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Volume 10,Issue 2,2003 Table of Contents
2003, 10(2):79-83.
DOI: 10.3872/j.issn.1007-385X.2003.2.001
Abstract:
Objective:To investigate the combined inhibitive effect of TNP-470 and rhES on the growth of lung adenocarcinoma LA795 in T739 mice. Methods: The purified rhES was acquired by using methanol to induce the recombinant pichia pastoris. GS115 and heparin affinity chromatography. The T739 mice inoculated with LA795 cells were randomized into three groups, 10 mice per group, one group was injected with PBS for 14 days, the other two groups were respectively treated with rhES and TNP-470+rhES. To observe the tumor growth in different groups, and the tumor volume was measured with caliper. The microvessel density(MVD) of tumors were measured by using immunohistochemistry. Results: The purified rhES was acquired. In compared with PBS group, the tumor growth of other two groups was inhibited significantly. And the tumor volume of TNP-470+rhES group are smaller than the rhES group (P<0.01). immunohistochemistry showed the combined therapy has more inhibitory effect on angiogenesis. Conclusion: The combined treatment with TNP-470 and rhES showed more than additive effect in tumor growth inhibition when compared with treatment with individual antiangiostatic protein rhES.
Objective:To investigate the combined inhibitive effect of TNP-470 and rhES on the growth of lung adenocarcinoma LA795 in T739 mice. Methods: The purified rhES was acquired by using methanol to induce the recombinant pichia pastoris. GS115 and heparin affinity chromatography. The T739 mice inoculated with LA795 cells were randomized into three groups, 10 mice per group, one group was injected with PBS for 14 days, the other two groups were respectively treated with rhES and TNP-470+rhES. To observe the tumor growth in different groups, and the tumor volume was measured with caliper. The microvessel density(MVD) of tumors were measured by using immunohistochemistry. Results: The purified rhES was acquired. In compared with PBS group, the tumor growth of other two groups was inhibited significantly. And the tumor volume of TNP-470+rhES group are smaller than the rhES group (P<0.01). immunohistochemistry showed the combined therapy has more inhibitory effect on angiogenesis. Conclusion: The combined treatment with TNP-470 and rhES showed more than additive effect in tumor growth inhibition when compared with treatment with individual antiangiostatic protein rhES.
JIANG Qian-li
,
WANG Jian-min
,
WEN Li-min
,
ZHANG Yu-sheng
,
JIANG Shan
,
HU Xiao-xia
,
HU Xiao-xia
2003, 10(2):84-87.
DOI: 10.3872/j.issn.1007-385X.2003.2.002
Abstract:
Objective: To explore therapeutic effect of a novel yeast cytosine deaminase (YCD) suicide gene in vivo on P388/DBA murine leukemia model. Methods: P388-YCD-eGFP clone was selected after retrovirus transduction by limiting dilution, P388-eGFP and wild type (wt) P388 were used as control. (1) Tumorigenesis study: DBA mice were inoculated with P388-YCD-eGFP, P388-eGFP or wt P388, 5×10 6/each i.p. (n=5). (2) 5-FC therapy study: After inoculation with P388-YCD-eGFP, P388-eGFP and wt P388 respectively (n=5)，each mouse was given 5-FC with a dose of 5 μmol/d×2 w. (3) 5-FC killing effect study: 2 groups (n=5) of mice inoculated with P388-YCD-eGFP were treated with 5-FC×2 d or PBS. Results: Mice in YCD, eGFP and wtP388 group developed leukemia and survived for (8±1.0) d, (7.6±0.89) d and (7.8±1.64) d (P>0.05), respectively. After 5-FC-2w-treatment, mice in YCD group survived (17.8±1.89) d (P<0.05) vs (7.8±1.10) d (eGFP) and (7.7±1.15) d (wtP388) group, respectively. Mice of YCD group with 5-FC-2 d-treatment survived for (13.3±2.36) d，while PBS group (8.0±1.15) d. Flow cytometry and pathological examinations suggested mice of in YCD group died of leukemia relapse. Conclusion: YCD/5-FC is an efficient suicide gene system in vivo.
Objective: To explore therapeutic effect of a novel yeast cytosine deaminase (YCD) suicide gene in vivo on P388/DBA murine leukemia model. Methods: P388-YCD-eGFP clone was selected after retrovirus transduction by limiting dilution, P388-eGFP and wild type (wt) P388 were used as control. (1) Tumorigenesis study: DBA mice were inoculated with P388-YCD-eGFP, P388-eGFP or wt P388, 5×10 6/each i.p. (n=5). (2) 5-FC therapy study: After inoculation with P388-YCD-eGFP, P388-eGFP and wt P388 respectively (n=5)，each mouse was given 5-FC with a dose of 5 μmol/d×2 w. (3) 5-FC killing effect study: 2 groups (n=5) of mice inoculated with P388-YCD-eGFP were treated with 5-FC×2 d or PBS. Results: Mice in YCD, eGFP and wtP388 group developed leukemia and survived for (8±1.0) d, (7.6±0.89) d and (7.8±1.64) d (P>0.05), respectively. After 5-FC-2w-treatment, mice in YCD group survived (17.8±1.89) d (P<0.05) vs (7.8±1.10) d (eGFP) and (7.7±1.15) d (wtP388) group, respectively. Mice of YCD group with 5-FC-2 d-treatment survived for (13.3±2.36) d，while PBS group (8.0±1.15) d. Flow cytometry and pathological examinations suggested mice of in YCD group died of leukemia relapse. Conclusion: YCD/5-FC is an efficient suicide gene system in vivo.
2003, 10(2):88-92.
DOI: 10.3872/j.issn.1007-385X.2003.2.003
Abstract:
Objective: The Explore the effective method of tumor gene therapy and studying the antitumor effect of NDV HN gene and apoptin VP3 gene.Methods: The carcinoma of colon model in BALB/c nude mice was constructed and the pVHN and pVVP3 DNA-liposome complexes were injected into the tumor. The therapeutic effect of NDV HN gene and apoptin VP3 gene to BALB/c nude mice bearing HCT tumor was observed. and the expressions of the Bcl-2 and PCNA of the tumors were detected with immunohistochemistry. Results: Compared with the control group ,the tumors in the group of combination therapy were decreased obviously,and the rate of suppressing tumor reached 70 percent. The results of pathological tissue section showed that apoptosis was induced greatly in the group of combination therapy,and the tumor cells in some range disappeared.The results of immunohistochemistry showed that the Bcl-2 and PCNA protein were expressed in control group and all of therapy groups , the Bcl-2 expression rates of control group and the therapy group with pVHN were marked difference (P<0.05) and the PCNA expression rates of control group and the therapy group with pVHN and pVVP3 were very marked difference (P<0.01) .Conclusion: NDV HN gene and apoptin VP3 gene are able to suppress the proliferation of the tumor cells in BALB/c nude mice bearing HCT tumor and induce apoptosis of the tumor cells. The apoptosis that induced by NDV HN gene and apoptin VP3 gene may correlated with down-regulation of Bcl-2 protein.
Objective: The Explore the effective method of tumor gene therapy and studying the antitumor effect of NDV HN gene and apoptin VP3 gene.Methods: The carcinoma of colon model in BALB/c nude mice was constructed and the pVHN and pVVP3 DNA-liposome complexes were injected into the tumor. The therapeutic effect of NDV HN gene and apoptin VP3 gene to BALB/c nude mice bearing HCT tumor was observed. and the expressions of the Bcl-2 and PCNA of the tumors were detected with immunohistochemistry. Results: Compared with the control group ,the tumors in the group of combination therapy were decreased obviously,and the rate of suppressing tumor reached 70 percent. The results of pathological tissue section showed that apoptosis was induced greatly in the group of combination therapy,and the tumor cells in some range disappeared.The results of immunohistochemistry showed that the Bcl-2 and PCNA protein were expressed in control group and all of therapy groups , the Bcl-2 expression rates of control group and the therapy group with pVHN were marked difference (P<0.05) and the PCNA expression rates of control group and the therapy group with pVHN and pVVP3 were very marked difference (P<0.01) .Conclusion: NDV HN gene and apoptin VP3 gene are able to suppress the proliferation of the tumor cells in BALB/c nude mice bearing HCT tumor and induce apoptosis of the tumor cells. The apoptosis that induced by NDV HN gene and apoptin VP3 gene may correlated with down-regulation of Bcl-2 protein.
2003, 10(2):93-96.
DOI: 10.3872/j.issn.1007-385X.2003.2.004
Abstract:
Objective:To investigate the role of HN in NDV antitumor. Method: Nucleic recombinant plasmid pVHN was constructed with pVAX1 and NDV HN gene and transfected into OS732 cells in vitro. 72 h after transfection, OS732 cells were collected and the content of cell surface sialic acid was measured. 6 weeks old BALB/c mice were implanted with S180 sarcoma through the injection of 2×10 6 S180 cells. On 7th day and 17th day after tumor transplantation,100 μg pVHN recombinant plasmid was administered intratumorly in vivo respectively. Control group was treated with pVAX1 as the same method. Serum of BALB/c mice bearing S180 sarcoma was separated and measured the content of sialic acid. T lymphocyte subsets were detected by FACS combined with rat anti mouse CD3+, CD4+, CD8+ antibodies. Results:pVHN was constructed correctly and expressed in OS732 cell. pVHN reduced the surface sialic acid of OS732 cell significantly in vitro. Expression of pVHN in vivo augmented CD8+ T lymphocyte subset and decreased serum sialic acid content. Conclusion: HN gene plays an important role in the reduction of sialic acid and the augmentation of CD8+ T lymphocyte subset
Objective:To investigate the role of HN in NDV antitumor. Method: Nucleic recombinant plasmid pVHN was constructed with pVAX1 and NDV HN gene and transfected into OS732 cells in vitro. 72 h after transfection, OS732 cells were collected and the content of cell surface sialic acid was measured. 6 weeks old BALB/c mice were implanted with S180 sarcoma through the injection of 2×10 6 S180 cells. On 7th day and 17th day after tumor transplantation,100 μg pVHN recombinant plasmid was administered intratumorly in vivo respectively. Control group was treated with pVAX1 as the same method. Serum of BALB/c mice bearing S180 sarcoma was separated and measured the content of sialic acid. T lymphocyte subsets were detected by FACS combined with rat anti mouse CD3+, CD4+, CD8+ antibodies. Results:pVHN was constructed correctly and expressed in OS732 cell. pVHN reduced the surface sialic acid of OS732 cell significantly in vitro. Expression of pVHN in vivo augmented CD8+ T lymphocyte subset and decreased serum sialic acid content. Conclusion: HN gene plays an important role in the reduction of sialic acid and the augmentation of CD8+ T lymphocyte subset
TANG Liang
,
MAN Xiao-bo
,
CAO Hui-fang
,
QIU Xiu-hua
,
LIU Shu qing
,
TAN Ye-xiong
,
WU Meng-chao
,
WANG Hong-yang
2003, 10(2):97-100.
DOI: 10.3872/j.issn.1007-385X.2003.2.005
Abstract:
Objective：To detect the effect of Mitomycin-C (MMC) on expression level and cellular distribution of NF-κB after MMC stimulation to further understand the mechanism of the NF-κB signal transduction in the response of SK-HEP-1 cell to the chemical drug. Methods：The cellular distribution and protein level of NF-κB was detected by Immunohistochemistry and Western blot method in SK-HEP-1 cell treated with different concentration of MMC for different duration. The mRNA expression of NF-κB gene was determined by RT-PCR in SK-HEP-1 cell treated with 50 μg/ml concentration MMC for different time after continuing treatment and 2 h treatment.Results：MMC stimulated the NF-κB influx into the nucleus from plasma and elevated the protein level of NF-κB and reached the peak at 8th hour. On effect of continual treatment of 50 μg/ml concentration MMC the expression level of NF-κB gene was elevated until the peak at 2nd h and then descended. However after treatment of MMC for 2 h and then withdrawal, the expression of NF-κB was elevated until 12th h. Conclusions：MMC could drive the NF-κB to come into the nucleus and elevate the expression of the gene. It was suggested that the NF-κB signal transduction pathway had relation to the response of SK-HEP-1 cell to MMC treatment.
Objective：To detect the effect of Mitomycin-C (MMC) on expression level and cellular distribution of NF-κB after MMC stimulation to further understand the mechanism of the NF-κB signal transduction in the response of SK-HEP-1 cell to the chemical drug. Methods：The cellular distribution and protein level of NF-κB was detected by Immunohistochemistry and Western blot method in SK-HEP-1 cell treated with different concentration of MMC for different duration. The mRNA expression of NF-κB gene was determined by RT-PCR in SK-HEP-1 cell treated with 50 μg/ml concentration MMC for different time after continuing treatment and 2 h treatment.Results：MMC stimulated the NF-κB influx into the nucleus from plasma and elevated the protein level of NF-κB and reached the peak at 8th hour. On effect of continual treatment of 50 μg/ml concentration MMC the expression level of NF-κB gene was elevated until the peak at 2nd h and then descended. However after treatment of MMC for 2 h and then withdrawal, the expression of NF-κB was elevated until 12th h. Conclusions：MMC could drive the NF-κB to come into the nucleus and elevate the expression of the gene. It was suggested that the NF-κB signal transduction pathway had relation to the response of SK-HEP-1 cell to MMC treatment.
GAO Shang-xian
,
SONG Jing
,
LIU Xing-xia
,
KANG Guo-hua
,
WANG Qun
,
ZHANG Ping
,
LI Wei
,
ZHANG Li-ning
,
LI Shou-ti
2003, 10(2):101-104.
DOI: 10.3872/j.issn.1007-385X.2003.2.006
Abstract:
Objective:To investigate the in vivo anti-tumor effect and its mechanism of NCPP. Methods:Ehrlich′s ascites carcinoma cells were injected into the peritoneal cavity of mice. On the day before the injection and then, every other day, NCPP and Corynebacterum Parvum( CPP ) were given in the same way, total 5 times. The survival rates of the tumor-carrying mice were analyzed. 10 days after the injection of NCPP and CPP, data were collected, including the phagocytic index and its rate of the macrophages, the level of the hydrogen peroxide and nitric oxide produced by the macrophages, splenic natural killing (NK) cell′s activity, and the proliferation of T cells. And splenic index was evaluated on the 14th day after the injection of NCPP and CPP. Results: Compared with the control group, the survival rates of tumor-bearing mice were significantly increased in both NCPP and CPP groups; NCPP and CPP groups induced higher levels of splenic index (>2 in everage); higher levels of macrophage activity (P<0.01), and splenic NK cell activity (P<005), However, there was no difference in all the parameters mentioned as above between NCPP group and CPP group. Conclusions: This study shows that NCPP plays the same role as CPP in the tumor inhibition and splenic stimulation,mainly through the activiting macrophage and NK cells.
Objective:To investigate the in vivo anti-tumor effect and its mechanism of NCPP. Methods:Ehrlich′s ascites carcinoma cells were injected into the peritoneal cavity of mice. On the day before the injection and then, every other day, NCPP and Corynebacterum Parvum( CPP ) were given in the same way, total 5 times. The survival rates of the tumor-carrying mice were analyzed. 10 days after the injection of NCPP and CPP, data were collected, including the phagocytic index and its rate of the macrophages, the level of the hydrogen peroxide and nitric oxide produced by the macrophages, splenic natural killing (NK) cell′s activity, and the proliferation of T cells. And splenic index was evaluated on the 14th day after the injection of NCPP and CPP. Results: Compared with the control group, the survival rates of tumor-bearing mice were significantly increased in both NCPP and CPP groups; NCPP and CPP groups induced higher levels of splenic index (>2 in everage); higher levels of macrophage activity (P<0.01), and splenic NK cell activity (P<005), However, there was no difference in all the parameters mentioned as above between NCPP group and CPP group. Conclusions: This study shows that NCPP plays the same role as CPP in the tumor inhibition and splenic stimulation,mainly through the activiting macrophage and NK cells.
2003, 10(2):105-109.
DOI: 10.3872/j.issn.1007-385X.2003.2.007
Abstract:
Objective:To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability.Methods:Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was κ. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.
Objective:To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability.Methods:Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was κ. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.
CHEN Yong-jing
,
SHI Qing
,
GE Yan
,
XU Kuan-feng
,
GU Tao
,
SUN Jian-jun
,
LI Wen-xiang
,
ZHANG Xue-guang
2003, 10(2):110-114.
DOI: 10.3872/j.issn.1007-385X.2003.2.008
Abstract:
Objective:To clone human PD-L1(B7-H1) gene and construct recombinant retrovirus vector carrying the target gene which can be expressed stably in mammal cell line L929. Methods: PD-L1 gene was amplified by PCR (polymerase chain reaction) from the human heart cDNA library and confirmed by DNA-sequence analysis. Digested with the restriction endonucleases PstI and EcoRI, the PD-L1 gene was inserted into retrovirus vector pGEZ-Term. The recombinant retrovirus vector together with its two helper virus vectors cotransfected into the package cell 293T in the context of LipfectAMINE. Then the supernatant of 293T was used to infect L929 cells. L929 cell line stably expressing PD-L1 protein was selected in the presence of Zeocin(500 μg/ml). Results: The full-length PD-L1(B7-H1) gene was successfully cloned; and the recombinant retrovirus vector carrying PD-L1 gene for expression was constructed; by transfecting package cell line 293T, recombinant PD-L1 retrovirus with infective capability was packaged and after 2 weeks of selection, the infected L929 cells formed monoclonal colonies in selective medium. Results of RT-PCR and flow cytometry indicated that L929 transgenetic cells could stably express human PD-L1 protein on the membrance of cells. Conclusion: Cloning of human PD-L1(B7-H1) gene and construction of the recombinant retrovirus vector and L929 cell line stably expressing PD-L1 protein could contribute to further biological function research and monoclonal antibody preparation.
Objective:To clone human PD-L1(B7-H1) gene and construct recombinant retrovirus vector carrying the target gene which can be expressed stably in mammal cell line L929. Methods: PD-L1 gene was amplified by PCR (polymerase chain reaction) from the human heart cDNA library and confirmed by DNA-sequence analysis. Digested with the restriction endonucleases PstI and EcoRI, the PD-L1 gene was inserted into retrovirus vector pGEZ-Term. The recombinant retrovirus vector together with its two helper virus vectors cotransfected into the package cell 293T in the context of LipfectAMINE. Then the supernatant of 293T was used to infect L929 cells. L929 cell line stably expressing PD-L1 protein was selected in the presence of Zeocin(500 μg/ml). Results: The full-length PD-L1(B7-H1) gene was successfully cloned; and the recombinant retrovirus vector carrying PD-L1 gene for expression was constructed; by transfecting package cell line 293T, recombinant PD-L1 retrovirus with infective capability was packaged and after 2 weeks of selection, the infected L929 cells formed monoclonal colonies in selective medium. Results of RT-PCR and flow cytometry indicated that L929 transgenetic cells could stably express human PD-L1 protein on the membrance of cells. Conclusion: Cloning of human PD-L1(B7-H1) gene and construction of the recombinant retrovirus vector and L929 cell line stably expressing PD-L1 protein could contribute to further biological function research and monoclonal antibody preparation.
2003, 10(2):115-118.
DOI: 10.3872/j.issn.1007-385X.2003.2.009
Abstract:
Objective: To clone human T cell receptor (TCR) ζ chain gene and express its encoding protein by baculoviral expression system in insect cells. Methods: TCR ζ chain cDNA was cloned from normal human peripheral blood mononuclear cells (PBMC) by RT-PCR and inserted into baculoviral transfer vector. This vector was co-transfected with baculovirus into insect sf 9 cells. The recombinant protein expressed was identified by SDS-PAGE and flow cytometry with mouse anti-human ζ chain monoclonal antibody. Results: Human TCR ζ chain cDNA cloned and inserted into baculoviral vector specifically expressed protein in the insect sf 9 cells, accounting for about 11% of the total protein yield in the supernatant of cell lysate. The molecular weight of the recombinant protein determined by SDS-PAGE was identical to what we anticipated. The insect cells transfected with recombinant baculovirus were demonstrated by intracellular labeling flow cytometry to express ζ chain protein. Conclusion: Human T cell receptor ζ chain gene was successfully cloned from human PBMC, and its encoding protein was highly expressed in insect cells. The TCR ζ chain protein obtained by bioengineering technique is useful for in depth biological function study.
Objective: To clone human T cell receptor (TCR) ζ chain gene and express its encoding protein by baculoviral expression system in insect cells. Methods: TCR ζ chain cDNA was cloned from normal human peripheral blood mononuclear cells (PBMC) by RT-PCR and inserted into baculoviral transfer vector. This vector was co-transfected with baculovirus into insect sf 9 cells. The recombinant protein expressed was identified by SDS-PAGE and flow cytometry with mouse anti-human ζ chain monoclonal antibody. Results: Human TCR ζ chain cDNA cloned and inserted into baculoviral vector specifically expressed protein in the insect sf 9 cells, accounting for about 11% of the total protein yield in the supernatant of cell lysate. The molecular weight of the recombinant protein determined by SDS-PAGE was identical to what we anticipated. The insect cells transfected with recombinant baculovirus were demonstrated by intracellular labeling flow cytometry to express ζ chain protein. Conclusion: Human T cell receptor ζ chain gene was successfully cloned from human PBMC, and its encoding protein was highly expressed in insect cells. The TCR ζ chain protein obtained by bioengineering technique is useful for in depth biological function study.
CAO Hui-fang
,
MAN Xiao-bo
,
JIANG Hong
,
XU Zhi-fei
,
TANG Liang
,
QIU Xiu-hua
,
TAN Ye-xiong
,
WU Meng -chao
,
WANG Hong-yang
2003, 10(2):119-121.
DOI: 10.3872/j.issn.1007-385X.2003.2.010
Abstract:
Objective: To quantitatively detect the expression level of spp1 gene in lung carcinoma. Method: The RT-PCR and real-time fluorescence quantitative RT-PCR method was used. Result: The spp1 gene expressed higher in tumor tissue than that in normal tissue in all 24 cases of lung carcinoma. In 9 cases spp1 expression level was about or more than 100 times as that of normal lung tissue. Conclusion: The spp1 gene expressed higher in lung carcinoma tissue than that of normal lung tissue and indicated that it might be an important marker in the diagnosis of the lung cancer.
Objective: To quantitatively detect the expression level of spp1 gene in lung carcinoma. Method: The RT-PCR and real-time fluorescence quantitative RT-PCR method was used. Result: The spp1 gene expressed higher in tumor tissue than that in normal tissue in all 24 cases of lung carcinoma. In 9 cases spp1 expression level was about or more than 100 times as that of normal lung tissue. Conclusion: The spp1 gene expressed higher in lung carcinoma tissue than that of normal lung tissue and indicated that it might be an important marker in the diagnosis of the lung cancer.
WU Jie
,
ZHANG Hong-bin
,
XIAN Jiang
,
YANG Tai-cheng
,
YANG Chuan-hong
,
WANG Jie
,
ZHENG Wen-ling
,
LAI Huang-wen
,
CHEN Hui-peng
2003, 10(2):122-125.
DOI: 10.3872/j.issn.1007-385X.2003.2.011
Abstract:
Objective: One strategy for improving the selectively and toxicity profile of antitumor agents is to design drug carrier systems. Thus a reagent targeting KDR expressed on tumor vasculature was prepared by a peptide binding to KDR specifically which screened from C7 peptide library coupled covalently to NHS-d-Biotin and BSA. Methods: A high affinity peptide specific for KDR was screened by phage display. ELISA, Gradient-ELISA, Competitive-ELISA and Blocking-ELISA were used to detect whether synthesized peptide bind to KDR. To explore whether peptide could be used to deliver agent to target site, synthesized peptide was chemically conjugated with large molecule-BSA and NHS-d-Biotin easily detected by avidin. The binding activity to KDR of chemical compound was determined by Cell-ELISA and Cell-immunohistology. Results:Synthesized P5 peptide having a dissociation constant (Kd) of about 168.6 nM with KDR was approximately 3 fold lower than Kd of VEGF with KDR. P5 blocked VEGF-KDR interaction while did not competes effectively with VEGF for binding KDR. The chemical conjugate, P5-BSA-Biotin, binds specifically to soluble KDR as well as KDR expressed on human endothelial cells. Conclusion:The peptide P5 that home to KDR expressed on tumor vasculature may also be useful in targeting therapies specifically to tumors.
Objective: One strategy for improving the selectively and toxicity profile of antitumor agents is to design drug carrier systems. Thus a reagent targeting KDR expressed on tumor vasculature was prepared by a peptide binding to KDR specifically which screened from C7 peptide library coupled covalently to NHS-d-Biotin and BSA. Methods: A high affinity peptide specific for KDR was screened by phage display. ELISA, Gradient-ELISA, Competitive-ELISA and Blocking-ELISA were used to detect whether synthesized peptide bind to KDR. To explore whether peptide could be used to deliver agent to target site, synthesized peptide was chemically conjugated with large molecule-BSA and NHS-d-Biotin easily detected by avidin. The binding activity to KDR of chemical compound was determined by Cell-ELISA and Cell-immunohistology. Results:Synthesized P5 peptide having a dissociation constant (Kd) of about 168.6 nM with KDR was approximately 3 fold lower than Kd of VEGF with KDR. P5 blocked VEGF-KDR interaction while did not competes effectively with VEGF for binding KDR. The chemical conjugate, P5-BSA-Biotin, binds specifically to soluble KDR as well as KDR expressed on human endothelial cells. Conclusion:The peptide P5 that home to KDR expressed on tumor vasculature may also be useful in targeting therapies specifically to tumors.
2003, 10(2):126-129.
DOI: 10.3872/j.issn.1007-385X.2003.2.012
Abstract:
Objective:To observe antitumor immunity of CD4+ T cells subset in CIKs.Methods: After large scale of amplification in vitro, CD4+ T cells subset in CIKs was isolated by magnetic beads separation columns. Distribution of Th1/ Th2 in CD4+ T cells subset in CIKs was analysized by intracellular cytokine staining. Cytotoxicity of purified CD4+ T cells subset in CIKs against raji cells and apoptosis of raji cells after 4 h and 20 h coculture were determined by LDH method and fluorescent staining method.Results: Purity of enriched CD4+ T cells subset in CIKs reached 96%. Comparing with PBMCs, significant increase in Th1 subset and Th0 subset were observed but no statistical differences were found in Th2 subset. Few raji cells were lysed by CD4+ T cells subset in CIKs after 4 h co-incubation. But after 20 h co-incubation, the same effective lysis of raji cells as CD4- T cells subset was obtained in CD4+ T cells subset in CIKs. Fluorescent staining showed that CD4+ T cells subset in CIKs induced apoptosis of raji after 4 h coculture. Conclusion: The present study suggested that CD4+ T cells in CIKs were not only regulatory cells capable of modulating host immune system, but also immune effectors capable of inducing apoptosis in tumor cells.
Objective:To observe antitumor immunity of CD4+ T cells subset in CIKs.Methods: After large scale of amplification in vitro, CD4+ T cells subset in CIKs was isolated by magnetic beads separation columns. Distribution of Th1/ Th2 in CD4+ T cells subset in CIKs was analysized by intracellular cytokine staining. Cytotoxicity of purified CD4+ T cells subset in CIKs against raji cells and apoptosis of raji cells after 4 h and 20 h coculture were determined by LDH method and fluorescent staining method.Results: Purity of enriched CD4+ T cells subset in CIKs reached 96%. Comparing with PBMCs, significant increase in Th1 subset and Th0 subset were observed but no statistical differences were found in Th2 subset. Few raji cells were lysed by CD4+ T cells subset in CIKs after 4 h co-incubation. But after 20 h co-incubation, the same effective lysis of raji cells as CD4- T cells subset was obtained in CD4+ T cells subset in CIKs. Fluorescent staining showed that CD4+ T cells subset in CIKs induced apoptosis of raji after 4 h coculture. Conclusion: The present study suggested that CD4+ T cells in CIKs were not only regulatory cells capable of modulating host immune system, but also immune effectors capable of inducing apoptosis in tumor cells.
2003, 10(2):130-133.
DOI: 10.3872/j.issn.1007-385X.2003.2.013
Abstract:
Objective:To investigate the cytotoxicity of ICE gene transfection in Combination with Antitumor Chemicals killing Hepatocellular Carcinoma Cells in vitro.Methods:The recombinant plasmid pLXSN-hICE was transferred to virus packing cell PA317 by electroporation method. And then the retrovirus containing human ICE cDNA generated by these PA317 cells were used to transfect human hepatocellular carcinoma cell line HepG2. The apoptosis of transferred cells were examined by gel electrophoresis. The influence of chemotherapeutic drug Carbo-platin to the proliferation of hepatocellular carcinoma cell line SMMC7721 and its derivative cells(SMMC7721-hICE,SMMC7721-antisence hICE,SMMC7721-neo)was observed by incorporation of 3H-TDR. Results: Electrophoresis of DNA displayed the apoptosis ladder of HepG2 transfected by ICE gene. The proliferation of SMMC7721-hICE was significantly suppressed in vitro induced by Carbo-alatin compared to the other three cell lines. Conclusion:ICE gene transfection could greatly increase the susceptibility of SMMC7721 cells to apoptotic cell death following chemotherapy. These findings suggest that combining ICE gene transfection with utilizing antitumor drugs would represent a novel approach for the effective treatment of hepatocellular carcinoma.
Objective:To investigate the cytotoxicity of ICE gene transfection in Combination with Antitumor Chemicals killing Hepatocellular Carcinoma Cells in vitro.Methods:The recombinant plasmid pLXSN-hICE was transferred to virus packing cell PA317 by electroporation method. And then the retrovirus containing human ICE cDNA generated by these PA317 cells were used to transfect human hepatocellular carcinoma cell line HepG2. The apoptosis of transferred cells were examined by gel electrophoresis. The influence of chemotherapeutic drug Carbo-platin to the proliferation of hepatocellular carcinoma cell line SMMC7721 and its derivative cells(SMMC7721-hICE,SMMC7721-antisence hICE,SMMC7721-neo)was observed by incorporation of 3H-TDR. Results: Electrophoresis of DNA displayed the apoptosis ladder of HepG2 transfected by ICE gene. The proliferation of SMMC7721-hICE was significantly suppressed in vitro induced by Carbo-alatin compared to the other three cell lines. Conclusion:ICE gene transfection could greatly increase the susceptibility of SMMC7721 cells to apoptotic cell death following chemotherapy. These findings suggest that combining ICE gene transfection with utilizing antitumor drugs would represent a novel approach for the effective treatment of hepatocellular carcinoma.
2003, 10(2):134-136.
DOI: 10.3872/j.issn.1007-385X.2003.2.014
Abstract:
Objective:To study the propagation and phenotypes changes of killer cell (CD3AK cell activated by CD3 mAb in vitro.Methods: Lymph nodes taken from lung cancer patient is dissociated into single cell suspension by mechanical method and cultured in culture medium added CD3 mAb and a little dose IL-2. We analyze cell immunophenotype by flow cytometry and proliferation by trypan blue exclusion test per 2 days. Results: Immunophenotypic analysis showed that CD3AK expressing CD3, CD8, CD56, CD25 increased, and reached a peak value which is 2.33 times than before culturing in the 8 th day. Conclusion: CD3 mAb added to the culture medium can obviously activate CD3AK cell and stimulate proliferation and keep its killer activity.
Objective:To study the propagation and phenotypes changes of killer cell (CD3AK cell activated by CD3 mAb in vitro.Methods: Lymph nodes taken from lung cancer patient is dissociated into single cell suspension by mechanical method and cultured in culture medium added CD3 mAb and a little dose IL-2. We analyze cell immunophenotype by flow cytometry and proliferation by trypan blue exclusion test per 2 days. Results: Immunophenotypic analysis showed that CD3AK expressing CD3, CD8, CD56, CD25 increased, and reached a peak value which is 2.33 times than before culturing in the 8 th day. Conclusion: CD3 mAb added to the culture medium can obviously activate CD3AK cell and stimulate proliferation and keep its killer activity.
联系方式
- 《中国肿瘤生物治疗杂志》
- 1994年创刊
- 主办单位：Chinese Society of Immunology, Chinese Anti-cancer Association
- 邮编：200433
- 电话：021-81871002-22
- 电子邮箱：cjcb@biother.cn
- 网址:http://www.biother.cn
- 刊号：ISSN 1007-385X
- CN 31-1725/R
- 国内定价： ￥20元/册
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Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.
Chinese Journal Of Cancer Biotheray ® 2025 All Rights Reserved
Supported by:Beijing E-Tiller Technology Development Co., Ltd.