细胞接触诱导骨髓间充质干细胞获得窦房结细胞表型的初步研究

Sinoatrial node cells phenotype expression is induced by direct bone marrow stem cells contact with sinoatrial node cells

目的通过建立体外骨髓间充质干细胞（BMSCs）与纯化培养的乳鼠窦房结细胞共培养体系，探讨窦房结微环境对BMSCs分化的诱导作用。方法自新生乳鼠的心脏分离窦房结细胞，并差速贴壁纯化培养，免疫荧光检测超极化激活环核苷酸门控阳离子通道基因4（HCN4）和缝隙连接蛋白45（Cx45）的表达。自成年大鼠的骨髓分离BMSCs，传2代后，用脂质体介导pEGFP-N1转染标记BMSCs，再与纯化培养的窦房结细胞以1：5比例进行直接接触共培养，并用窦房结细胞条件培养液对转染后的BMSCs进行培养作为对照。1周后应用免疫荧光检测BMSCs的HCN4和Cx45表达。结果接触共培养组中，可见部分表达绿色荧光蛋白的BMSCs同时表达HCN4和Cx45，而条件液培养组中未见HCN4和Cx45的表达。结论直接接触共培养体系可诱导BMSCs初步分化为窦房结样细胞。

Objective To observe the differentiation of bone mesenchymal stem cells （BMSCs） cocultured with purified sinoatrial node cells （SNC） of neonate rats. Methods SNC from neonatal SD rat were cultured and purified with differential attachment method and labeled with BrdU. Rat BMSCs were isolated by a Percoll＇s gradient solution and cultured in DMEM. After 2 passages, these BMSCs were transfeeted with pEGFP-N1 by Lipofeetamin and labeled with GFP. EGFP-BMSC were co-cultured with SNC in a rate of 1 ： 5 for 1 week. EGFP-BMSC cultured in SNC culture medium served as controls. SNC marker hyperpolarization activated cyclic nueleotide gated cation channel 4 （ HCN4 ） and eonnexin 45 （ Cx45 ） expressions were determined by immunofluoreseence staining. Result Positive immunofluoreseence staining against HCN4 and Cx45 were detected in EGFP-BMSC co-cultured with SNC but not in EGFP-BMSC cultured in SNC culture medium. Conclusion Direct cell-to-cell contact between BMSCs and SNC cells may induce BMSCs differentiation into sinus node-like cells.