Plenti6.3/V5-TERT慢病毒载体的构建并成功建立永生化大鼠骨髓间充质干细胞系的研究

目的 为实现慢病毒感染后可用抗生素筛选TERT基因稳定整合的骨髓间充质干细胞,首先对pCI-neo-TERT质粒进行了改造和构建.方法 将pCI-neo-TERT酶切后得到TERT片段,并进行PCR扩增,将得到的TERT片段连入Plenti6.3/V5,酶切鉴定载体构建正确后,将改造和构建载体分别与辅助质粒psPAX2、pMD2.G共转染HEK-293T包装细胞,收获病毒上清并感染靶细胞,使用相应抗生素筛选细胞,验证抗性基因的插入.结果 成功构建了重组慢病毒载体Plenti6.3/V5-TERT,通过调整病毒上清的用量可以使靶细胞达到很高的感染效率;用相应抗生素筛选细胞证明了抗性基因的表达.结论 我们成功构建了慢病毒载体Plenti6.3/V5-DEST-TERT,从而使病毒感染后用抗生素筛选稳定整合TERT的骨髓间充质干细胞成为可能.

Construction of Plenti6.3/V5-TERT lentiviral vector and successful immortalization of rat bone marrow mesenchymal stem cells

Objective TERT plays a vital role in senescence and cell proliferation in BMSCs. Methods pCI-neo-TERT was generously provided by Dr. Weinberg. It was reconstructed into a lentivral vector by infecting HEK-293T. The expression of TERT and c-myc were detected by western blot assay. Bone marrow mesenchymal stem cells（BMMSCs） were isolated from bone marrow of SD rats by density gradient centrifugation. The third passage cells were divided into 3 groups ： control group, pCI-neo-TERT transducted group and Plenti6. 3/VS- DEST-TERT transducted group. The character of the TERT transdected ceils were detected by CD44 and CD29 with flow cytometer. Results The lentiviral vecor Plenti6. 3/VS-DEST-TERT was successful reconstructed and overexpressed in the transducted cells. The growth curve showed that the growth rate of plenti-TERT-BMMSCs PD ： （43.5 ± 0. 24） hours ] was higer than pCIneo-TERT-BMMSCs PD ： （ 67. 8 ± 0. 52 ） hours ] and MSC PD ： （ 94. 3± 0. 26） hours ] （P 〈 0.05 ）. Conclusions We construct the lentiviral vecor Plenti6. 3/VS-DEST-TERT and immortalize rat BMMSCs.