转人B7-H3基因鳞癌细胞瘤苗诱导抗肿瘤免疫应答的体外研究

发布时间：2019-06-01 19:53

【摘要】： 目的: 肿瘤细胞提呈抗原给T细胞时,由于表面缺乏共刺激分子的表达,不能起始有效的抗肿瘤免疫应答。为了将共刺激分子导入肿瘤细胞以制备肿瘤疫苗,我们将表达B7-H3基因的真核表达载体pEGFP-C1-B7-H3转染入口腔鳞癌细胞Tca8113中,检测B7-H3基因在转染口腔鳞癌细胞Tca8113中的表达,进一步研究转人B7-H3基因鳞癌细胞疫苗体外诱导抗肿瘤免疫应答的能力。方法: 采用脂质体介导法将真核表达质粒pEGFP-C1-B7-H3转染入人鳞癌细胞Tca8113中,转染24h,14d,30d后,荧光显微镜下观察绿色荧光蛋白的表达,待转染成功后,用RT-PCR检测基因B7-H3在转染细胞中的表达;用Western blot检测其蛋白的表达,经G418筛选后获得稳定高表达克隆。以丝裂霉素C(MMC)处理后,制成肿瘤细胞疫苗,经体外与人外周血淋巴细胞共同培养后,测定淋巴细胞特异性杀伤活性及对淋巴细胞产生细胞因子的影响。结果: 用Lipofectamine2000细胞转染试剂盒把pEGFP-C1-B7-H3载体转入口腔鳞癌细胞株Tca8113中后,于24h、14d、30d时,在10×20倍倒置显微镜下用荧光观察细胞(光波波长488nm或513nm),同时与未转染质粒的口腔鳞癌细胞Tca8113做对照。转染B7-H3的Tca8113细胞,经过细胞传代培养后仍然能够检测到高表达的B7-H3基因,用Trizol从转染了pEGFP-C1-B7-H3的Tca8113细胞中提取总RNA,然后用RT-PCR检测B7-H3的表达,PCR产物电泳结果表明,扩增产物的大小约215bp,与预期的大小一致。转染空载体pEGFP-C1的Tca8113细胞中,没有检测到215bp的PCR产物。用Western blot方法抽提B7-H3/Tca8113基因转染细胞,裂解已转染pEGFP-C1-B7-H3的Tca8113细胞(B7-H3/Tca8113)的基因转染细胞膜蛋白中相对分子质量约84～90KD大小的特异性条带;而mock/Tca8113没有特异性条带。经过G418筛选后挑取单克隆法建立了B7-H3/Tca8113细胞株。转染B7-H3的Tca8113细胞能够高表达B7-H3蛋白。经MMC处理后,与野生型的Tca8113细胞相比,上述瘤苗对T细胞体外培养试验显示该基因转染细胞与T细胞共培养可促进其体外增殖,诱导淋巴细胞产生针对Tca8113的特异性杀伤作用,能显著增强淋巴细胞分泌IFN-γ的能力。结论: 用脂质体法把真核表达载体pEGFP-C1-B7-H3转染到口腔鳞癌细胞Tca8113中,用挑选单克隆法建立了稳定表达B7-H3蛋白的细胞株,用RT-PCR及Western blot鉴定B7-H3基因在该细胞中有表达。B7-H3/Tca8113经MMC处理后得到TCV-hB7-H3,将其与T细胞体外培养试验显示该基因转染细胞与T细胞共培养可促进其体外增殖,诱导淋巴细胞产生针对Tca8113的特异性杀伤作用,能显著增强淋巴细胞分泌IFN-γ的能力。转B7-H3基因人鳞癌细胞疫苗能诱导有效的抗鳞癌免疫反应。为以后在体内试验中研究B7-H3对免疫细胞的调节和肿瘤基因治疗奠定了基础。
[Abstract]:Aim: when tumor cells present antigen to T cells, they can not initiate effective anti-tumor immune response due to the lack of costimulatory molecules on the surface. In order to introduce costimulatory molecules into tumor cells to prepare tumor vaccine, we transferred the eukaryotic expression vector pEGFP-C1-B7-H3 expressing B7-H3 gene into Tca8113. The expression of B7-H3 gene in oral squamous cell carcinoma cell line Tca8113 was detected, and the ability of anti-tumor immune response induced by human B7-H3 gene squamous cell carcinoma cell vaccine in vitro was further studied. Methods: the eukaryotic expression plasmid pEGFP-C1-B7-H3 was transformed into human squamous cell carcinoma cell line Tca8113 by Liposome mediated method. The expression of green fluorescent protein was observed under fluorescence microscope for 24 hours and 30 days after transfection, and after successful transfection, the expression of green fluorescent protein was observed under fluorescence microscope, and the expression of green fluorescent protein was observed under fluorescence microscope. The expression of gene B7-H3 in transfected cells was detected by RT-PCR. The expression of its protein was detected by Western blot, and the stable and high expression clone was obtained after G418 screening. The tumor cell vaccine was prepared by mitomycin C (MMC) treatment. After co-culture with human peripheral blood lymphocytes in vitro, the specific killing activity of lymphocytes and the effect on cytokines produced by lymphocytes were measured. Results: the pEGFP-C1-B7-H3 vector was transferred into oral squamous cell carcinoma cell line Tca8113 with Lipofectamine2000 cell transfer kit. At 24 h, 14 d and 30 d, the cells were observed by fluorescence under 10 脳 20 times inverted microscope (light wave wavelength 488nm or 513nm). At the same time, it was compared with oral squamous cell carcinoma cell line Tca8113, which was not infected with plasmid. The high expression of B7-H3 gene was still detected in Tca8113 cells transfected with B7-H3 after subculture. The total RNA, was extracted from Tca8113 cells transfected with pEGFP-C1-B7-H3 by Trizol, and the expression of B7-H3 was detected by RT-PCR. The results of PCR product electrophoresis showed that the size of the amplified product was about 215 BP, which was consistent with the expected size. No PCR products of 215bp were detected in Tca8113 cells transfected with empty vector pEGFP-C1. The B7-H3/Tca8113 gene transfected cells were extracted by Western blot and the specific bands of the relative molecular weight of the membrane proteins of Tca8113 cells (B7-H3/Tca8113) which had been transfected with pEGFP-C1-B7-H3 were lysed into the membrane proteins of Tca8113 cells (B7-H3/Tca8113), which were about the size of 84~90KD. However, mock/Tca8113 had no specific band. B7-H3/Tca8113 cell line was established by single clone method after G418 screening. B7-H3 protein was highly expressed in Tca8113 cells transfected with B7-H3. After MMC treatment, compared with wild type Tca8113 cells, the co-culture of T cells and T cells by the above tumor vaccine showed that the co-culture of T cells and T cells could promote the proliferation of T cells in vitro and induce lymphocytes to produce specific killing effect against Tca8113. It can significantly enhance the ability of lymphocytes to secrete IFN- 纬. Conclusion: the eukaryotic expression vector pEGFP-C1-B7-H3 was transferred into oral squamous cell carcinoma cell line Tca8113 by lipid method, and the cell line stably expressing B7-H3 protein was established by selected single clone method. The expression of B7-H3 gene in the cells was identified by RT-PCR and Western blot. TCV-hB7-H3, was obtained by MMC treatment of B7-H3/Tca8113. The culture test with T cells in vitro showed that co-culture of T cells and T cells could promote the proliferation of T cells in vitro, induce lymphocytes to produce specific killing effect against Tca8113, and significantly enhance the ability of lymphocytes to secrete IFN- 纬. Human squamous cell carcinoma cell vaccine with B7-H3 gene can induce effective anti-squamous cell carcinoma immune response. It lays a foundation for the study of the regulation of B7-H3 on immune cells and tumor gene therapy in vivo.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R730.5

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