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Volume 8,Issue 1,2001 Table of Contents
2001, 8(1):6-9.
DOI: 10.3872/j.issn.1007-385X.2001.1.003
Abstract:
研究CpG联合非复制重组痘苗病毒在肿瘤免疫治疗中的效果。方法 :构建非复制重组痘苗病毒v△ 11β75 ,联合应用免疫刺激寡核苷酸和重组痘苗病毒v△ 11β75进行肿瘤免疫治疗。 结果 :重组痘苗病毒v△ 11β75在人源 143细胞中的不能正常繁殖 ,Southern blot证实痘苗病毒天坛株HindⅢC ,K片段间基因的缺失。在Wistar大鼠的Walker′s肿瘤模型中 ,联合应用CpG ODN和非复制痘苗病毒修饰的WRC2 5 6细胞裂解物进行免疫治疗 ,可以延长荷瘤鼠的生存期 ,肿瘤生长速度降低。结论 :CpG ODN可以增强非复制痘苗病毒修饰的肿瘤细胞裂解物的抗肿瘤治疗效果 ,为肿瘤免疫治疗提供了一种新的途径。
研究CpG联合非复制重组痘苗病毒在肿瘤免疫治疗中的效果。方法 :构建非复制重组痘苗病毒v△ 11β75 ,联合应用免疫刺激寡核苷酸和重组痘苗病毒v△ 11β75进行肿瘤免疫治疗。 结果 :重组痘苗病毒v△ 11β75在人源 143细胞中的不能正常繁殖 ,Southern blot证实痘苗病毒天坛株HindⅢC ,K片段间基因的缺失。在Wistar大鼠的Walker′s肿瘤模型中 ,联合应用CpG ODN和非复制痘苗病毒修饰的WRC2 5 6细胞裂解物进行免疫治疗 ,可以延长荷瘤鼠的生存期 ,肿瘤生长速度降低。结论 :CpG ODN可以增强非复制痘苗病毒修饰的肿瘤细胞裂解物的抗肿瘤治疗效果 ,为肿瘤免疫治疗提供了一种新的途径。
2001, 8(1):10-12.
DOI: 10.3872/j.issn.1007-385X.2001.1.004
Abstract:
Objective:Express and purify the recombant protein in prokaryotic system, preparing the mouse anti human angiostatin polyclony antibody.Methods: The cDNA of angiostatin was amplified by PCR and was subcloned into vector pQE-30, and transformed into the E.coli BL21(DE3). At last angiostatin was purified and used to immune the mouse for preparing polyclony antibody .Results: The recombinant clones were picked out by the methods of restrictional enzyme cut and sequence analysis. Recombinant angiostatin was highly expressed when the strain was induced with 1mmol/L IPTG. Angiostatin was successfully purified and the polyclony antibody was also successfully prepared. Conclusion: The recombinant protein and the polyclony antibody can be used in further studies.
Objective:Express and purify the recombant protein in prokaryotic system, preparing the mouse anti human angiostatin polyclony antibody.Methods: The cDNA of angiostatin was amplified by PCR and was subcloned into vector pQE-30, and transformed into the E.coli BL21(DE3). At last angiostatin was purified and used to immune the mouse for preparing polyclony antibody .Results: The recombinant clones were picked out by the methods of restrictional enzyme cut and sequence analysis. Recombinant angiostatin was highly expressed when the strain was induced with 1mmol/L IPTG. Angiostatin was successfully purified and the polyclony antibody was also successfully prepared. Conclusion: The recombinant protein and the polyclony antibody can be used in further studies.
2001, 8(1):13-16.
DOI: 10.3872/j.issn.1007-385X.2001.1.005
Abstract:
Objective:Express and purify the recombant protein in prokaryotic system, preparing the mouse anti human angiostatin polyclony antibody.Methods: The cDNA of angiostatin was amplified by PCR and was subcloned into vector pQE-30, and transformed into the E.coli BL21(DE3). At last angiostatin was purified and used to immune the mouse for preparing polyclony antibody .Results: The recombinant clones were picked out by the methods of restrictional enzyme cut and sequence analysis. Recombinant angiostatin was highly expressed when the strain was induced with 1mmol/L IPTG. Angiostatin was successfully purified and the polyclony antibody was also successfully prepared. Conclusion: The recombinant protein and the polyclony antibody can be used in further studies.
Objective:Express and purify the recombant protein in prokaryotic system, preparing the mouse anti human angiostatin polyclony antibody.Methods: The cDNA of angiostatin was amplified by PCR and was subcloned into vector pQE-30, and transformed into the E.coli BL21(DE3). At last angiostatin was purified and used to immune the mouse for preparing polyclony antibody .Results: The recombinant clones were picked out by the methods of restrictional enzyme cut and sequence analysis. Recombinant angiostatin was highly expressed when the strain was induced with 1mmol/L IPTG. Angiostatin was successfully purified and the polyclony antibody was also successfully prepared. Conclusion: The recombinant protein and the polyclony antibody can be used in further studies.
2001, 8(1):17-19.
DOI: 10.3872/j.issn.1007-385X.2001.1.006
Abstract:
Objective: To investigate the correlation between the expression of vascular endothelial growth factor(VEGF) and angiogenesis in tumor. Methods: VEGF 165 sense and antisense gene recombinants were introduced into human gastric cancer cells, respectively. Then the growth of transfected cells in nude mice and the microvascular density and histological change were examined. Results: The growth rate of tumor in nude mice inoculated with sense VEGF cells was markedly higher than that in nude mice inoculated with antisense-VEGF cells. In histological examination, the microvascular density in tumor caused by sense-VEGF cells was greatly higher than that in tumor caused by antisense VEGF cells. Conclusion: As starting angiogenesis, VEGF might promote the growth of tumor, and the inhibition of VEGF production might prevent solid tumor from growing.
Objective: To investigate the correlation between the expression of vascular endothelial growth factor(VEGF) and angiogenesis in tumor. Methods: VEGF 165 sense and antisense gene recombinants were introduced into human gastric cancer cells, respectively. Then the growth of transfected cells in nude mice and the microvascular density and histological change were examined. Results: The growth rate of tumor in nude mice inoculated with sense VEGF cells was markedly higher than that in nude mice inoculated with antisense-VEGF cells. In histological examination, the microvascular density in tumor caused by sense-VEGF cells was greatly higher than that in tumor caused by antisense VEGF cells. Conclusion: As starting angiogenesis, VEGF might promote the growth of tumor, and the inhibition of VEGF production might prevent solid tumor from growing.
2001, 8(1):20-22.
DOI: 10.3872/j.issn.1007-385X.2001.1.007
Abstract:
Objective: To generate an adenovirus, Ad-CD, expressing green fluorescence protein and bacterial cytosine deaminase, and use it for tumor suicide gene therapy. Methods: CD gene was cloned into pAdTrack-CMV, pAdTrack-CMV-CD and pAdEasy-1 were homologous recominated in bacteria. 293 cells were transfected by the newly-recombinated plasmid pAd-CD and generated adenoviruses. LoVo cells were infected with the adenovirus, and the tumor-killing effect after 5-FC was administered.Results: pAdTrack-CMV-CD and the newly-recombinated plasmid pAd-CD were tested by restriction endonuclease digestions. Followed by GFP, Ad-CD were produced in 293 and LoVo cells infected by Ad-CD were killed after 5-FC was administered.Conclusion: Adenovirus plasmid homologous recombinant in bacteria is a simple method for adenovirus generation. In vitro adenoviral carried CD gene is efficient for tumor cell suicide gene therapy and has a bystand-effect, which is not depended on cell direct contact
Objective: To generate an adenovirus, Ad-CD, expressing green fluorescence protein and bacterial cytosine deaminase, and use it for tumor suicide gene therapy. Methods: CD gene was cloned into pAdTrack-CMV, pAdTrack-CMV-CD and pAdEasy-1 were homologous recominated in bacteria. 293 cells were transfected by the newly-recombinated plasmid pAd-CD and generated adenoviruses. LoVo cells were infected with the adenovirus, and the tumor-killing effect after 5-FC was administered.Results: pAdTrack-CMV-CD and the newly-recombinated plasmid pAd-CD were tested by restriction endonuclease digestions. Followed by GFP, Ad-CD were produced in 293 and LoVo cells infected by Ad-CD were killed after 5-FC was administered.Conclusion: Adenovirus plasmid homologous recombinant in bacteria is a simple method for adenovirus generation. In vitro adenoviral carried CD gene is efficient for tumor cell suicide gene therapy and has a bystand-effect, which is not depended on cell direct contact
2001, 8(1):23-26.
DOI: 10.3872/j.issn.1007-385X.2001.1.008
Abstract:
Objective: To investigate the correlation between the expression of vascular endothelial growth factor(VEGF) and angiogenesis in tumor. Methods: VEGF 165 sense and antisense gene recombinants were introduced into human gastric cancer cells, respectively. Then the growth of transfected cells in nude mice and the microvascular density and histological change were examined. Results: The growth rate of tumor in nude mice inoculated with sense VEGF cells was markedly higher than that in nude mice inoculated with antisense-VEGF cells. In histological examination, the microvascular density in tumor caused by sense-VEGF cells was greatly higher than that in tumor caused by antisense VEGF cells. Conclusion: As starting angiogenesis, VEGF might promote the growth of tumor, and the inhibition of VEGF production might prevent solid tumor from growing.
Objective: To investigate the correlation between the expression of vascular endothelial growth factor(VEGF) and angiogenesis in tumor. Methods: VEGF 165 sense and antisense gene recombinants were introduced into human gastric cancer cells, respectively. Then the growth of transfected cells in nude mice and the microvascular density and histological change were examined. Results: The growth rate of tumor in nude mice inoculated with sense VEGF cells was markedly higher than that in nude mice inoculated with antisense-VEGF cells. In histological examination, the microvascular density in tumor caused by sense-VEGF cells was greatly higher than that in tumor caused by antisense VEGF cells. Conclusion: As starting angiogenesis, VEGF might promote the growth of tumor, and the inhibition of VEGF production might prevent solid tumor from growing.
2001, 8(1):27-30.
DOI: 10.3872/j.issn.1007-385X.2001.1.009
Abstract:
Objective: To generate an adenovirus, Ad-CD, expressing green fluorescence protein and bacterial cytosine deaminase, and use it for tumor suicide gene therapy. Methods: CD gene was cloned into pAdTrack-CMV, pAdTrack-CMV-CD and pAdEasy-1 were homologous recominated in bacteria. 293 cells were transfected by the newly-recombinated plasmid pAd-CD and generated adenoviruses. LoVo cells were infected with the adenovirus, and the tumor-killing effect after 5-FC was administered.Results: pAdTrack-CMV-CD and the newly-recombinated plasmid pAd-CD were tested by restriction endonuclease digestions. Followed by GFP, Ad-CD were produced in 293 and LoVo cells infected by Ad-CD were killed after 5-FC was administered.Conclusion: Adenovirus plasmid homologous recombinant in bacteria is a simple method for adenovirus generation. In vitro adenoviral carried CD gene is efficient for tumor cell suicide gene therapy and has a bystand-effect, which is not depended on cell direct contact
Objective: To generate an adenovirus, Ad-CD, expressing green fluorescence protein and bacterial cytosine deaminase, and use it for tumor suicide gene therapy. Methods: CD gene was cloned into pAdTrack-CMV, pAdTrack-CMV-CD and pAdEasy-1 were homologous recominated in bacteria. 293 cells were transfected by the newly-recombinated plasmid pAd-CD and generated adenoviruses. LoVo cells were infected with the adenovirus, and the tumor-killing effect after 5-FC was administered.Results: pAdTrack-CMV-CD and the newly-recombinated plasmid pAd-CD were tested by restriction endonuclease digestions. Followed by GFP, Ad-CD were produced in 293 and LoVo cells infected by Ad-CD were killed after 5-FC was administered.Conclusion: Adenovirus plasmid homologous recombinant in bacteria is a simple method for adenovirus generation. In vitro adenoviral carried CD gene is efficient for tumor cell suicide gene therapy and has a bystand-effect, which is not depended on cell direct contact
2001, 8(1):31-33.
DOI: 10.3872/j.issn.1007-385X.2001.1.010
Abstract:
Objective: To improve the preparation of adherent lymphokine-activated killer cells (A-LAK cells) and to study the synergistic anti-tumor effect of phenylacetate(PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant derived from SMMC7721 cells treated with PA previously and the changes of proliferation and anti-tumor activity of A-LAK cells were observed. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) as well as standard LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensely suppressed the proliferation and cytotoxic activity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK could be simply prepared by using PME, and have the synergistic anti-tumor effect with phenylacetate.
Objective: To improve the preparation of adherent lymphokine-activated killer cells (A-LAK cells) and to study the synergistic anti-tumor effect of phenylacetate(PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant derived from SMMC7721 cells treated with PA previously and the changes of proliferation and anti-tumor activity of A-LAK cells were observed. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) as well as standard LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensely suppressed the proliferation and cytotoxic activity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK could be simply prepared by using PME, and have the synergistic anti-tumor effect with phenylacetate.
2001, 8(1):34-36.
DOI: 10.3872/j.issn.1007-385X.2001.1.011
Abstract:
Objective: To investigate the gene regulation of the promoter of human telomerase reverse transcriptase (htert) by transcription factor c-myc. Methods: The various plasmids were transfected into cell lines NIH3T3, COS-7 and HepG 2 by Lipofect respectively. The plasmids included c-myc expressing plasmid and its control vector, the plasmid TERTLuc(800) harboring 800 bp of htert promoter and TERTLuc(800)DM, which was mutated in c-myc binding sites of TERTLuc(800). The reporter gene luciferase activities were measured 48 hours after transfection. Results: The plasmid TERTLuc(800), generated stronger activity in hepatoma cell line HepG 2. Transcription factor c-myc tran-activated the htert promoter in a dose-dependent manner. In addition, mutation of the c-myc binding sites in the htert promoter decreased its activity by 50%. c-myc up-regulated the htert promoter activity through the c-myc binding sites. Conclusion: Our results indicate that c-myc can trans-activate the htert promoter activity.
Objective: To investigate the gene regulation of the promoter of human telomerase reverse transcriptase (htert) by transcription factor c-myc. Methods: The various plasmids were transfected into cell lines NIH3T3, COS-7 and HepG 2 by Lipofect respectively. The plasmids included c-myc expressing plasmid and its control vector, the plasmid TERTLuc(800) harboring 800 bp of htert promoter and TERTLuc(800)DM, which was mutated in c-myc binding sites of TERTLuc(800). The reporter gene luciferase activities were measured 48 hours after transfection. Results: The plasmid TERTLuc(800), generated stronger activity in hepatoma cell line HepG 2. Transcription factor c-myc tran-activated the htert promoter in a dose-dependent manner. In addition, mutation of the c-myc binding sites in the htert promoter decreased its activity by 50%. c-myc up-regulated the htert promoter activity through the c-myc binding sites. Conclusion: Our results indicate that c-myc can trans-activate the htert promoter activity.
LUO Kun
,
JIN Ningyi
,
WU Congmei
,
GUO Zhiru
,
QUN Yunlong
,
GUO Yan
,
XIA Zhiping
,
AN Ruguo
,
YIN Zhen
2001, 8(1):37-40.
DOI: 10.3872/j.issn.1007-385X.2001.1.012
Abstract:
Objective: To research influence of consecutive Immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy priming with recombinant fowlpox virus vUTALG and boosting with nucleic acid vaccine plasmid pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4 cells from splenic lymphocytes increased singnificantly and the proliferation response of splenocytes to Con A, LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity response in mice.
Objective: To research influence of consecutive Immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy priming with recombinant fowlpox virus vUTALG and boosting with nucleic acid vaccine plasmid pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4 cells from splenic lymphocytes increased singnificantly and the proliferation response of splenocytes to Con A, LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity response in mice.
2001, 8(1):41-44.
DOI: 10.3872/j.issn.1007-385X.2001.1.013
Abstract:
Objective: To improve the preparation of adherent lymphokine-activated killer cells (A-LAK cells) and to study the synergistic anti-tumor effect of phenylacetate(PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant derived from SMMC7721 cells treated with PA previously and the changes of proliferation and anti-tumor activity of A-LAK cells were observed. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) as well as standard LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensely suppressed the proliferation and cytotoxic activity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK could be simply prepared by using PME, and have the synergistic anti-tumor effect with phenylacetate.
Objective: To improve the preparation of adherent lymphokine-activated killer cells (A-LAK cells) and to study the synergistic anti-tumor effect of phenylacetate(PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant derived from SMMC7721 cells treated with PA previously and the changes of proliferation and anti-tumor activity of A-LAK cells were observed. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) as well as standard LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensely suppressed the proliferation and cytotoxic activity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK could be simply prepared by using PME, and have the synergistic anti-tumor effect with phenylacetate.
2001, 8(1):45-48.
DOI: 10.3872/j.issn.1007-385X.2001.1.014
Abstract:
Objective: To investigate in vitro the biological characteristics of AdCD80-infected human lung cancer cells on the basis of generation of replication-deficient hB7-1(CD80) recombinant adenovirus. Methods: Human CD80 gene was transduced into lung cancer cells mediated by recombinant adenovirus and then the expression of the gene was detected by PCR and agarose gel electrophoresis. The biological characteristics of the above cells were analysed with electron microscope, FACS and etc. Results: The titers of rAd reached to 10 10 PFU/ml and more than 90% Anip973 lung cancer cells could be infected by 30 MOI rAd. The growth curve and cloning efficiency of rAdCD80-infected Anip973 cells showed no significant difference compared with that of the control cells. The cell proliferation cycle of rAdCD80-infected 973 cells showed no change through FACS test. Having been infected by rAdCD80, the surface structure and ultrastructure of 973 cells had a little change. Conclusion: These results will lay foundation for tumor vaccines.
Objective: To investigate in vitro the biological characteristics of AdCD80-infected human lung cancer cells on the basis of generation of replication-deficient hB7-1(CD80) recombinant adenovirus. Methods: Human CD80 gene was transduced into lung cancer cells mediated by recombinant adenovirus and then the expression of the gene was detected by PCR and agarose gel electrophoresis. The biological characteristics of the above cells were analysed with electron microscope, FACS and etc. Results: The titers of rAd reached to 10 10 PFU/ml and more than 90% Anip973 lung cancer cells could be infected by 30 MOI rAd. The growth curve and cloning efficiency of rAdCD80-infected Anip973 cells showed no significant difference compared with that of the control cells. The cell proliferation cycle of rAdCD80-infected 973 cells showed no change through FACS test. Having been infected by rAdCD80, the surface structure and ultrastructure of 973 cells had a little change. Conclusion: These results will lay foundation for tumor vaccines.
2001, 8(1):52-54.
DOI: 10.3872/j.issn.1007-385X.2001.1.016
Abstract:
Objective: To research influence of consecutive Immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy priming with recombinant fowlpox virus vUTALG and boosting with nucleic acid vaccine plasmid pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4 cells from splenic lymphocytes increased singnificantly and the proliferation response of splenocytes to Con A, LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity response in mice.
Objective: To research influence of consecutive Immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy priming with recombinant fowlpox virus vUTALG and boosting with nucleic acid vaccine plasmid pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4 cells from splenic lymphocytes increased singnificantly and the proliferation response of splenocytes to Con A, LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity response in mice.
2001, 8(1):55-58.
DOI: 10.3872/j.issn.1007-385X.2001.1.017
Abstract:
研究HBV启动子调控下的GFP报告基因在肿瘤细胞中的表达。方法 :采用DNA重组技术 ,将GFPs65T和HBV的EⅡmCMV启动子 (增强子 )亚克隆到哺乳动物表达载体pcDNA3 中 ,构建了pcDNA3 GFP ,pcDNA3 GFP EⅡ ,pcDNA3 GFP EⅡ w3种含GFP的重组质粒 ,在Lipofectin介导下分别转染Hela和Bel740 2肿瘤细胞 ,经G418抗性筛选 ,挑选阳性克隆并扩大培养用于荧光和Westernblotting检测 ,利用GELDoc2 0 0 0数字成像系统对GFP蛋白表达进行定量分析。结果 :酶切和电泳表明重组质粒构建正确 ;获得了稳定转染各质粒的阳性细胞克隆 ;经Westernblotting检测 ,亲本和空载细胞的GFP值在本底范围 ,GFP转染的各细胞 ,均可检测到 2 7kD的GFP目的蛋白 ;EⅡmCMV启动子调控的Hela GFP EⅡ ,Hela GFP EⅡ w表达的GFP量均低于Hela GFP ;pcDNA3 GFP EⅡ w在Bel740 2细胞中的表达量明显高于其在Hela细胞中的表达量。结论 :HBV启动子调控下的GFP报告基因能够在肝癌Bel740 2细胞中表达并具有一定的专一性。
研究HBV启动子调控下的GFP报告基因在肿瘤细胞中的表达。方法 :采用DNA重组技术 ,将GFPs65T和HBV的EⅡmCMV启动子 (增强子 )亚克隆到哺乳动物表达载体pcDNA3 中 ,构建了pcDNA3 GFP ,pcDNA3 GFP EⅡ ,pcDNA3 GFP EⅡ w3种含GFP的重组质粒 ,在Lipofectin介导下分别转染Hela和Bel740 2肿瘤细胞 ,经G418抗性筛选 ,挑选阳性克隆并扩大培养用于荧光和Westernblotting检测 ,利用GELDoc2 0 0 0数字成像系统对GFP蛋白表达进行定量分析。结果 :酶切和电泳表明重组质粒构建正确 ;获得了稳定转染各质粒的阳性细胞克隆 ;经Westernblotting检测 ,亲本和空载细胞的GFP值在本底范围 ,GFP转染的各细胞 ,均可检测到 2 7kD的GFP目的蛋白 ;EⅡmCMV启动子调控的Hela GFP EⅡ ,Hela GFP EⅡ w表达的GFP量均低于Hela GFP ;pcDNA3 GFP EⅡ w在Bel740 2细胞中的表达量明显高于其在Hela细胞中的表达量。结论 :HBV启动子调控下的GFP报告基因能够在肝癌Bel740 2细胞中表达并具有一定的专一性。
2001, 8(1):59-60.
DOI: 10.3872/j.issn.1007-385X.2001.1.018
Abstract:
目的：探讨阳离子脂质体介导的基因转染效率与肺癌细胞增殖特性的关系。方法：采用pCMVβ报告基因质粒及自杀基因E.colicd的真核表达载体评价脂质体介导的基因转染效率。肺癌细胞增殖特性的检测包括绘制细胞生长曲线、计算细胞群体倍增时间、流式细胞仪测量肺癌细胞的细胞周期分布。结果：肺癌细胞的类型对脂质介导的基因转染效率具有显著影响。细胞增殖速度越快，处于分裂期（G2／M）的细胞越多，则脂质介导的基因转染效率越高。结论：脂质介导的基因转染对细胞增殖的依赖,提示 由快速增殖细胞构成的组织更适合阳离子脂质介导的基因治疗。
目的：探讨阳离子脂质体介导的基因转染效率与肺癌细胞增殖特性的关系。方法：采用pCMVβ报告基因质粒及自杀基因E.colicd的真核表达载体评价脂质体介导的基因转染效率。肺癌细胞增殖特性的检测包括绘制细胞生长曲线、计算细胞群体倍增时间、流式细胞仪测量肺癌细胞的细胞周期分布。结果：肺癌细胞的类型对脂质介导的基因转染效率具有显著影响。细胞增殖速度越快，处于分裂期（G2／M）的细胞越多，则脂质介导的基因转染效率越高。结论：脂质介导的基因转染对细胞增殖的依赖,提示 由快速增殖细胞构成的组织更适合阳离子脂质介导的基因治疗。
2001, 8(1):63-64.
DOI: 10.3872/j.issn.1007-385X.2001.1.020
Abstract:
Objective: To investigate in vitro the biological characteristics of AdCD80-infected human lung cancer cells on the basis of generation of replication-deficient hB7-1(CD80) recombinant adenovirus. Methods: Human CD80 gene was transduced into lung cancer cells mediated by recombinant adenovirus and then the expression of the gene was detected by PCR and agarose gel electrophoresis. The biological characteristics of the above cells were analysed with electron microscope, FACS and etc. Results: The titers of rAd reached to 10 10 PFU/ml and more than 90% Anip973 lung cancer cells could be infected by 30 MOI rAd. The growth curve and cloning efficiency of rAdCD80-infected Anip973 cells showed no significant difference compared with that of the control cells. The cell proliferation cycle of rAdCD80-infected 973 cells showed no change through FACS test. Having been infected by rAdCD80, the surface structure and ultrastructure of 973 cells had a little change. Conclusion: These results will lay foundation for tumor vaccines.
Objective: To investigate in vitro the biological characteristics of AdCD80-infected human lung cancer cells on the basis of generation of replication-deficient hB7-1(CD80) recombinant adenovirus. Methods: Human CD80 gene was transduced into lung cancer cells mediated by recombinant adenovirus and then the expression of the gene was detected by PCR and agarose gel electrophoresis. The biological characteristics of the above cells were analysed with electron microscope, FACS and etc. Results: The titers of rAd reached to 10 10 PFU/ml and more than 90% Anip973 lung cancer cells could be infected by 30 MOI rAd. The growth curve and cloning efficiency of rAdCD80-infected Anip973 cells showed no significant difference compared with that of the control cells. The cell proliferation cycle of rAdCD80-infected 973 cells showed no change through FACS test. Having been infected by rAdCD80, the surface structure and ultrastructure of 973 cells had a little change. Conclusion: These results will lay foundation for tumor vaccines.
2001, 8(1):65-66.
DOI: 10.3872/j.issn.1007-385X.2001.1.022
Abstract:
目的观察点突变重组人β-干扰素（IFN-β）对人肿瘤细胞体内外生长的抑制作用，为其临床应用提供实验资料。方法分别将人结肠癌LoVo、肺腺癌A549细胞置于含200IU/ml,1000IU/ml及5000IU/mlIFN-β培养基中培养24h后，于0.3%琼脂糖培养基中培养10～14d，观察软琼脂中集落形成情况。将裸鼠皮下接种LoVo，A549细胞，72h后分组，每次注射20万IU/kg或100万IU/kg,500万IU/kgIFN-β，每天1次，连续10d，同时设置生理盐水对照组、标准品组和注射用环磷酰胺治疗组。荷瘤后第14天处死小鼠，称取瘤重。以上实验均重复3批。结果在体外，经重组人新型β-干扰素处理的LoVo细胞及A549细胞的集落数均明显低于未经处理的细胞（0.05>P>0.01）,与标准品组无明显差异(P>0.05)。荷瘤裸鼠经治疗后，瘤重随着重组人β-干扰素剂量的升高而降低，即重组人β-干扰素可呈剂量依赖性地抑制LoVo,A549细胞在体内的生长，在疗效方面与标准品间无显著差异(P>0.05)。结论重组人β-干扰素在体内外能明显抑制人结肠癌和肺腺癌的生长。
目的观察点突变重组人β-干扰素（IFN-β）对人肿瘤细胞体内外生长的抑制作用，为其临床应用提供实验资料。方法分别将人结肠癌LoVo、肺腺癌A549细胞置于含200IU/ml,1000IU/ml及5000IU/mlIFN-β培养基中培养24h后，于0.3%琼脂糖培养基中培养10～14d，观察软琼脂中集落形成情况。将裸鼠皮下接种LoVo，A549细胞，72h后分组，每次注射20万IU/kg或100万IU/kg,500万IU/kgIFN-β，每天1次，连续10d，同时设置生理盐水对照组、标准品组和注射用环磷酰胺治疗组。荷瘤后第14天处死小鼠，称取瘤重。以上实验均重复3批。结果在体外，经重组人新型β-干扰素处理的LoVo细胞及A549细胞的集落数均明显低于未经处理的细胞（0.05>P>0.01）,与标准品组无明显差异(P>0.05)。荷瘤裸鼠经治疗后，瘤重随着重组人β-干扰素剂量的升高而降低，即重组人β-干扰素可呈剂量依赖性地抑制LoVo,A549细胞在体内的生长，在疗效方面与标准品间无显著差异(P>0.05)。结论重组人β-干扰素在体内外能明显抑制人结肠癌和肺腺癌的生长。
18 Construction and
Expression of Recombinant Retroviral Vector Containing D-Amino Acid Oxidase cDNA
2001, 8(1):67-69.
DOI: 10.3872/j.issn.1007-385X.2001.1.023
Abstract:
Galα(1,3)Gal是非灵长类哺乳动物细胞表面的一种主要的异种抗原 ,由α(1,3)半乳糖基转移酶合成。人体内没有这种酶 ,也不产生这种抗原 ,但却产生大量的抗Galα(1,3)Gal抗体 (anti Gal)。如果在人的肿瘤细胞上重新表达Galα(1,3)Gal,它与anti Gal结合后会引发一系列的免疫反应 ,产生抗肿瘤的作用。本文综述了这方面的研究
Galα(1,3)Gal是非灵长类哺乳动物细胞表面的一种主要的异种抗原 ,由α(1,3)半乳糖基转移酶合成。人体内没有这种酶 ,也不产生这种抗原 ,但却产生大量的抗Galα(1,3)Gal抗体 (anti Gal)。如果在人的肿瘤细胞上重新表达Galα(1,3)Gal,它与anti Gal结合后会引发一系列的免疫反应 ,产生抗肿瘤的作用。本文综述了这方面的研究
2001, 8(1):70-71.
DOI: 10.3872/j.issn.1007-385X.2001.1.024
Abstract:
分子伴侣是广泛分布在原核生物细胞、真核生物细胞内 ,帮助新生肽链进行正确折叠、非共价组装的一类蛋白。来自于不同纯系小鼠肉瘤细胞的gp96 (一种分子伴侣 )能够在不同的纯系小鼠之间建立起特异的细胞免疫应答 ,某些分子伴侣如HSP70等可作为抗原提呈分子呈现在肿瘤细胞表面 ,可作为肿瘤细胞表面的靶抗原激活抗肿瘤的细胞免疫。总之 ,分子伴侣或与分子伴侣结合的多肽复合体抗原由于具有可超越MHCⅠ类抗原分子限制的特点可开发成肿瘤疫苗用于治疗。
分子伴侣是广泛分布在原核生物细胞、真核生物细胞内 ,帮助新生肽链进行正确折叠、非共价组装的一类蛋白。来自于不同纯系小鼠肉瘤细胞的gp96 (一种分子伴侣 )能够在不同的纯系小鼠之间建立起特异的细胞免疫应答 ,某些分子伴侣如HSP70等可作为抗原提呈分子呈现在肿瘤细胞表面 ,可作为肿瘤细胞表面的靶抗原激活抗肿瘤的细胞免疫。总之 ,分子伴侣或与分子伴侣结合的多肽复合体抗原由于具有可超越MHCⅠ类抗原分子限制的特点可开发成肿瘤疫苗用于治疗。
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Supported by:Beijing E-Tiller Technology Development Co., Ltd.