Nucleofector^(TM)电转仪转染外源基因至骨髓基质细胞优化方案

Optimization protocol of gene PDX-1 transfer into rat bone marrow stromal cell by nucleofection

目的用Nucleofector^(TM)电转仪介导真核表达载体pEGFP-C1及pEGFP-C1-PDX-1转染大鼠骨髓来源细胞,并对其转染条件进行优化以获得较高效率。方法将已构建好的重组载体以Nucleofector^(TM)电转仪转染不同方法获得的骨髓来源细胞,改变DNA的量、转染后血清浓度,在荧光显微镜下观察荧光并计算转染效率;转染后48h进行逆转录-聚合酶链反应(RT-PCR)检测目的基因的表达情况。结果相同条件下转染骨髓来源的神经干细胞(NSC)较转染骨髓基质细胞(MSC)可获得更高的转染效率;质粒2～10μg获得最佳的转染效率;提高转染后培养基的血清浓度可提高转染后细胞存活率及转染效率;RT-PCR检测转染后骨髓来源nestin+细胞有PDX-1表达。结论通过优化转染条件提高了pEGFP-C1-PDX-1转染大鼠骨髓来源nestin+细胞的效率,为成为组织工程的种子细胞提供了实验依据。

Objective To transfect eukaryotic expression vectors pEGFP-C1 and pEGFP-C1- PDX-1 into cells from bone marrow stromal cells by Nucleofector^TM and to optimize the conditions of transfection. Methods The recombinant plasmid was transfected into bone marrow stromal cells and nestin ＋ cells from bone marrow stromal cells. The contents of DNA and medium serum concentration after transfection were changes. The expression of PDX-1 gene in the transfected cells was detected by RT-PCR. Results The nucleofecter was more efficient in NSC than MSC by same program; Satisfactory efficiency of transfection was obtained when the content of DNA was 2-10 μg and medium serum concentration was 20%. The PDX-1 in the transfected cells was expressed. Conclusion The efficiency of transfection was enhanced by optimizing the transfection conditions. It is possible to use the nestin ＋ cells derived from bone marrow stromal cells as seed cells in tissue-engineering.