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

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.