Mobile website
Volume 12,Issue 4,2005 Table of Contents
LIU Xing-tian
,
ZHANG Li-ning
,
WANG Yu-kun
,
WANG Xiao-yan
,
GAO Li-fen
,
WANG Qun
,
LI Na
,
ZHANG Pin
,
GAO Fei
2005, 12(4):244-248.
DOI: 10.3872/j.issn.1007-385X.2005.4.002
Abstract:
Objective:To primarily explore the immunological mechanism of SGVHD and provide a platform for study of graft-versus leukemia.Methods: Lethally irradiated C3H/HeJ mice were reconstructed with syngeneic bone marrow cells and induced with cyclosporine (CsA) for 21d. After cessation of CsA , the clinical symptoms of SGVHD(such as weight loss, diarrhea, hunching, runting) were observed. The histological changes of murine liver, colon, skin, thymus and ear were examined by routine pathological methods. Meanwhile, the expression of cytokines in mice colon and liver were examined by RT-PCR. Results: 67% (19/28) of mice developed GVHD-like clinical symptom in SGVHD-induced group and their pathological detection of liver and colon showed tissue necrosis and increased lymphocyte infiltration .The levels of costimulatory molecule (CD137) and Th1-type cytokines（IL-12, IFN-γ and TNF-α）mRNA were enhanced in liver and colon of SGVHD mice, compared with control mice.Conclusion: The CsA can induce syngeneic BMT recipients of C3H/HeJ mice to develop SGVHD, and costimulatory molecules and Th1 type cytokines played an important role in development of SGVHD.
Objective:To primarily explore the immunological mechanism of SGVHD and provide a platform for study of graft-versus leukemia.Methods: Lethally irradiated C3H/HeJ mice were reconstructed with syngeneic bone marrow cells and induced with cyclosporine (CsA) for 21d. After cessation of CsA , the clinical symptoms of SGVHD(such as weight loss, diarrhea, hunching, runting) were observed. The histological changes of murine liver, colon, skin, thymus and ear were examined by routine pathological methods. Meanwhile, the expression of cytokines in mice colon and liver were examined by RT-PCR. Results: 67% (19/28) of mice developed GVHD-like clinical symptom in SGVHD-induced group and their pathological detection of liver and colon showed tissue necrosis and increased lymphocyte infiltration .The levels of costimulatory molecule (CD137) and Th1-type cytokines（IL-12, IFN-γ and TNF-α）mRNA were enhanced in liver and colon of SGVHD mice, compared with control mice.Conclusion: The CsA can induce syngeneic BMT recipients of C3H/HeJ mice to develop SGVHD, and costimulatory molecules and Th1 type cytokines played an important role in development of SGVHD.
ZHUANG Ran
,
OUYANG Wei-ming
,
XIE Xin
,
ZHANG Xin-hai
,
LAN Tian
,
LI Lin
,
FANG Liang
,
JIN Bo-quan
2005, 12(4):249-252.
DOI: 10.3872/j.issn.1007-385X.2005.4.003
Abstract:
Objective: To clone and express human MAGE-C2 gene and to investigate the expression pattern in transfected eukaryote cells. Methods: MAGE-C2 cDNA was amplified by RT-PCR from total RNA of human colorectal adnocarcinoma cell line SW480. Expression vectors of complete open reading frame (ORF) sequence of MAGE-C2 were constructed by PCR and gene cloning technique. After sequencing, the vectors were transfected into E. coli BL21 and 293T cell, respectively. Recombinant GST-MAGE-C2 fusion protein was expressed via IPTG induction. GST-MAGE-C2 fusion protein was purified through glutathione agarose column. Results:The sequence of cloned MAGE-C2 was identical with that reported in GenBank. GFP-MAGE-C2 fusion protein was localized on nuclear identified by fluorescent microscope. SDS-PAGE and Western blot analysis that purified GST-MAGE-C2 fusion protein exhibited a band with Mr. 70 000. Conclusion: The MAGE-C2 gene was cloned and expressed successfully, which not only provides the immunogen for further preparation of anti-MAGE-C2 antibodies, but also applies to research the mechanism of tumor's pathogenesis and cellular immunity response to MAGE.
Objective: To clone and express human MAGE-C2 gene and to investigate the expression pattern in transfected eukaryote cells. Methods: MAGE-C2 cDNA was amplified by RT-PCR from total RNA of human colorectal adnocarcinoma cell line SW480. Expression vectors of complete open reading frame (ORF) sequence of MAGE-C2 were constructed by PCR and gene cloning technique. After sequencing, the vectors were transfected into E. coli BL21 and 293T cell, respectively. Recombinant GST-MAGE-C2 fusion protein was expressed via IPTG induction. GST-MAGE-C2 fusion protein was purified through glutathione agarose column. Results:The sequence of cloned MAGE-C2 was identical with that reported in GenBank. GFP-MAGE-C2 fusion protein was localized on nuclear identified by fluorescent microscope. SDS-PAGE and Western blot analysis that purified GST-MAGE-C2 fusion protein exhibited a band with Mr. 70 000. Conclusion: The MAGE-C2 gene was cloned and expressed successfully, which not only provides the immunogen for further preparation of anti-MAGE-C2 antibodies, but also applies to research the mechanism of tumor's pathogenesis and cellular immunity response to MAGE.
2005, 12(4):253-257.
DOI: 10.3872/j.issn.1007-385X.2005.4.004
Abstract:
Objective:To investigate the effect of 67 kD laminin receptor antisense oligonucleotides (67kD LN-R ASODN) on the invasion and metastasis abilities of ovarian carcinoma cell line HRA.Methods:The ASODN of 67 kD laminin receptor and its control, sense oligonucleotides (SODN) were synthesized and transfected into the target cells. RT-PCR and FCM methods were performed to evaluate the 67 kD LN-R gene and protein expression levels to identify the efficiency of ASODN. The invasiveness of transfected cells was measured quantitatively by matrigel invasion assays (transwell chamber). Results: The 67 kD LN-R gene and protein expression and invasiveness of HRA cells treated with ASODN of different final concentration were significantly decreased compared with that transfected with SODN and the controls（P<0.05）. Conclusion:67 kD LN-R ASODN has a significant inhibitory effect on the invasiveness of human ovarian carcinoma cell line HRA in a dose-dependent manner. It may become a new gene therapeutic agent for ovarian carcioma.
Objective:To investigate the effect of 67 kD laminin receptor antisense oligonucleotides (67kD LN-R ASODN) on the invasion and metastasis abilities of ovarian carcinoma cell line HRA.Methods:The ASODN of 67 kD laminin receptor and its control, sense oligonucleotides (SODN) were synthesized and transfected into the target cells. RT-PCR and FCM methods were performed to evaluate the 67 kD LN-R gene and protein expression levels to identify the efficiency of ASODN. The invasiveness of transfected cells was measured quantitatively by matrigel invasion assays (transwell chamber). Results: The 67 kD LN-R gene and protein expression and invasiveness of HRA cells treated with ASODN of different final concentration were significantly decreased compared with that transfected with SODN and the controls（P<0.05）. Conclusion:67 kD LN-R ASODN has a significant inhibitory effect on the invasiveness of human ovarian carcinoma cell line HRA in a dose-dependent manner. It may become a new gene therapeutic agent for ovarian carcioma.
4 The Bladder Cancer-Specificity and Activity of the Expression of Smac Gene Promoted by UPIb Promoter
2005, 12(4):258-261.
DOI: 10.3872/j.issn.1007-385X.2005.4.005
Abstract:
Objective: To investigate the bladder cancer-specificity and activity of the expression of Smac gene promoted by UpIb promoter. Methods:Smac mRNA and protein were detected in BIU87 cell line and many other non-bladder cancer cell lines before and after transfection with pcDNA3-UpIb promoter-Smac including UpIb promoter and Smac gene by RT-PCR and immunohistochemical method, respectively.Results:The expression of Smac mRNA in BIU87 cell line increased about 2.1 times after transfection . The Smac protein positive cell rate of BIU-87 cell line was about 25.6% before transfection and it increased to about 70.5% after transfection. There was no significant difference about the expression of Smac mRNA and protein in non-bladder cancer cell lines before and after transfection. Conclusions: UpIb promoter could promote the expression of Smac gene with bladder cancer-specificity and considerable activity, which laid the foundation of target gene therapy for bladder cancer.
Objective: To investigate the bladder cancer-specificity and activity of the expression of Smac gene promoted by UpIb promoter. Methods:Smac mRNA and protein were detected in BIU87 cell line and many other non-bladder cancer cell lines before and after transfection with pcDNA3-UpIb promoter-Smac including UpIb promoter and Smac gene by RT-PCR and immunohistochemical method, respectively.Results:The expression of Smac mRNA in BIU87 cell line increased about 2.1 times after transfection . The Smac protein positive cell rate of BIU-87 cell line was about 25.6% before transfection and it increased to about 70.5% after transfection. There was no significant difference about the expression of Smac mRNA and protein in non-bladder cancer cell lines before and after transfection. Conclusions: UpIb promoter could promote the expression of Smac gene with bladder cancer-specificity and considerable activity, which laid the foundation of target gene therapy for bladder cancer.
2005, 12(4):262-265.
DOI: 10.3872/j.issn.1007-385X.2005.4.006
Abstract:
Objective: To investigate the effect of NDGA, the lipoxygenase inhibitor, on colon cancer cell line HT- 29 in vitro from the aspects of cell growth inhibition, cell apoptosis and its impact on the expression of telomerase. Methods: We applied respectively i) MTT to draw the growth curve. ii) inverted phase contrast microscope to observe morphologic change of cells, iii) scanning electron microscope to observe changes of cell′s ultra- microstructure and apoptotic body. iv) RT-PCR to detect the changes of the expression of human telomerase reverse transeriptase (hTERT).Results:Different concentrations of NDGA were used to dispose cancer cells separately, with the rise of the drugis concentration, the form of cells became round, the volume waned, cells abscised from the inner surface of the bottle. and growth inhibition became increasing abvious. Also through scaming electron microscope, apoptic bodies could be found colon cancer cell line HT- 29 showed positive expression of hTERTmRNA, which became weaker following the rising of the drug's concentration. Conclusions: NDGA which, displays the relying effect of doses, can inhibit the growth of colon cancer cell line HT- 29 and induce its apoptosis, telomerase plays an active role in this course.
Objective: To investigate the effect of NDGA, the lipoxygenase inhibitor, on colon cancer cell line HT- 29 in vitro from the aspects of cell growth inhibition, cell apoptosis and its impact on the expression of telomerase. Methods: We applied respectively i) MTT to draw the growth curve. ii) inverted phase contrast microscope to observe morphologic change of cells, iii) scanning electron microscope to observe changes of cell′s ultra- microstructure and apoptotic body. iv) RT-PCR to detect the changes of the expression of human telomerase reverse transeriptase (hTERT).Results:Different concentrations of NDGA were used to dispose cancer cells separately, with the rise of the drugis concentration, the form of cells became round, the volume waned, cells abscised from the inner surface of the bottle. and growth inhibition became increasing abvious. Also through scaming electron microscope, apoptic bodies could be found colon cancer cell line HT- 29 showed positive expression of hTERTmRNA, which became weaker following the rising of the drug's concentration. Conclusions: NDGA which, displays the relying effect of doses, can inhibit the growth of colon cancer cell line HT- 29 and induce its apoptosis, telomerase plays an active role in this course.
2005, 12(4):266-271.
DOI: 10.3872/j.issn.1007-385X.2005.4.007
Abstract:
Objective:To investigate effect of exogenous wild-type p53 gene on the cell growth and tumorigenicity of human gallbladder cancer cell lines.Methods:After identification of the genetic status of p53 gene of GBC-SD cell lines with the immunocytochemistry staining and the direct sequencing technique of PCR products, eukaryotic expressing plasmid pCMV-p53 was introduced by lipofectamine-mediated into GBC-SD cell lines. Growing transfected cells were selected by G418. The presence and expression of exogenous p53 gene was detected by PCR, RT-PCR and Western blot. The cellular proliferating ability was assessed using the cell growth curve and cloning assay. The xenograft in nude mice was performed to examine the effect of tumorigenicity.Results: P53 protein overexpression was showed in GBC-SD cell lines. A transversion of TAC→AAC at codon 126 of exon 5 was confirmed. PCR, RT-PCR and Western blot showed exogenous p53 gene had successfully transfected into GBC-SD cells and obtained high expression. The growth and proliferation of the cells were greatly decreased, and the tumorigenicity was significantly inhibited after transfection wtp53.Conclusion:The expression of exogenous wild-type p53 gene could effectively inhibit the growth of gallbladder cancer GBC-SD cells in vitro and in vivo.
Objective:To investigate effect of exogenous wild-type p53 gene on the cell growth and tumorigenicity of human gallbladder cancer cell lines.Methods:After identification of the genetic status of p53 gene of GBC-SD cell lines with the immunocytochemistry staining and the direct sequencing technique of PCR products, eukaryotic expressing plasmid pCMV-p53 was introduced by lipofectamine-mediated into GBC-SD cell lines. Growing transfected cells were selected by G418. The presence and expression of exogenous p53 gene was detected by PCR, RT-PCR and Western blot. The cellular proliferating ability was assessed using the cell growth curve and cloning assay. The xenograft in nude mice was performed to examine the effect of tumorigenicity.Results: P53 protein overexpression was showed in GBC-SD cell lines. A transversion of TAC→AAC at codon 126 of exon 5 was confirmed. PCR, RT-PCR and Western blot showed exogenous p53 gene had successfully transfected into GBC-SD cells and obtained high expression. The growth and proliferation of the cells were greatly decreased, and the tumorigenicity was significantly inhibited after transfection wtp53.Conclusion:The expression of exogenous wild-type p53 gene could effectively inhibit the growth of gallbladder cancer GBC-SD cells in vitro and in vivo.
2005, 12(4):272-274.
DOI: 10.3872/j.issn.1007-385X.2005.4.008
Abstract:
Objective: To evaluate the cytotoxic effect of PEM induced by HSPgp96 on anti-tumor in vitro. Methods:PEM separated from mice induced by thioglycolate were divided into three groups randomly: Culture medium in control; LPS-induced group; HSPgp96-induced group. The production of NO, the cytotoxic effect to H22 cells and the morphologic change of PEM were investigated separately by enzyme method, MTT assay and scanning electron microscope. Results:In vitro, HSPgp96 can increased NO production from PEM of mice and significantly enhance the cytotoxic effect of PEM to H22 cells as well as LPS. Conclusion:HSPgp96 can effectively induce the cytotoxic effect of PEM on anti-tumor in which NO is one of the capital effective molecules in vitro.
Objective: To evaluate the cytotoxic effect of PEM induced by HSPgp96 on anti-tumor in vitro. Methods:PEM separated from mice induced by thioglycolate were divided into three groups randomly: Culture medium in control; LPS-induced group; HSPgp96-induced group. The production of NO, the cytotoxic effect to H22 cells and the morphologic change of PEM were investigated separately by enzyme method, MTT assay and scanning electron microscope. Results:In vitro, HSPgp96 can increased NO production from PEM of mice and significantly enhance the cytotoxic effect of PEM to H22 cells as well as LPS. Conclusion:HSPgp96 can effectively induce the cytotoxic effect of PEM on anti-tumor in which NO is one of the capital effective molecules in vitro.
2005, 12(4):275-279.
DOI: 10.3872/j.issn.1007-385X.2005.4.009
Abstract:
Objective:To effectively express human M-CSF soluble receptor in tobacco plants and study its anti-M-CSF activity. Methods:The plant expression plasmid expressing recombinant human M-CSF soluble receptor with C-terminal His-tag and endoplasmic reticulum retention sequence was constructed. Then the target gene was introduced into tobacco genome by agrobacterium tumefaciens-mediated transformation. PCR and Western blot were used to select the transgenic tobacco plants. ELISA was used to evaluate the expression level while colony forming assay was used to test its biological activity. Results:More than 50 transgenic tobacco plants were developed. PCR results showed the insertion of target gene into tobacco genome and Western blot results showed the expression of recombinant protein. The recombinant M-CSFsR was effectively expressed, however, the expression levels varied among different transgenic tobacco plants, with the maximum reaching 1.92% of total soluble protein in leaf tissues. The tobacco-derived M-CSFsR could inhibit the colony formation of J6-1 cells. Conclusion:Bioactive recombinant human M-CSFsR was expressed in transgenic tobacco plants at high level.
Objective:To effectively express human M-CSF soluble receptor in tobacco plants and study its anti-M-CSF activity. Methods:The plant expression plasmid expressing recombinant human M-CSF soluble receptor with C-terminal His-tag and endoplasmic reticulum retention sequence was constructed. Then the target gene was introduced into tobacco genome by agrobacterium tumefaciens-mediated transformation. PCR and Western blot were used to select the transgenic tobacco plants. ELISA was used to evaluate the expression level while colony forming assay was used to test its biological activity. Results:More than 50 transgenic tobacco plants were developed. PCR results showed the insertion of target gene into tobacco genome and Western blot results showed the expression of recombinant protein. The recombinant M-CSFsR was effectively expressed, however, the expression levels varied among different transgenic tobacco plants, with the maximum reaching 1.92% of total soluble protein in leaf tissues. The tobacco-derived M-CSFsR could inhibit the colony formation of J6-1 cells. Conclusion:Bioactive recombinant human M-CSFsR was expressed in transgenic tobacco plants at high level.
2005, 12(4):280-284.
DOI: 10.3872/j.issn.1007-385X.2005.4.010
Abstract:
Objective:To investigate the expression of VEGF and its receptor (KDR) in prostate cancer cell lines. Methods: Immunocytochemical staining and Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR)were employed to test the expression of VEGF and its receptor KDR in five human prostate cancer cell lines, LNCaP, PC-3, PC-3M, DU-145, 22-RV1. ELISA was employed to test VEGF concentration in culture supernatant of prostate cancer cell lines. Results:All five prostate cancer cell lines expressed VEGF and KDR. The expression of protein and mRNA in PC-3, DU-145 and LNCaP cell lines was higher than that in PC-3M and 22RV1 cell lines (P< 0. 01). The result of the VEGF concentration in cell culture supernatants is as same as that of immunocytochemical staining.Conclusion: Although they were expressed differentially, both VEGF and KDR were present in prostate cancer cell lines. The expression of VEGF and KDR might be related with the pathogenesis and progress of prostate cancer.
Objective:To investigate the expression of VEGF and its receptor (KDR) in prostate cancer cell lines. Methods: Immunocytochemical staining and Western blot and reverse transcriptase-polymerase chain reaction(RT-PCR)were employed to test the expression of VEGF and its receptor KDR in five human prostate cancer cell lines, LNCaP, PC-3, PC-3M, DU-145, 22-RV1. ELISA was employed to test VEGF concentration in culture supernatant of prostate cancer cell lines. Results:All five prostate cancer cell lines expressed VEGF and KDR. The expression of protein and mRNA in PC-3, DU-145 and LNCaP cell lines was higher than that in PC-3M and 22RV1 cell lines (P< 0. 01). The result of the VEGF concentration in cell culture supernatants is as same as that of immunocytochemical staining.Conclusion: Although they were expressed differentially, both VEGF and KDR were present in prostate cancer cell lines. The expression of VEGF and KDR might be related with the pathogenesis and progress of prostate cancer.
2005, 12(4):285-288.
DOI: 10.3872/j.issn.1007-385X.2005.4.011
Abstract:
Objective:To prepare and cryopreserve umbilical cord blood(UCB) units for clinical hematopoietic stem cell transplantation. Methods:HSC of UCB units that met our standards were collected, separated cryopreserved and tested. Results:4 500 (72.2%) of 6 232 cord blood units were suitable for preservation. Average net volume, numbers of total nucleated cells, percentage of CD34+ cells and CFU-C of the UCB units were 90.7±22.2 ml, 10.5±3.4×108, 024±0.15%, 78.9±44.14/105 NC, respectively. After separation, the cell recovery of nucleated cells, CD34+ cells and CFU-C were 82.1±6.52%, 92.5±6.15% and 90±9.67%, respectively. The cell viability after separating was over 95%.After thawing, the recovery of nucleated cells, CD34+ cells and CFU-C were 84.2±10.1%, 96.5±21.8% and 115.1±23.3%, respectively. The cell viability was 93.8±4.4%. Among the 569 searching cases , 532 (93%) cases could found 4/6 of HLA-A, B and DR loci matched UCB units in our bank, including 39 (6.58%) found 6 loci matched and 237 (41.65%) found 5 loci matched. Conclusion:These results indicated that we can supply high quality cord blood HSC for clinical transplantation.
Objective:To prepare and cryopreserve umbilical cord blood(UCB) units for clinical hematopoietic stem cell transplantation. Methods:HSC of UCB units that met our standards were collected, separated cryopreserved and tested. Results:4 500 (72.2%) of 6 232 cord blood units were suitable for preservation. Average net volume, numbers of total nucleated cells, percentage of CD34+ cells and CFU-C of the UCB units were 90.7±22.2 ml, 10.5±3.4×108, 024±0.15%, 78.9±44.14/105 NC, respectively. After separation, the cell recovery of nucleated cells, CD34+ cells and CFU-C were 82.1±6.52%, 92.5±6.15% and 90±9.67%, respectively. The cell viability after separating was over 95%.After thawing, the recovery of nucleated cells, CD34+ cells and CFU-C were 84.2±10.1%, 96.5±21.8% and 115.1±23.3%, respectively. The cell viability was 93.8±4.4%. Among the 569 searching cases , 532 (93%) cases could found 4/6 of HLA-A, B and DR loci matched UCB units in our bank, including 39 (6.58%) found 6 loci matched and 237 (41.65%) found 5 loci matched. Conclusion:These results indicated that we can supply high quality cord blood HSC for clinical transplantation.
SUN Yan
,
WANG Hong-jun
,
ZHANG Wei
,
WU Yang
,
ZHANG Zhi-qiang
,
Feng Li
,
WANG Li-qiang
,
WU Yi-min
2005, 12(4):289-291.
DOI: 10.3872/j.issn.1007-385X.2005.4.012
Abstract:
Objective:To extract Usnic acid from usnea longisima ach, and to evaluate the ability of Usnic acid to inhibit growth of human prostate cancer cells PC-3M in vitro. Methods:The cells were observed by microscope.The cytotoxicity to cancer cells was determined by MTT assay. DNA synthesis was tested by 3H-TdR in corporation assay in the experiments. Results: There were significant differences between PC-3M cells lest group and control group. The Usnic acid had streng thened cytotoxicity, and the rate of 3H-TdR incorporation was lower than that of the control in a dose-dependent manner (r=0.9799, r=-0.9525). Conclusion: The results suggested that the Usnic acid has inhibitory effects on growth of human PC-3M cells in vitro.
Objective:To extract Usnic acid from usnea longisima ach, and to evaluate the ability of Usnic acid to inhibit growth of human prostate cancer cells PC-3M in vitro. Methods:The cells were observed by microscope.The cytotoxicity to cancer cells was determined by MTT assay. DNA synthesis was tested by 3H-TdR in corporation assay in the experiments. Results: There were significant differences between PC-3M cells lest group and control group. The Usnic acid had streng thened cytotoxicity, and the rate of 3H-TdR incorporation was lower than that of the control in a dose-dependent manner (r=0.9799, r=-0.9525). Conclusion: The results suggested that the Usnic acid has inhibitory effects on growth of human PC-3M cells in vitro.
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
您是第位访问者
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.