大鼠骨髓间充质干细胞L-型钙通道的表达及电流检测

EXPRESSION AND ELECTROPHYSIOLOGICAL CHARACTERISTICS OF L-TYPE CALCIUM CHANNEL IN RAT BONE MESENCHYMAL STEM CELLS

目的 探讨L-型钙通道基因和蛋白在骨髓间充质干细胞（MSCs）中的表达及离子电流，以进一步研究其在MSCs增殖和分化中的作用。方法 分离、培养和纯化大鼠骨髓间充质干细胞，细胞免疫荧光化学方法检测表面分子CD14、CD29、CD34，CD44、CD45、CD106的阳性率和L-型钙通道蛋白的表达；采用RT-PCR技术观察MSCs细胞中钙离子通道不同亚基α1C、α1D、α1G、α1H、α1S mRNA的表达；全细胞膜片钳技术记录单个细胞离子电流。结果 细胞免疫荧光化学方法检测CD29、CD44、CD106阳性率在93％左右，CD14、CD34、CD45表达阴性。RT-PCR结果显示，可见L-型钙通道α1C亚基的mRNA高表达；但未见L-型钙通道α1D、α1S亚基和T-型钙通道的α1G、α1H亚基mRNA的表达。L-型钙通道蛋白（α1C）呈阳性表达。在36例细胞中16例细胞记录到电压依赖性内向电流。此电流激活电位-30mV，最大激活电位为0-10mV，可被nifedipine（10μmol／L）阻断，细胞外液中以10mmol／L Ba^2＋作为负载离子，记录的电流强度明显大于2mmol／L Ca^2＋时，证实为L-型钙电流。结论 MSCs不仅有L-型钙通道的基因和蛋白的表达，并且具有功能电流。

Objective To explore the electrophysiological characteristics and the expression of mRNA and protein of L- type calcium channel in rat bone mesenchymal stem cells（MSCs）. Methods MSCs were isolated, cultured and purified. RT- PCR was used to detect the mRNA expression of α1C, α1D, α1H, α1S. The protein expression of L-type calcium channel（α1C） was testified by immunohistochamical. Ion currents were recorded in MSCs using whole-cell patch clamp technques. Results CD29, CD44, CD106 expressed in about 93 % MSCs and CD14, CD34, CD45 expressed negatively. A high expression of mRNA in α1C was detected by RT-PCR but no expressions were observed in α1D, α1G, α1H, α1S. Immunofluorescent double labeling showed an expression of α1C subunits in MSCs. Moreover, inward currents were recorded in 16 of 36 cells using whole-cell patch clamp techniques. The currents were activated around - 30 mV and peaked at 0 to 10 mV and were blocked by nifedipine（10 μmol/L）. These cells had larger currents with Ba^2＋ （10 mmol/L） in bath solution than with Ca^2＋ （2 mmol/L）. Conclusion The results indicated that adult rat MSCs expresse functional L-type calcium channels. It is possible that this channel plays a role on the proliferation and differentiation of MSCs.