目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B...目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B7-1的识别。以天然表达B7-1的Daudi细胞、Raji细胞及8266细胞为观察对象,分别加入(5、10、20、40)μg/m L B7-1 mAb处理,采用噻唑蓝(MTT)法检测mAb对肿瘤细胞体外增殖的影响,TranswellTM法分析mAb对肿瘤细胞体外迁移的影响,流式细胞术分析mAb对肿瘤细胞凋亡的诱导作用。结果成功制备1株能稳定分泌小鼠抗人B7-1 mAb的杂交瘤,命名为5G10。经流式细胞术分析,该抗体与Daudi、Raji、8266、U266、BEL-7402及MCF-7肿瘤细胞的阳性结合率分别为(96.30±2.12)%、(95.70±1.79)%、(96.80±2.48)%、(23.20±2.35)%、(1.68±0.35)%、(0.55±0.04)%;与对照组相比,20μg/m L 5G10 mAb明显抑制Daudi细胞、Raji细胞及8266细胞的增殖,肿瘤细胞迁移率明显降低,细胞凋亡率明显增加。结论小鼠抗人B7-1 mAb能特异性识别和结合不同肿瘤细胞膜表面的B7-1,并能显著抑制天然表达B7-1的肿瘤细胞的体外生长、迁移并促进其凋亡。展开更多
Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA w...Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA was identified to be consistent with the data from other researchers. pCD mB7 1 plasmid was transfected into B16(F0) cells, and effective expression of mB7 1 in these tumor cells could be detected till the 6th month by RT PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7 1 gene were more effective than B16 wt and B16 neo in inducing specific cytotoxity of lymphocytes against B16 wt cells. It is suggested that expression of B7 1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.展开更多
Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transducti...Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced BL-4 cells can be used as a good tumor vaccine. The combination of the two kinds of vaccine may have potential application value in human cancer treatment.展开更多
To find a new way for gene therapy against tumors with weak immunogenicity, the effect of mB7 1 costimulation alone, or combined with IL 6, in inducing antitumor immunity in vitro was investigated. It was found...To find a new way for gene therapy against tumors with weak immunogenicity, the effect of mB7 1 costimulation alone, or combined with IL 6, in inducing antitumor immunity in vitro was investigated. It was found that mB7 1 cDNA transfected B16 cells (B16 mB7 1) induced the expansion of effector lymphocytes and the generation of specific lytic activity more effectively than wild type B16 melanoma cells (B16 wt) or mock transfected B16 cells (B16 neo) did. ( P <0.01), IL 6 could effectively stimulate lymphocytes proliferation, but failed to enhance its cytotoxicity, while the combination of mB7 1 and IL 6 increased both lymphocyte proliferative response and T cell mediated cytotoxicity more significantly than B7 1 or IL 6 did alone ( P <0.01) . It was inferred that the costimulatory molecule B7 1 is required for the activation and proliferation of T lymphocytes; the expression of mB7 1 in tumor cells could increase their immunogenicity and induce effective antitumor immune response, and the combination of B7 1 and IL 6 could induce more effective antitumor immunity, indicating that cooperation of IL 6 and mB7 1 plays a role in T lymphocyte activation.展开更多
Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Met...Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Methods: ①B7. 1 gene was subcloned from the cloning vector using PCR. Both of tlie PCR products and eukaryocytic expressing vector pHook were digested with Apa I , Sal 1 and linked using T4 DNA ligase. Tlie ligased products were transduced into DH5a. B7. 1 gene containing clones were selected by digestion with Apa I and Sal I which were further conformed by sequencing. ②HL60 cells were transformed by B7. 1 with lipofectamine and detected by FACS. ③Tumor formation, mice survival time and the immunotherapeutic and immunoprotective effects by immunization with B7. 1+ cells were evaluated. Results: ①The PCR products were about 620 bp. Six clones were all digested by Apa I and Sal I to produce 620 bp gene fragment which was in tlie same way as 67. 1. It demonstrated t/iat t/ie recombinant vector had been constructed successfully. Further sequencing confirmed the validity of the construction. There was no nucleotide mutation. ② B7. 1 was availably expressed on HL60 cells. ③ B7. 1+ HL60 cells delayed the growth of tumor and prolonged the survival time of leukemic mice, distinctively. So did tlie immunoprotection of B7. 1 + HL60 cells. Conclusion: The human B7. 1(CD80) eukaryocytic expressing vector can be successfully constructed by molecular cloned methods and can be stably and availably expressed on tlie membrane of B7. 1 negative acute myelocytic leukemia (AML) cell line HL60. Furthermore, the costimulatory molecular vaccine has significant effection of immunotherapy and immunoprotection and gives the rationale for clinical application.展开更多
文摘目的制备小鼠抗人B7-1(CD80)单克隆抗体(mAb),研究其对天然表达B7-1的恶性肿瘤细胞体外生长和迁移的影响。方法以人B7-1基因转染的L929-B7-1细胞免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备mAb,并通过流式细胞术检测mAb对肿瘤细胞膜型B7-1的识别。以天然表达B7-1的Daudi细胞、Raji细胞及8266细胞为观察对象,分别加入(5、10、20、40)μg/m L B7-1 mAb处理,采用噻唑蓝(MTT)法检测mAb对肿瘤细胞体外增殖的影响,TranswellTM法分析mAb对肿瘤细胞体外迁移的影响,流式细胞术分析mAb对肿瘤细胞凋亡的诱导作用。结果成功制备1株能稳定分泌小鼠抗人B7-1 mAb的杂交瘤,命名为5G10。经流式细胞术分析,该抗体与Daudi、Raji、8266、U266、BEL-7402及MCF-7肿瘤细胞的阳性结合率分别为(96.30±2.12)%、(95.70±1.79)%、(96.80±2.48)%、(23.20±2.35)%、(1.68±0.35)%、(0.55±0.04)%;与对照组相比,20μg/m L 5G10 mAb明显抑制Daudi细胞、Raji细胞及8266细胞的增殖,肿瘤细胞迁移率明显降低,细胞凋亡率明显增加。结论小鼠抗人B7-1 mAb能特异性识别和结合不同肿瘤细胞膜表面的B7-1,并能显著抑制天然表达B7-1的肿瘤细胞的体外生长、迁移并促进其凋亡。
文摘Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA was identified to be consistent with the data from other researchers. pCD mB7 1 plasmid was transfected into B16(F0) cells, and effective expression of mB7 1 in these tumor cells could be detected till the 6th month by RT PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7 1 gene were more effective than B16 wt and B16 neo in inducing specific cytotoxity of lymphocytes against B16 wt cells. It is suggested that expression of B7 1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.
基金a grant from the "95 National Key Project of China" (No.96-906-01-20).
文摘Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced BL-4 cells can be used as a good tumor vaccine. The combination of the two kinds of vaccine may have potential application value in human cancer treatment.
文摘To find a new way for gene therapy against tumors with weak immunogenicity, the effect of mB7 1 costimulation alone, or combined with IL 6, in inducing antitumor immunity in vitro was investigated. It was found that mB7 1 cDNA transfected B16 cells (B16 mB7 1) induced the expansion of effector lymphocytes and the generation of specific lytic activity more effectively than wild type B16 melanoma cells (B16 wt) or mock transfected B16 cells (B16 neo) did. ( P <0.01), IL 6 could effectively stimulate lymphocytes proliferation, but failed to enhance its cytotoxicity, while the combination of mB7 1 and IL 6 increased both lymphocyte proliferative response and T cell mediated cytotoxicity more significantly than B7 1 or IL 6 did alone ( P <0.01) . It was inferred that the costimulatory molecule B7 1 is required for the activation and proliferation of T lymphocytes; the expression of mB7 1 in tumor cells could increase their immunogenicity and induce effective antitumor immune response, and the combination of B7 1 and IL 6 could induce more effective antitumor immunity, indicating that cooperation of IL 6 and mB7 1 plays a role in T lymphocyte activation.
基金Supported by Grant from the Key Clinical Department Development Item of China,Health Ministry(20012131)
文摘Objective: To construct the human B7. 1 (CD80) eukaryocytic expressing vector and its expression on HL60 cells to investigate the immunotherapeutic and immunoprotective effects of B7. 1 molecule on acute leukemia. Methods: ①B7. 1 gene was subcloned from the cloning vector using PCR. Both of tlie PCR products and eukaryocytic expressing vector pHook were digested with Apa I , Sal 1 and linked using T4 DNA ligase. Tlie ligased products were transduced into DH5a. B7. 1 gene containing clones were selected by digestion with Apa I and Sal I which were further conformed by sequencing. ②HL60 cells were transformed by B7. 1 with lipofectamine and detected by FACS. ③Tumor formation, mice survival time and the immunotherapeutic and immunoprotective effects by immunization with B7. 1+ cells were evaluated. Results: ①The PCR products were about 620 bp. Six clones were all digested by Apa I and Sal I to produce 620 bp gene fragment which was in tlie same way as 67. 1. It demonstrated t/iat t/ie recombinant vector had been constructed successfully. Further sequencing confirmed the validity of the construction. There was no nucleotide mutation. ② B7. 1 was availably expressed on HL60 cells. ③ B7. 1+ HL60 cells delayed the growth of tumor and prolonged the survival time of leukemic mice, distinctively. So did tlie immunoprotection of B7. 1 + HL60 cells. Conclusion: The human B7. 1(CD80) eukaryocytic expressing vector can be successfully constructed by molecular cloned methods and can be stably and availably expressed on tlie membrane of B7. 1 negative acute myelocytic leukemia (AML) cell line HL60. Furthermore, the costimulatory molecular vaccine has significant effection of immunotherapy and immunoprotection and gives the rationale for clinical application.
