SHH基因转染大鼠骨髓间充质干细胞的实验研究

The feasibility of transfection of Sonic hedgehog gene into bone marrow mesenchymal stem cells

目的探讨SHH基因转染大鼠骨髓间充质干细胞（MSC）的可行性。方法利用电转法将SHH基因转染人大鼠骨髓间充质干细胞，在荧光显微镜下观察转染效率；分别绘制未转染及转染SHH基因后细胞生长曲线，观察转染对细胞的影响；采用PCR、RT—PCR、Western．blot检测转染后骨髓间充质干细胞中外源性SHH基因的表达情况。结果生长曲线显示转染后骨髓间充质干细胞生长曲线指数生长期及平台期延后，在转染后第12天与未转染达到一致。转染后48h（即将用于移植前），经荧光倒置显微镜观察转染效率为30％，PCR检测转染后骨髓间充质干细胞中SHH表达较未转染细胞明显升高，实时定量PCR检测其升高约7倍左右，通过Western．blot检测转染后SHH蛋白产物表达明显升高。结论电转法能够有效将外源性SHH基因转染人大鼠MSC并能获得有效表达，为进一步大鼠缺血性心脏病模型的在体基因治疗提供了前提基础。

Objective To evaluate the feasibility of transfection of Sonic hedgehog gene （SHH） into bone marrow mesenchymal stem cells（BMMSC）. Methods After the SHH gene was transfected into BMMSC by electroporation apparatus, the transfection rate was evaluated by fluorescence inverted micro scope. The growth curves of untransfected and transfected BMMSC were drawn, respectively, to observe the influence of transfection on cells. The expression of SHH gene in the BMMSC was detected by PCR, RT PCR, Westernblot analyses. Results Through fluorescence inverted microscope, the observed transfection rate was appropriately 30%, PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells, and it is quantified by qPCR for appropriately 7 times. Westernblot further demon strated that the SHH protein expression in transfected cells had a distinct increase. However, it was ob served that the exponential phase of BMMSCSHH growth curve delayed. The growth curves of both overlap 12 days after transfection. Conclusions This electroporation method can transfect exogenous SHH gene in to BMMSC sufficiently with the effective protein expression in BMMSCSHH. It is the foundation of further research of genetic therapy for isehemic heart disease.