在hUCMSCs中过表达Shox2和HCN4以构建生物起搏表型细胞的研究

Construction of biological pacemaker phenotype cells by overexpressing Shox2 and HCN4 in hUCMSCs

目的:拟利用慢病毒载体将Shox2和HCN4转染至永生化人脐带间充质干细胞(hUCMSCs),探讨体外过表达Shox2和HCN4的hUCMSCs是否具有起搏样细胞表型。方法:进行hUCMSCs体外传代培养。分别构建携带Shox2和HCN4基因的慢病毒载体及空载体,通过慢病毒介导Shox2和HCN4基因转染至hUCMSCs。采用CCK-8实验检测各组细胞活力。应用RT-qPCR和Western blot方法检测起搏相关因子mRNA和蛋白表达水平。结果:(1)慢病毒过表达载体构建成功,Shox2-MSCs组和HCN4-MSCs组转染效率约为50%~55%,Shox2-HCN4-MSCs组转染效率约40%~45%,形态较前也稍有变化,折光性较好;(2)实验组细胞生长速度较对照组变缓(P<0.05);(3)过表达组中HCN4、Tbx18和Tbx3的mRNA表达量显著高于对照组(P<0.05),Nkx2.5和Cx43的mRNA表达量显著低于对照组(P<0.01);(4)与对照组相比,单基因过表达组Cx45蛋白水平显著上调,Nkx2.5和Cx43蛋白水平显著下调(P<0.01),双基因过表达组比单基因过表达组上调(或下调)更显著(P<0.01)。结论:过表达Shox2和(或)HCN4的hUCMSCs具有起搏样细胞表型;共转染有促进hUCMSCs向起搏样细胞表型转化的叠加效果。

AIM:In this study,we intend to use lentiviral vectors to transfect Shox2 and HCN4 gene into immortalized human umbilical cord mesenchymal stem cells(hUCMSCs),and to explore whether the transfected hUCMSCsin vitro have pacing cell-like phenotype.METHODS:The hUCMSCs were cultured and passaged in vitro. Lentiviral vectors carrying Shox2 and HCN4 genes and empty vectors were constructed,and Shox2 and HCN4 genes were transfected to hUCMSCs by lentivirus. Cell viability in each group was detected by CCK-8 assay. The mRNA and protein expression levels of related genes were detected by RT-qPCR and Western blot.RESULTS:(1)The lentiviral overexpression vector was successfully constructed. The transfection efficiency of Shox2-MSCs group and HCN4-MSCs group was about 50%~55%,Shox2-HCN4-MSCs group was about 40%~45%.(2)The growth rate of cells in the experimental group was slower than that in the control group(P<0. 05).(3)The mRNA expression levels of HCN4,Tbx18 and Tbx3 in the overexpression group were significantly higher than those in the control group(P<0. 05),and the expression levels of Nkx2. 5 and Cx43 were significantly lower than those in the control group(P<0. 01).(4)Compared with the control group,the protein level of Cx45 in the single-gene overexpression group was significantly up-regulated and the protein levels of Nkx2. 5 and Cx43 were significantly down-regulated(P<0. 01). The upregulation and downregulation were more significant in the double overexpression group(P<0. 01).CONCLUSION:hUCMSCs overexpressing Shox2 and/or HCN4 have pacemakerlike cell phenotype,and the co-transfection has the additive effect of promoting the transformation of hUCMSCs into pacemaker-like cells.