不同转染试剂对慢病毒包装及包装后病毒感染豚鼠成纤维细胞效率的影响  

Effects of different transfection reagents on lentiviral packaging efficiency and the lentiviral infection efficiency of guinea-pig fibroblasts after packaging

作　　者：何承文[1] 何玉龙 吴月红[1,2] 邵风慧 

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室,银川750021 [2]浙江理工大学生命科学学院,杭州310018 [3]内蒙古阜丰生物科技有限公司,呼和浩特010070

出　　处：《黑龙江畜牧兽医》2015年第10期34-38,295,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31260287;31201867;31460585)

摘　　要：为了研究不同转染试剂对慢病毒载体包装效率及包装后病毒感染细胞的能力,试验利用磷酸钙、脂质体2000、脂质体LTX和Quick Shuttle-293转染编码绿色荧光蛋白(Green Fluorescent Protein,GFP)基因的重组慢病毒载体进入293T细胞包装病毒,包装的病毒感染豚鼠成纤维细胞,通过流式细胞仪检测转染试剂的转染效率和病毒感染效率,同时利用MTT法检测了4种转染试剂的细胞毒性。结果表明:磷酸钙在转染效率、转染后细胞活力及包装后病毒对豚鼠成纤维细胞的感染效率都最低,分别为(54.8±2.9)%、(26.7±6.0)%和(7.6±1.1)%;脂质体2000和脂质体LTX对293T细胞转染效率差异不显著(P>0.05),但脂质体LTX转染后细胞活力显著高于脂质体2000(P<0.05),即脂质体LTX的细胞毒性显著低于脂质体2000。而Quick Shuttle-293法转染后的转染效率、转染后细胞活力和其包装后的病毒对靶细胞的感染率分别为(95.5±1.8)%、(88.5±5.7)%和(93.3±1.0)%,都显著高于其他三种转染试剂(P<0.05)。说明在本试验条件下,Quick Shuttle-293是一种高效、低毒的慢病毒转染包装试剂,可用于通过病毒载体介导的基因转染技术制备豚鼠诱导多能性干细胞(i PS)。To study the effects of different transfection reagents on the packaging efficiency of lentiviral vector and the ability of the lentivirus to infect the cells,the recombinant lentiviral vector encoding green fluorescent protein( GFP) gene was transfected into 293 T cells by using calcium phosphate,liposome 2000,liposome LTX and Quick Shuttle- 293,respectively,and subsequently the guinea pig fibroblasts were infected by the lentivirus. The transfection efficiency of the transfection reagents and the efficiency of lentiviral infection were detected by flow cytometry,and the cytotoxicity of four transfection reagents was detected by MTT assay at the same time. The results showed that The transfection efficiency,cell viability after transfection and the lentiviral infection efficiency of guinea pig fibroblasts were the lowest using calcium phosphate,they were( 54. 8 ± 2. 9) %,( 26. 7 ± 6. 0) % and( 7. 6 ± 1. 1) %,respectively. There were no significant differences( P > 0. 05) in the transfection efficiency of 293 T cells using liposome 2000 and liposome LTX,but the cell viability after transfection by using liposomes LTX was significantly higher than( P < 0. 05) that using liposome 2000,namely,the cytotoxicity of liposome LTX was significantly lower than that of liposome 2000. The transfection efficiency,cell viability after transfection and the lentiviral infection efficiency of target cells by using Quick Shuttle- 293 was( 95. 5 ± 1. 8) %,( 88. 5 ± 5. 7) % and( 93. 3 ± 1. 0) %,respectively,which were significantly higher( P < 0. 05) than using the other three transfection reagents. The results indicate that under the experimental conditions described in the present study,Quick Shuttle-293 is an efficient,low- toxicity lentiviral transfection reagent,which can be used to prepare the induced pluripotent stem( i PS) cells of guinea pig by using a lentiviral vector- mediated gene transfer technology.

关 键 词：基因转染 慢病毒 绿色荧光蛋白 豚鼠 成纤维细胞 

分 类 号：Q25[生物学—细胞生物学]