人骨髓间充质干细胞的分离培养及其膜表面电压依赖型钙通道的研究

Isolation and culture of human marrow mesenchymal stem cells and their voltage-operated Ca2＋ channels on the membrane surface

目的 分离培养人骨髓间充质干细胞（hMSC）,用膜片钳技术观察其膜表面电压依赖型钙通道.方法 取人骨髓血,用Percoll（1.073 g/ml）密度梯度离心及贴壁筛选结合的方法,体外培养扩增hMSC.台盼蓝拒染法计数活细胞数.流式细胞仪分析其免疫表型及细胞周期.用膜片钳技术观察hMSC膜表面电压依赖型钙通道的表达情况.结果 hMSC传代后形态上均为紧密排列的成纤维细胞样结构,细胞活力检测达99%.流式细胞检测表明,hMSC表达CD44、CD105,不表达CD31、CD34和CD45.细胞周期检测显示传代后的hMSC处于G1/G0期的细胞为81.52%,处于G2、M和S期的细胞约占18.48%,G2/G1为1.67.膜片钳检测显示,在20个记录中,有6个细胞可记录到对nifedipine敏感的内向钙电流,当激活电压大约在-40 mv时,最大内向峰值电流（Ica-Peak） 在-0 mV为（96.67±13.50）pA,当加入5 μmol/L nifedipine,峰值钙电流为（47.75±11.24）pA,显著受到抑制（P＜0.05）.结论 体外分离培养扩增的hMSC细胞活力强,主要为静止期细胞,其细胞膜表面存在电压依赖性钙通道.

Objective To isolate and culture human marrow mesenchymal stem cells （hMSCs）,and to detect the voltage-operated Ca2＋ channels （VOCCs） on the membrane surface of hMSCs using patch clamp technique. Methods Human bone marrow mononuclear cells were collected by gradient centrifugation on Percoll at a density of 1.073 g/ml combined with wall adherence method. The hMSCs were further cultured and expanded in vitro. Trypan blue exclusion staining method was used to count the number of living cells. The cellular phenotypes and cell cycle of hMSCs were identified by fluorescent activated cell sorting （FACS）, and the expression of VOCCs on the membrane surface of hMSCs was observed with patch clamp technique. Results The typical morphology of passaged hMSCs featured a dense array of fibroblastlike structure figure. Up to 99% of hMSCs was viable. Flow cytometry demonstrated that hMSCs expressed CD44 and CD105 but not CD34, CD45 and CD31. Cell cycles of hMSCs showed that 81.52 % of hMSCs was in G1 and G0 stages, and 18.48 % in G2, M and S stages, with a G2/G1 ratio of 1.67. Patch clamp examination displayed nifedipine-sensitive calcium influx in 6 out of 20 hMSCs. The peak calcium influx （ICa-Peak） was （96.67±13.50） pA at -0 mV when activated voltage was about -40 mV. However, the Ica-Peak was restrained to （47.75± 11.24） pA with 5 μ mol/L nifedipine （P〈0.05）. Conclusion hMSCs isolated and cultured in vitro were highly viable and mostly were at G0 or G1 stages. VOCCs were shown to be present on the membrane surface of hMSCs.