胰十二指肠同源框基因-1在骨髓来源nestin阳性细胞的转染及表达

目的 将真核表达载体pEGFP-C1-PDX-1转染大鼠骨髓来源nestin阳性细胞,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转染骨髓来源nestin阳性细胞,改变DNA的量、转染后培养基中的血清浓度,在荧光显微镜下观察荧光并计算转染效率;转染后48 h用RT-PCR检测目的 基因的表达情况.结果 质粒2～10 μg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阳性细胞有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阳性细胞的效率,为其成为组织工程的种子细胞提供了实验依据.

PDX-1 gene transfection and expression in bone marrow stromal cells-derived nestin-positive cells

OBJECTIVE: To introduce the eukaryotic expression vector pEGFP-C1-PDX-1 into nestin-positive cell derived from bone marrow stromal cells by nucleofection and optimize the conditions for transfection. METHODS: The recombinant plasmid was transfected into bone marrow stromal cells-derived nestin-positive cells with varied DNA quantities or the serum concentration in the medium. The expression of PDX-1 gene in the transfected cells was detected by RT-PCR. RESULTS: Satisfactory efficiency of transfection was achieved with the DNA quantity of 2-10 microg and medium serum concentration of 20%. PDX-1 expression was detected in the transfected cells by RT-PCR. CONCLUSION: The optimized transfection conditions result in enhanced efficiency of PDX-1 gene transfection into nestin-positive cells derived from bone marrow stromal cells, which may serve as the seed cells in tissue-engineering.