【作者】 杨宏宇； 邹多宏； 储眉； 郝艳红； 张芳婷；

【Author】 Yang Hong-yu, Zhou Duo-hong, Chu Mei, He Yan-hong, Zhang Fang-ting Department of Oral and Maxillofacial Surgery,Shenzhen Hospital,Peking University

【机构】 北京大学深圳医院口腔颌面外科；

【摘要】 <正>目的:转入B7-H3基因鳞癌细胞系的建立及瘤苗制备。方法:从淋巴细胞中提取RNA,用RT-PCR 方法将B7-H3的编码序列cDNA扩增。然后将该基因的编码序列插入到真核荧光蛋白载体pEGFP-C1中, 构建成最终的表达载体pEGFP-C1-B7-H3。采用脂质体介导法将真核表达质粒pEGFP-B7-H3导入人鳞癌细胞Tca8113中(B7-H3／Tca8113),RT-PCR检测B7-H3基因在该细胞中的表达,Western blot检测其蛋白的表达。经G418筛选后,用有限稀释法建立稳定高表达的带有B7-H3基因的Tca8113鳞癌细胞系,以丝裂霉素C(MMC)(800μg／ml)处理后,制成肿瘤细胞疫苗(Tca8113-TCV)。结果:RT-PCR方法检测到目的基因更多还原

【Abstract】 Objective: To construct the cell line of Tca8113/hB7-H3 and make cancer vaccines. Methods: The total RNA was isolated from PBMC lymph-cell and the B7-H3cDNA was amplified from the mRNA by RT-PCR technique, and then the PCR products of B7-H3 was inserted into pEGFP-Cl, to form the expression vector.The eukaryotic expression vector pEGFP-B7-H3 was transfected into human squamous cell carcinoma cell line Tca8113 by lipofectamine 2000.The expression of GFP-B7-H3 protein and B7-H3 was detected by Western blot and RT-PCR, respectively..Then the transfected cells were selected in medium containing G418 (800μg/ml) and termed as Tca8113/hB7-H3, through definite dilution method, to construct the cell line of Tca8113/hB7-H3. The TCV-hB7-H3 was obtained by treating them with mitomycin (MMC). Results: A 215 bp fragment of RT-PCR product was detected by using the B7-H3 specific primers. DAB shows that a differential belt which was consisted of about 65 000 ~ 86000bp protein molecule.Tca8113/hB7-H3 cells expressed B7-H3 protein with highest cell surface level. Through definite dilution method, we construct the ceil line of Tca8113/hB7-H3. The TCV-hB7-H3 was obtained by treating them with mitomycin (MMC). Conclusion: The cell line of Tca8113/hB7-H3 was constructed and cancer vaccines were made too. This gives a good level to research cancer in vivo or vitro and lays a base of making further study its antitumour function.更多还原