SV40 LT基因诱导人骨髓间充质干细胞的实验研究

Immortalization of human bone marrow mesenchymal stem cells by transfected with SV40 LT genes

目的利用猿猴病毒40大T抗原(SV40 LT)基因异位表达建立永生化人骨髓间充质干细胞(hM-SCs)系。方法使用含有SV40大T片段的重组质粒载体转染人原代骨髓间充质干细胞,经G418筛选,连续传代培养,通过形态学、免疫组织化学及PCR技术等方法对细胞系进行鉴定。结果转染后骨髓间充质干细胞基本保持了原代细胞的表型特征、形态均一、多为梭形,呈漩涡状或放射状排列,有特征性表型。该系已培养5个月,超过36代。细胞免疫组化染色显示有SV40 LT的表达。结论成功地构建了SV40 LT基因永生化的人骨髓间充质干细胞系,为其在神经系统疾病治疗中的应用奠定了实验基础。

Objective To immortalize human bone marrow mesenchymal stem ceils （hMSCs）by ectopic expression of simian virus 40 large T antigen （ SV40 LTag） without malignant transformation. Methods A vector that contained SV40 LT fragment and drug resistance gene were used to transfect normal human bone marrow mesenchymal stem cells. The transfected cells were screened with 400μg/ml G418. An under inverted microscope was used to observe the morphology and growth of the cells. The expression of transfected DNA fragments were detected in expanded cell lines by immunocytochemistry and PCR. Results The cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping. The existence and expression of SV40 LTag was detected in the transfected cells by immunocytochemistry and PCR. Immunochemistry results showed that the cultured cell positive for CD29 and CD44, and negative for CD34 and CD45. Conclusion Ectopic expres- sion of SV40 LT can effectively immortalize human bone marrow mesenchymal stem cells. It may provide stable cell resource for the basic researches and cell-transplantation therapies for basic and clinical research in neuroscience.