PSMA-CAR慢病毒表达载体的包装研究  

Study on packaging of lentivirus expression vectors of PSMA-CAR

作　　者：王丙萍 江松 高维实[2] WANG Bing-ping;JIANG Song;GAO Wei-shi(Cancer Institute,Inner Mongolia People's Hospital,Hohhot 010010,Inner Mongolia,China;Department of Abdominal Tumor Surgery,Inner Mongolia People's Hospital,Hohhot 010010,Inner Mongolia,China;Inner Mongolia Medico!University,Hohhot 010010,Inner Mongolia,China)

机构地区：[1]内蒙古自治区人民医院肿瘤研究所,内蒙古呼和浩特010010 [2]内蒙古医科大学,内蒙古呼和浩特010010 [3]内蒙古自治区人民医院腹部肿瘤外科,内蒙古呼和浩特010010

出　　处：《生物医学工程与临床》2021年第5期617-624,共8页Biomedical Engineering and Clinical Medicine

基　　金：内蒙古自治区自然科学基金资助项目(2021LHMS08060)。

摘　　要：目的前列腺特异性膜抗原-嵌合抗原受体(PSMA-CAR)慢病毒表达载体的包装方法研究。方法采用慢病毒三质粒病毒包装系统(Del8.9+VSVG、PsAX2+OG)及包装细胞(HEK-293T、HEK-293FT)在转染试剂[聚乙烯亚胺(PEI)、Lipofectamine 2000、磷酸钙]的介导下,包装慢病毒颗粒。通过检测转染效率筛选最佳包装细胞、最佳包装质粒组合、最佳转染试剂和剂量。结果 HEK-293FT细胞的转染效率高于HEK-293T细胞的转染效率。PsAX2+OG组合的转染效率高于Del8.9+VSVG组合的转染效率。PEI方法转染时,包装质粒相同,空载体的转染效率明显要高很多;目的质粒相同,采用包装质粒为PsAX2+OG组合时,转染效率更高;数据统计表明,质粒总量∶PEI的比例在1∶9时转染效率更高。Lipofectamine 2000方法转染时,包装质粒和包膜质粒相同,空载体的转染效率更高;目的质粒和包装、包膜质粒相同,质粒总量∶Lipofectamine 2000的比例在1.0∶2.0和1.0∶2.5时转染效率显著高于其他两组比例。磷酸钙转染法时,随着加入的磷酸钙的增多,转染效率呈递减趋势。结论采用包装质粒PsAX2和包膜质粒OG时转染效率相对更高。质粒总量和加入的PEI量比例在1∶9时转染效率更高;质粒总量和加入的Lipofectamine 2000量比例在1.0∶2.0和1.0∶2.5时转染效率更高。PEI包装法和Lipofectamine 2000包装法转染效率相当,Lipofectamine 2000包装法相对高一些,但在考虑包装成本的前提下后续大量包装PSMA-CAR慢病毒颗粒时,应选择PEI包装法。Objective To study the packaging method of PSMA-CAR lentivirus expression vectors. Methods The lentivirus three-plasmid virus packaging system(Del8.9 + VSVG, PsAX2 + OG) and packaging cells(HEK-293 T, HEK-293 FT) were used to package lentivirus particles under mediation of transfection reagents[polyethylenimine(PEI), Lipofectamine 2000, calcium phosphate]. The best packaging cells, best packaging plasmid combination, best transfection reagents and dosages were screened by testing transfection efficiency. Results The transfection efficiency of HEK-293 FT cells was higher than that of HEK-293 T cells. The transfection efficiency of PsAX2 + OG was higher than that of Del8.9 + VSVG. For PEI transfection,with the same packaging plasmid, transfection efficiency of empty vector was significantly higher;the target plasmid was the same, when the packaging plasmid was PsAX2 + OG, the transfection efficiency was higher;the statistics analyze results showed that transfection efficiency was higher when total amount of plasmid ∶ PEI was 1 ∶ 9. For Lipofectamine 2000 method,when transfection, packaging plasmid and envelope plasmid were the same, the transfection efficiency of empty vector was higher. When target plasmid, packaging and envelope plasmid were the same, the total amount of plasmid ∶ Lipofectamine2000 were 1.0 ∶ 2.0 and 1.0 ∶ 2.5, the transfection efficiency were significantly higher than that of other two proportion. For calcium phosphate transfection method, with the increase of calcium phosphate added, the transfection efficiency decreased.Conclusion It is demonstrated that with the use of package plasmid PsAX2 and envelope plasmid OG, a relatively higher transfection efficiency can be obtained. The transfection efficiency is higher when ratio of total plasmid and added PEI at 1 ∶ 9.The transfection efficiency is higher when ratio of total plasmid and Lipofectamine 2000 were 1.0 ∶ 2.0 and 1.0 ∶ 2.5. The transfection efficiency of PEI packaging method is as same as Lipofectamine 2000 packaging meth

关 键 词：嵌合抗原受体(CAR) 前列腺特异性膜抗原(PSMA) 慢病毒载体 病毒包装 转染效率 

分 类 号：Q782[生物学—分子生物学] R394[医药卫生—医学遗传学]