Shox2基因修饰犬骨髓间充质干细胞功能检测 .docVIP

DOI：10.16016/j.1000-5404.201501205 Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化 罗首鸣，冯媛媛，杨攀，宋治远（400038重庆第三军医大学西南医院心血管内科，重庆市介入心脏病学研究所） 目的探讨矮小同源盒基因亚型2（Shox2）外源基因对犬骨髓间充质干细胞(canine mesenchymal stem cells,cMSCs)体外诱导分化。方法分离培养犬骨髓间充质干细胞，用包装好的两种慢病毒载体（pLentis-mShox2-RFP及pLentis-RFP）分别转染第代cMSCs，收集转染阳性的cMSCs与乳鼠心肌细胞(NRVMs)共培养。实验分组：A组RFP-cMSCs组B组RFP-cMSCs + NRVMs共培养组C组Shox2-RFP-cMSCs组D组Shox2-RFP-cMSCs +NRVMs共培养组。共培养诱导分化5d后，利用嘌呤霉素对cMSCs进行纯化，运用全细胞膜片钳检测诱导出的内向电流的动力学特征，运用RT-PCR、Western-blot检测HCN4、Tbx3、Cx45/43、Nkx2.5等标志性基因mRNA和蛋白的表达情况，并行免疫荧光检测。结果Western blot及PCR结果示D组细胞HCN4、Tbx3、Cx45等窦房结细胞特异性基因的表达较对照组明显增高（P＜0.05），A、B两组细胞Cx43及Nkx2.5较C、D组细胞明显增高（P＜0.05）；免疫荧光示在Shox2基因调控作用下，cMSCs有HCN4通道生成；膜片钳检测结果显示C组细胞产生了具有明显时间-电压依赖性的内向电流，且对细胞外Cs+敏感，符合起搏电流If的动力学特征；D组细胞可见If电流检出率明显增加，电流密度和幅值也增加，而A和B两组未检测出此电流。结论mShox2基因修饰的cMSC诱导产生起搏离子流If高表达窦房结标志性基因，Shox2；共培养；；If电流canine mesenchymal stem cells were transfected with Shox2 Luo Shouming,Feng Yuanyuan,Yang Pan,Song Zhiyuan（Department of Cardiovascular Diseases,Southwest Hospital,Third Military Medical University,Chongqing,400038,China) [Abstract] Objective To explore the effect of short stature homobox2 (Shox2) gene on the differentiation of the canine bone marrow mesenchymal (cMSCs) in vitro. Methods The cMSCs were isolated by gradient centrifugation, transfected with the lentiviral vector pLentis-mShox2-RFP and pLentis-RFP respectively.The whole experiment was divided into four groups: the cMSCs transfected with RFP (Group A as control), the cMSCs transfected with RFP and co-cultured with NRVMs(Group B), the cMSCs transfected with mShox2-RFP (Group C), and the cMSCs transfected with mShox2-RFP and co-cultured with NRVMs (Group D).For each group, the patch-clamp technique were introduced to detect the If current characteristics, while RT-PCR、Western-blot and immunofluorescence were used respectively to test the mRNA levels and the protein expressions of HCN4, Tbx3, Cx45/43, Nkx2.5, etc. Results Western-blot and PCR results suggested that the expressions of HCN4, Tbx3 and C