF-1γ/rhGM-CSF fusion gene. The molecular weight of the expressed protein was about 25 000 and was recognized by GM-CSF specific antibody. The fusion protein had a stronger effect in stimulating bone marrow cell colony-formation than GM-CSF (P<005) and in chemoattracting immature dendritic cells than SDF-1 (P<0.05). Conclusion: SDF-1γ/rhGM-CSF fusion protein can promote bone marrow cell colony-formation and chemoattraction of immature dendritic cells, which might be used for promoting hematopoiesis and immune function of tumor patients after chemotherapy.
ZHANG Jun-ping
,
MAO Guang-hua
,
SHI Tian-liang
,
YANG Xiao-ling
,
XIAO Yan
,
ZHANG Li-bin
,
FENG Hui-jing
,
HAN Ya-ping
,
ZHI Ting
,
WANG Jiang-tao
,
JIA Lin-zi
2011, 18(4):424-429.
DOI: 10.3872/j.issn.1007-385X.2011.4.016
Abstract:
Objective : To evaluate the safety and therapeutic effect of dentritic cell (DC)-cytokine induced killer cells (CIKs) combined with chemotherapy in treatment of advanced non-small cell lung cancer (NSCLC) patients. Methods: Fifty patients with advanced NSCLC (stage Ⅲ to Ⅳ), who were admitted to Tumor Hospital of Shanxi Province from August 2008 to January 2010, were treated by DC-CIK combined with chemotherapy (docetaxel+cisplatin) and were taken as the combined treatment group; another fifty advanced NSCLC patients who were treated with chemotherapy alone (docetaxel+cisplatin) during the same period were taken as controls. The immune function, therapeutic effect, 1-year survival, life quality, and side effects were compared between the two groups. Furthermore, the safety and therapeutic effects of DC-CIK therapy were observed. Results: DC-CIK cells from NSCLC patients were successfully induced, the ratios of CD3+CD8+ and CD3＋CD56+ cells in DC-CIK cells were significantly increased after culture (P＜0.05). There were no obvious changes of T cell subsets in the peripheral blood after combined therapy, and the therapy increased IFN-γ level (P<0.05). In the chemotherapy group, the ratios of CD3+CD4+, CD3+CD8+, CD3-CD56+ cells and IL-2, TNF-α levels were significantly decreased after cell culture (P＜0.05); and the ratios of CD3+CD8+, CD3+CD56+ cells in DC-CIK was increased (P＜0.05). The disease control rate (DCR) of combined therapy group was higher than that in chemotherapy group (78.0% vs 56.0%, P<0.05); the 1-year survival rates of combined therapy group and chemotherapy group were 50% and 44%, respectively, showing no significant difference (P>0.05). The combined therapy group had less side effects（including bone marrow suppression, nausea and vomiting, and peripheral nerve toxicity）compared with the control chemotherapy group (P＜0.05). The physical condition and appetite of NSCLC patients in the combined therapy group were better than those in chemotherapy group.Conclusion: Treatment with DC-CIK cells combined with chemotherapy is safe and effective for advanced NSCLC, and it can also improve the remission rate, survival and quality of life of NSCLC patients.
Objective : To evaluate the safety and therapeutic effect of dentritic cell (DC)-cytokine induced killer cells (CIKs) combined with chemotherapy in treatment of advanced non-small cell lung cancer (NSCLC) patients. Methods: Fifty patients with advanced NSCLC (stage Ⅲ to Ⅳ), who were admitted to Tumor Hospital of Shanxi Province from August 2008 to January 2010, were treated by DC-CIK combined with chemotherapy (docetaxel+cisplatin) and were taken as the combined treatment group; another fifty advanced NSCLC patients who were treated with chemotherapy alone (docetaxel+cisplatin) during the same period were taken as controls. The immune function, therapeutic effect, 1-year survival, life quality, and side effects were compared between the two groups. Furthermore, the safety and therapeutic effects of DC-CIK therapy were observed. Results: DC-CIK cells from NSCLC patients were successfully induced, the ratios of CD3+CD8+ and CD3＋CD56+ cells in DC-CIK cells were significantly increased after culture (P＜0.05). There were no obvious changes of T cell subsets in the peripheral blood after combined therapy, and the therapy increased IFN-γ level (P<0.05). In the chemotherapy group, the ratios of CD3+CD4+, CD3+CD8+, CD3-CD56+ cells and IL-2, TNF-α levels were significantly decreased after cell culture (P＜0.05); and the ratios of CD3+CD8+, CD3+CD56+ cells in DC-CIK was increased (P＜0.05). The disease control rate (DCR) of combined therapy group was higher than that in chemotherapy group (78.0% vs 56.0%, P<0.05); the 1-year survival rates of combined therapy group and chemotherapy group were 50% and 44%, respectively, showing no significant difference (P>0.05). The combined therapy group had less side effects（including bone marrow suppression, nausea and vomiting, and peripheral nerve toxicity）compared with the control chemotherapy group (P＜0.05). The physical condition and appetite of NSCLC patients in the combined therapy group were better than those in chemotherapy group.Conclusion: Treatment with DC-CIK cells combined with chemotherapy is safe and effective for advanced NSCLC, and it can also improve the remission rate, survival and quality of life of NSCLC patients.
2011, 18(4):430-433.
DOI: 10.3872/j.issn.1007-385X.2011.4.017
Abstract:
Objective : To observe the proportions of CD3+CD4+TIL-17+ helper T cells (Th17 cells) and CD4+TCD25+Foxp3+ regulatory T cells (Tregs) in the peripheral blood of acute leukemia patients and the changes of related cytokines (including IL-17, TGF-β, IL-6), and to analyze their relationship. Methods: The ratios of Treg and Th17 cells in peripheral CD4+T cells were examined by flow cytometry. The serum levels of IL-17, TGF-β and IL-6 were measured by ELISA. Results: The ratio of Th17 cells in CD4+T cells was (1.39±0.24)%, which was significantly higher than that in the control group (\[0.26±0.11\]%, P<0.05); the ratio of Treg cells in CD4+T cells was (11.58±217)%, which was significantly higher than that in the control group (\[2.47±0.72\]%, P<0.05); and Treg cells were positively related with Th17 cells. The serum levels of TGF-β, IL-6, and IL-17 in acute leukemia patients were (26.06±243), (14.66±2.47), and (18.63±2.38) pg/ml, respectively, which were significantly higher than those in the control group (\[13.41±1.92\],\[1.44±0.29\], and \[10.34±1.71\] pg/ml, P<0.05). Conclusion: The ratios of Treg and Th17 cells are increased in the peripheral blood of acute leukemia patients, and they are positively correlated with each other. The serum levels of TGF-β, IL-6, and IL-17 are also elevated, which may affect the balance between Treg cells and Th17 cells.
Objective : To observe the proportions of CD3+CD4+TIL-17+ helper T cells (Th17 cells) and CD4+TCD25+Foxp3+ regulatory T cells (Tregs) in the peripheral blood of acute leukemia patients and the changes of related cytokines (including IL-17, TGF-β, IL-6), and to analyze their relationship. Methods: The ratios of Treg and Th17 cells in peripheral CD4+T cells were examined by flow cytometry. The serum levels of IL-17, TGF-β and IL-6 were measured by ELISA. Results: The ratio of Th17 cells in CD4+T cells was (1.39±0.24)%, which was significantly higher than that in the control group (\[0.26±0.11\]%, P<0.05); the ratio of Treg cells in CD4+T cells was (11.58±217)%, which was significantly higher than that in the control group (\[2.47±0.72\]%, P<0.05); and Treg cells were positively related with Th17 cells. The serum levels of TGF-β, IL-6, and IL-17 in acute leukemia patients were (26.06±243), (14.66±2.47), and (18.63±2.38) pg/ml, respectively, which were significantly higher than those in the control group (\[13.41±1.92\],\[1.44±0.29\], and \[10.34±1.71\] pg/ml, P<0.05). Conclusion: The ratios of Treg and Th17 cells are increased in the peripheral blood of acute leukemia patients, and they are positively correlated with each other. The serum levels of TGF-β, IL-6, and IL-17 are also elevated, which may affect the balance between Treg cells and Th17 cells.
2011, 18(4):434-436.
DOI: 10.3872/j.issn.1007-385X.2011.4.018
Abstract:
目的： 探讨过表达CDX2对结肠癌LoVo细胞裸鼠皮下移植瘤的抑制作用，为结肠癌生物治疗提供实验依据。 方法： 脂质体法介导pEGFP-C1-CDX2真核表达载体转染入结肠癌LoVo细胞，G418筛选出稳定表达CDX2的LoVo细胞（LoVo-CDX2细胞）；接种LoVo-CDX2细胞制备裸鼠皮下移植瘤模型，观察CDX2过表达对LoVo细胞移植瘤生长的影响。 结果： Western blotting结果显示，转染pEGFP-C1-CDX2组LoVo细胞中CDX2蛋白表达量高于pEGFP-C1组及未转染组。LoVo-CDX2、LoVo-C1和LoVo细胞接种裸鼠的第20天，LoVo-CDX2组移植瘤质量较LoVo组和LoVo-C1组移植瘤质量显著减轻\[ (0.62±0.22) vs (2.10±078)、(2.56±0.76）g，分别P＜0.05和P＜0.01]。 结论： CDX2过表达可以抑制结肠癌LoVo细胞裸鼠移植瘤的生长。
目的： 探讨过表达CDX2对结肠癌LoVo细胞裸鼠皮下移植瘤的抑制作用，为结肠癌生物治疗提供实验依据。 方法： 脂质体法介导pEGFP-C1-CDX2真核表达载体转染入结肠癌LoVo细胞，G418筛选出稳定表达CDX2的LoVo细胞（LoVo-CDX2细胞）；接种LoVo-CDX2细胞制备裸鼠皮下移植瘤模型，观察CDX2过表达对LoVo细胞移植瘤生长的影响。 结果： Western blotting结果显示，转染pEGFP-C1-CDX2组LoVo细胞中CDX2蛋白表达量高于pEGFP-C1组及未转染组。LoVo-CDX2、LoVo-C1和LoVo细胞接种裸鼠的第20天，LoVo-CDX2组移植瘤质量较LoVo组和LoVo-C1组移植瘤质量显著减轻\[ (0.62±0.22) vs (2.10±078)、(2.56±0.76）g，分别P＜0.05和P＜0.01]。 结论： CDX2过表达可以抑制结肠癌LoVo细胞裸鼠移植瘤的生长。
2011, 18(4):437-440.
DOI: 10.3872/j.issn.1007-385X.2011.4.019
Abstract:
越来越多的研究表明，基质金属蛋白酶（matrix metalloproteinases，MMPs）与肿瘤细胞上皮间质转化（epithelial-mesenchymal transition，EMT）的关系密切。MMPs是间质细胞表型的特有蛋白之一，既可以作为EMT发生的一个重要标志物，又可以诱发EMT。MMPs诱导肿瘤细胞发生EMT的机制是一个复杂的网络，可能涉及多个信号通路，如介导Rac1b激活ROS释放通路、活化TGF-β通路、降解E-cadherin促使β-catenin入核通路等。鉴于MMPs是诱导EMT促进肿瘤转移的一个关键因素，靶向MMPs已成为临床防治肿瘤的一个新策略。因此MMPs抑制剂则是当前研究热点，目前，多达50多种MMPs抑制剂被列入临床治疗肿瘤的候选药物，但其中大部分的疗效并未得到肯定。缺乏特异性则是其主要原因，故靶向MMP-EMT途径的肿瘤治疗仍然面临着很大的挑战，不仅需要鉴定出涉及肿瘤进展、促进EMT的主要或单一MMP，而且需要开发出具有高度特异性、选择性的MMPs抑制剂。
越来越多的研究表明，基质金属蛋白酶（matrix metalloproteinases，MMPs）与肿瘤细胞上皮间质转化（epithelial-mesenchymal transition，EMT）的关系密切。MMPs是间质细胞表型的特有蛋白之一，既可以作为EMT发生的一个重要标志物，又可以诱发EMT。MMPs诱导肿瘤细胞发生EMT的机制是一个复杂的网络，可能涉及多个信号通路，如介导Rac1b激活ROS释放通路、活化TGF-β通路、降解E-cadherin促使β-catenin入核通路等。鉴于MMPs是诱导EMT促进肿瘤转移的一个关键因素，靶向MMPs已成为临床防治肿瘤的一个新策略。因此MMPs抑制剂则是当前研究热点，目前，多达50多种MMPs抑制剂被列入临床治疗肿瘤的候选药物，但其中大部分的疗效并未得到肯定。缺乏特异性则是其主要原因，故靶向MMP-EMT途径的肿瘤治疗仍然面临着很大的挑战，不仅需要鉴定出涉及肿瘤进展、促进EMT的主要或单一MMP，而且需要开发出具有高度特异性、选择性的MMPs抑制剂。
2011, 18(4):441-447.
DOI: 10.3872/j.issn.1007-385X.2011.4.020
Abstract:
肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）是肿瘤微环境中的一种重要的炎症细胞。活化后的TAM通过分泌多种细胞因子和趋化因子促进肿瘤相关的血管生成、侵袭、浸润和转移；该过程也是免疫调控中的重要环节，TAM中核转录因子-κB（nuclear transcription factor- κB，NF-κB）、Toll样受体（Toll like receptor，TLR）等介导的信号通路的活化可以促进肿瘤的生长与增殖。上述功能受TAM所处肿瘤微环境的影响。对多种人类实体瘤和血液系统恶性肿瘤的研究发现，TAM与肿瘤预后不良相关。TAM向肿瘤组织的归巢能力使针对TAM的分子靶向治疗为肿瘤诊疗开辟了新思路，是抗肿瘤治疗的有效手段。
肿瘤相关巨噬细胞（tumor-associated macrophage，TAM）是肿瘤微环境中的一种重要的炎症细胞。活化后的TAM通过分泌多种细胞因子和趋化因子促进肿瘤相关的血管生成、侵袭、浸润和转移；该过程也是免疫调控中的重要环节，TAM中核转录因子-κB（nuclear transcription factor- κB，NF-κB）、Toll样受体（Toll like receptor，TLR）等介导的信号通路的活化可以促进肿瘤的生长与增殖。上述功能受TAM所处肿瘤微环境的影响。对多种人类实体瘤和血液系统恶性肿瘤的研究发现，TAM与肿瘤预后不良相关。TAM向肿瘤组织的归巢能力使针对TAM的分子靶向治疗为肿瘤诊疗开辟了新思路，是抗肿瘤治疗的有效手段。
2011, 18(4):448-451.
DOI: 10.3872/j.issn.1007-385X.2011.4.021
Abstract:
髓样来源的抑制性细胞（myeloid-derived suppressor cells，MDSC）是一群具有免疫抑制功能细胞的统称，通常认为它们是正常单核/巨噬细胞、DC细胞、粒细胞等处于分化的未成熟阶段，且可以分为单个核和分叶核两类。肿瘤模型小鼠中，单个核类MDSC典型的标记性分子是CD11b和Ly-6C，而分叶核类MDSC的典型标记分子是CD11b和Ly-6G；在肿瘤患者中，MDSC同时表达CD11和CD33等分子而不表达人白细胞抗原DR。MDSC由肿瘤微环境诱导产生，肿瘤产生过程中其在淋巴器官、血液以及病变部位聚集；针对不同的免疫细胞群，其通过分泌抑制性因子、接触性抑制以及诱导产生其他抑制性细胞等各种方式发挥免疫抑制作用，进而抑制天然免疫和适应性免疫。目前发现，MDSC抑制功能可以通过抗原特异性和抗原非特异性两种方式发挥作用，这是由其所处的局部微环境以及诱发其产生的肿瘤的性质来决定的。目前研究比较多的是其对NK细胞和T细胞的抑制作用，认为其主要可以通过产生精氨酸酶-1（arginase-1）、活性氧族（reactive oxygen species, ROS）以及抑制性的表面分子来实现对免疫系统的抑制作用，在肿瘤的生长以及转移过程中发挥重要的作用。
髓样来源的抑制性细胞（myeloid-derived suppressor cells，MDSC）是一群具有免疫抑制功能细胞的统称，通常认为它们是正常单核/巨噬细胞、DC细胞、粒细胞等处于分化的未成熟阶段，且可以分为单个核和分叶核两类。肿瘤模型小鼠中，单个核类MDSC典型的标记性分子是CD11b和Ly-6C，而分叶核类MDSC的典型标记分子是CD11b和Ly-6G；在肿瘤患者中，MDSC同时表达CD11和CD33等分子而不表达人白细胞抗原DR。MDSC由肿瘤微环境诱导产生，肿瘤产生过程中其在淋巴器官、血液以及病变部位聚集；针对不同的免疫细胞群，其通过分泌抑制性因子、接触性抑制以及诱导产生其他抑制性细胞等各种方式发挥免疫抑制作用，进而抑制天然免疫和适应性免疫。目前发现，MDSC抑制功能可以通过抗原特异性和抗原非特异性两种方式发挥作用，这是由其所处的局部微环境以及诱发其产生的肿瘤的性质来决定的。目前研究比较多的是其对NK细胞和T细胞的抑制作用，认为其主要可以通过产生精氨酸酶-1（arginase-1）、活性氧族（reactive oxygen species, ROS）以及抑制性的表面分子来实现对免疫系统的抑制作用，在肿瘤的生长以及转移过程中发挥重要的作用。
2011, 18(4):452-455.
DOI: 10.3872/j.issn.1007-385X.2011.4.022
Abstract:
缺氧是一种重要的病理状态，其与肿瘤生长、发展的关系越来越多受到关注，许多研究发现缺氧与肿瘤侵袭、转移的关系密切。细胞黏附分子（cell adhesion molecule，CAM）是参与细胞与细胞之间及细胞与细胞外基质之间相互作用的分子，可大致分为5类：钙黏素、整合素、选择素、免疫球蛋白超家族及透明质酸黏素。研究证实，影响肿瘤,缺氧状态的同时也能影响相关黏附分子的表达，特别是E-cadherin、整合素分子，以及一些其他黏附分子。深入研究缺氧和这些肿瘤黏附分子之间的关系，可以为肿瘤治疗提供新的思路。
缺氧是一种重要的病理状态，其与肿瘤生长、发展的关系越来越多受到关注，许多研究发现缺氧与肿瘤侵袭、转移的关系密切。细胞黏附分子（cell adhesion molecule，CAM）是参与细胞与细胞之间及细胞与细胞外基质之间相互作用的分子，可大致分为5类：钙黏素、整合素、选择素、免疫球蛋白超家族及透明质酸黏素。研究证实，影响肿瘤,缺氧状态的同时也能影响相关黏附分子的表达，特别是E-cadherin、整合素分子，以及一些其他黏附分子。深入研究缺氧和这些肿瘤黏附分子之间的关系，可以为肿瘤治疗提供新的思路。
2011, 18(4):456-462.
DOI: 10.3872/j.issn.1007-385X.2011.4.023
Abstract:
溶瘤病毒可在肿瘤细胞中增殖复制，并溶解瘤细胞，释放肿瘤抗原，激发机体自身的抗肿瘤免疫反应，它具有基因操控性、规模化、肿瘤选择性等生物学特征，从而成为个性化肿瘤疫苗的优良载体。但是，肿瘤存在很强的免疫抑制网络，单纯性溶瘤病毒难以激发理想的免疫应答。因此，根据需要加载不同的免疫调控分子，以增强机体的抗肿瘤免疫能力，已成为溶瘤病毒肿瘤疫苗研究的新思路。基于这一原理设计的OncoVexGM-CSF、JX-594、CG0070、KH901等免疫增强型肿瘤疫苗在临床试验中取得了较为显著的疗效，为肿瘤个性化疫苗的临床应用带来了曙光。尽管如此，溶瘤病毒肿瘤疫苗在疗效评价标准、作用机制、给药方式与治疗方案等方面还需要更为深入的探索。
溶瘤病毒可在肿瘤细胞中增殖复制，并溶解瘤细胞，释放肿瘤抗原，激发机体自身的抗肿瘤免疫反应，它具有基因操控性、规模化、肿瘤选择性等生物学特征，从而成为个性化肿瘤疫苗的优良载体。但是，肿瘤存在很强的免疫抑制网络，单纯性溶瘤病毒难以激发理想的免疫应答。因此，根据需要加载不同的免疫调控分子，以增强机体的抗肿瘤免疫能力，已成为溶瘤病毒肿瘤疫苗研究的新思路。基于这一原理设计的OncoVexGM-CSF、JX-594、CG0070、KH901等免疫增强型肿瘤疫苗在临床试验中取得了较为显著的疗效，为肿瘤个性化疫苗的临床应用带来了曙光。尽管如此，溶瘤病毒肿瘤疫苗在疗效评价标准、作用机制、给药方式与治疗方案等方面还需要更为深入的探索。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.