Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化  

Expression profiles of HCN4 in canine mesenchymal stem cells after Shox2 transfection

作　　者：罗首鸣 冯媛媛[1] 杨攀[1] 宋治远[1] 

机构地区：[1]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,重庆400038

出　　处：《第三军医大学学报》2015年第9期869-875,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81270246)~~

摘　　要：目的探讨矮小同源盒基因亚型2(short stature homobox 2,Shox2)外源基因对犬骨髓间充质干细胞(canine mesenchymal stem cells,c MSCs)体外诱导分化的影响。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p Lentis-m Shox2-RFP及p Lentis-RFP分别转染第3代c MSCs,收集转染阳性的c MSCs与乳鼠心肌细胞(rat ventricular myocytes,NRVMs)共培养。实验分4组:A组(RFP-c MSCs组)、B组(RFP-c MSCs+NRVMs共培养组)、C组(Shox2-RFP-c MSCs组)、D组(Shox2-RFPc MSCs+NRVMs共培养组)。共培养诱导分化5 d后,利用嘌呤霉素对c MSCs进行纯化,运用全细胞膜片钳检测诱导出内向电流的动力学特征,运用实时荧光定量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RTPCR检测结果显示,D组细胞Shox2、HCN4、Tbx3、Cx45窦房结细胞特异性基因的表达较A、B、C组明显增高(P<0.05),B组细胞Cx43及Nkx2.5较C、D组细胞明显增高(P<0.05);免疫荧光结果显示,在Shox2基因调控作用下,c MSCs有HCN4通道生成;膜片钳检测结果显示,C、D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,符合起搏离子流If的动力学特征;而A、B组未检测出此电流。结论经m Shox2基因修饰的c MSCs通过体外诱导,产生了起搏离子流If并高表达窦房结标志性基因,成功向具有一定自主搏动能力的窦房结样细胞分化。Objective To determine the effect of exogenous gene short stature homobox 2 （Shox2） on the differentiation of canine bone marrow mesenchymal （cMSCs） in vitro. Methods cMSCs were isolated by gradient centrifugation, and the cells at passage 3 were transfected with the lentiviral vector pLentis- mShox2-RFP and pLentis-RFP respectively. Then the cells were divided into 4 groups, that is, the cMSCs transfected with RFP （group A as control）, the cMSCs transfected with RFP and co-cultured with rat ventricular myocytes （NRVMs, group B ）, the cMSCs transfected with mShox2-RFP （group C ）, and the eMSCs transfected with mShox2-RFP and co-cultured with NRVMs （group D）. Patch-clamp technique were introduced to detect the If current characteristics, while qPCR, Western blotting and immunofluorescence assay were used respectively to test the mRNA and protein expression levels of HCN4, Tbx3, Cx45/43 and Nkx2.5, etc. Results Western blotting and PCR results suggested that the expression of HCN4, Tbx3 and Cx45, mainly expressed in sinus node cells, were significantly higher in the group D than in the other groups （ P 〈 0.05 ）. Meanwhile, the expression of Cx43 and Nkx2.5 was remarkably higher in the group B than the groups C and D （P 〈 0. 05 ）. Besides, immunofluorescence assay suggested that Shox2 promoted the generation of HCN4 in the cMSCs. Furthermore, the patch-clamp test indicated that the cMSCs in the groups C and D displayed a hyperpolarization-activated inward current that was highly sensitive to Cs＋ and showed obviously voltage and time dependences, which met the It current characteristics. But no such current was found in the cells from groups A and B. Conclusion cMSCs transfected with mShox2 generate If current and express highly the sinus-node-cell-like genes, which indicating that those cMSCs are successfully differentiated into sinus node-like cells with pacing function.

关 键 词：Shox2 共培养 间充质干细 起搏离子流 

分 类 号：R331.22[医药卫生—人体生理学] R394.33[医药卫生—基础医学]