MiR-375抑制的人诱导多能干细胞分化为起搏样细胞的研究  

Experimental research on the differentiation of miR-375-inhibited human-induced pluripotent stem cells into pacemaker cells

作　　者：章澜 杨媚[1] 王晞[1] 唐艳红[1] 宋思维[1] 陈玉婷[1] 王腾[1] 黄从新[1] ZHANG Lan;YANG Mei;WANG Xi;TANG Yan-hong;SONG Si-wei;CHEN Yu-ting;WANG Teng;HUANG Cong-xin(Department of Cardiology,Renmin Hospital of Wuhan University,Cardiovascular Research Insti­tute,Wuhan University,Hubei Key Laboratory of Cardiology,Wuhan 430060,Hubei,China)

机构地区：[1]武汉大学人民医院心内科武汉大学心血管病研究所心血管病湖北重点实验室,湖北武汉430060

出　　处：《中国心脏起搏与心电生理杂志》2022年第2期148-154,共7页Chinese Journal of Cardiac Pacing and Electrophysiology

基　　金：湖北省技术创新专项(2016ACA153)。

摘　　要：目的 探讨抑制miR-375表达能否使其修饰的人诱导多潜能干细胞(hiPSC)在向hiPSC源性心肌细胞(hiPSC-CMs)分化的过程中更高效地向起搏样细胞转化。方法 采用hiPSC时序性激活或抑制Wnt信号通路定向诱导分化到hiPSC-CMs,流式分析检测其分化效率。分别转染携带miR-375抑制剂和绿色荧光蛋白(GFP)的慢病毒rLV-ZsGreen-Puro-hsa-mir-375 inhibitor(miR-375组)和等量带GFP的空载慢病毒(GFP组),空白组不做转病毒处理(Null组)。分化14 d后在倒置显微镜下计算各组细胞每分钟搏动次数,采用实时荧光聚合酶链式反应和蛋白质免疫印迹检测三组起搏细胞发育相关转录因子(Shox2、Nkx2.5、Isl1)、起搏相关离子通道超极化激活环核苷酸门控通道4型(HCN4)mRNA和蛋白水平变化,采取免疫荧光法检测GFP组和miR-375组Shox2蛋白和心肌细胞特异性标志物心肌肌钙蛋白T(cTNT)的表达。结果 分化第7 d起可见细胞自发性搏动,流式分析结果cTNT阳性率超80%,hiPSCs成功诱导分化到hiPSC-CMs;与GFP组和Null组比较,miR-375组搏动速度及Shox2、HCN4、Isl1的mRNA和蛋白表达水平明显升高,Nkx2.5表达明显降低(P<0.05);荧光显微镜观察miR-375组Shox2蛋白表达强于GFP组,cTNT蛋白表达两组无显著差异。结论 通过抑制miR-375表达能增加hiPSCs向起搏样细胞的分化效率。Objective To investigate whether the inhibition of Mir-375 expression can make human induced pluripotent stem cells(hiPSC) transform into sinoatrial node pacemaker cells during the differentiation of human induced pluripotent stem cells-derived cardiomyocytes(hiPSC-CMs) more efficiently. Methods HiPSCs were differentiated into hiPSC-CMs by sequential activation or suppression of Wnt signaling pathway, the efficiency of differentiation was measured by flow analysis. Lentivirus rLV-Zsgreen-puro-hsa-Mir-375 inhibitor with green fluorescent protein(GFP) and Mir-375 inhibitor(Mir-375 group) and equivalent amount of no-load lentivirus with GFP(GFP group) were transfected into cells respectively, the blank group was not treated with virus transfer(Null group). After 14 days of differentiation, the beating frequecy in per minute were calculated under the inverted microscope, and the mRNA and protein levels of development-related transcription factors(Shox2,Nkx2.5,Isl1)and pace-related ion channel hyperpolarization activated cyclic nucleotide gated channel 4(HCN4) in the three groups were detected by real-time fluorescence polymerase chain reaction and western blot. The expression of Shox2 protein and cardiomyocyte specific marker cardiac troponin T(cTNT) in GFP and miR-375 group were detected by immunofluorescence assay. Results Spontaneous cell pulsation was observed on the 7 th day of differentiation, flow cytometry showed that the positive rate of cTNT was over 80%,which showed that hiPSCs were differentiated into hiPSC-CMs successfully.Compared with GFP group and NULL group,the pulsatile speed and mRNA and protein expression levels of Shox2,HCN4and Isl1 were significantly increased in Mir-3 7 5group,while the expression level of Nkx2.5was significantly decreased(P<0.05).Fluorescence microscopy showed Shox2protein expression in Mir-375group was stronger than that in GFP group while no significant difference in cTNT protein expression between two groups. Conclusion By inhibiting the expression of miR-375,hiPSCs modifie

关 键 词：心血管病学 微小核糖核酸-375 人诱导多潜能干细胞 慢病毒载体 矮小身材同源异型框基因2 心肌细胞 生物起搏 

分 类 号：R318.11[医药卫生—生物医学工程]