小梅山猪骨髓间充质干细胞Cx43基因修饰

Overexpression of connexin 43 in bone marrow mesenchymal stem cells in Xiao Meishan swines

摘要:

通过在小梅山猪骨髓间充质干细胞 (BMSCs) 中过表达缝隙连接蛋白43基因 (Cx43)，探究Cx43对小梅山猪BMSCs增殖及凋亡的影响，为构建转基因动物模型奠定基础。利用分子技术构建Cx43真核表达载体pEGFP-Cx43，Nucleofector TM法转染P3代BMSCs，检测细胞转染效率，经RT-PCR、免疫荧光和Western blotting鉴定Cx43的表达，流式细胞技术分析BMSCs的增殖能力和凋亡情况。酶切鉴定和测序结果表明，pEGFP-Cx43真核表达载体构建成功，转染BMSCs后，EGFP阳性细胞约占60%。转基因BMSCs中Cx43蛋白表达显著增加，细胞增殖能力显著提高、凋亡率显著下降。结果表明，在小梅山猪BMSCs中过表达Cx43不仅能促进细胞增殖而且抑制其凋亡，为下一步在体研究奠定了基础。

Abstract:

We studied the function of connexin 43 (Cx43) gene in Xiao Meishan swine bone marrow mesenchymal stem cells (BMSCs) resulting from overexpression. Cx43 eukaryotic expression vector (pEGFP-Cx43) was constructed and transfected into BMSCs by nucleofectorTM, after detecting the transfection efficiency; the expression of Cx43 was verified by RT-PCR, immunofluorescence and western blotting. Furthermore, we detected its cell cycle and apoptosis through flow cytometry. Our results show that pEGFP-Cx43 plasmid was successfully constructed, and green fluorescence in pEGFP-Cx43 transfected BMSCs was highly expressed with 60% transfection efficiency. In transgenic Xiao Meishan swines BMSCs, the expression level of Cx43 mRNA and protein were up-regulated. Meanwhile, the ability of cell proliferation was significantly increased, and the apoptosis rate was significantly reduced. Taken together, Cx43 overexpression could promote the proliferation of Xiao Meishan swine’s BMSCs and markedly reduce their apoptosis, which provides evidence for in vivo research.