配对相关的同源异型框1导入棕色脂肪干细胞构建生物起搏  

Paired-related homeobox 1 introduced into brown adipose-derived stem cells to trigger biological pacing

作　　者：尹琳 刘明鑫[1] 王凤媛 唐艳红[1] 王晞[1] 赵庆彦[1] 王腾[1] 陈玉婷[1] 黄从新[1] YIN Lin;LIU Mingxin;WANG Feng yuan;TANG Yanhong;WANG Xi;ZHAO Qingyan;WANG Teng;CHEN Yuting;HUANG Congxin(Departjnent of Cardiology,Renmin Hospital of Wuhan University,Cardiovascular Research Institute,Wuhan University,Hubei Key Laboratory of Cardiology,Hubei 430060,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省重点实验室

出　　处：《国际心血管病杂志》2019年第6期351-357,共7页International Journal of Cardiovascular Disease

基　　金：湖北省技术创新专项(2016ACA153);中央高校基本科研业务费专项资金(2042015kf0229)

摘　　要：目的:探讨过表达配对相关的同源异型框1(Prrx1)是否能诱导棕色脂肪干细胞(BADSC)向类窦房结细胞分化,构建生物起搏。方法:分离培养大鼠BADSC,分别转染带有GFP的空载腺病毒(Ad-GFP组)和带有Prrx1的腺病毒(Ad-Prrx1组)。光镜下观察细胞形态和荧光表达强度,Western blot、实时聚合酶链反应检测窦房结细胞相关的起搏蛋白(HCN4)和转录因子[TBX18、胰岛素基因增强子结合蛋白1(ISL-1)、Pitx2]的表达水平,膜片钳技术检测起搏电流I_f,免疫荧光技术检测细胞HCN4、TBX18和ISL-1的表达。结果:Ad-Prrx1组的起搏相关因子TBX18、ISL-1、HCN4的mRNA和蛋白表达水平均明显高于Ad-GFP组,而Pitx2的mRNA表达水平明显降低(P均<0.05)。膜片钳记录到BADSC的I_f电流,且该电流能被4 mmol/L CsCl阻断。免疫荧光显微镜下可见Ad-Prrx1组Prrx1与TBX18、ISL-1、HCN4在BADSC中共表达,而Ad-GFP组未发现有上述起搏相关蛋白共表达。结论:过表达Prrx1能诱导BADSC分化为类窦房结细胞。Objective:To investigate whether overexpression of paired-related homeobox 1(Prrx1)can successfully induce differentiation o￡brown adipose-derived stem cells(BADSCs)into sinus node-like cells.Methotls:BADSCs were isolated and cultured,and the adenovirus with GFP(Ad-GFP group)and Prrx1(Ad-Prrx1 group)were transfected respectively.Light microscope was used to observe cell morphology and fluorescence expression intensity.Western blot and real-time polymerase chain reaction were used to detect the expression level of hyperpolarization-activated cyclic nucleotide-gated potassium channel 4(HCN4)and transcription factors related to sinus node cells(Tbx18,Isl-1 and Pitx2).Wholecell patch-clamp technique was used to record the pacing current hyperpolarization-activated inward current(If).Immunofluorescence technique was used to detect the expression of HCN4,Tbx18 and Isl-1.Results:The mRNA and protein expression levels of Tbx18,Isl-1 and HCN4 in Ad-Prrx1 group were significantly higher than those in Ad-GFP group,while the mRNA expression level of Pitx2 was significantly lower(P all<0.05).Whole-cell patch clamps were able to record the If current in Ad-Prrx1 group rather than in Ad-GFP group.The If current could be blocked by 4 mmol/L CsCl.Immunofluorescence analysis showed that Prrx1 was coexpressed with TBX18,ISL-1,and HCN4 in Ad-Prrx1 group,which did not appear in Ad-GFP group.Conclusions:Overexpression of prrxl can induce BADSCs to differentiate into sinus node-like cells.

关 键 词：配对相关的同源异型框1 生物起搏 类窦房结细胞 TBX18 胰岛素基因增强子结合蛋白1 

分 类 号：R54[医药卫生—心血管疾病]