【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。更多还原

【Abstract】 Objective:To investigate whether overexpression of paired-related homeobox 1(Prrx1) can successfully induce differentiation of brown adipose-derived stem cells(BADSCs) into sinus node-like cells. Methods: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). Whole-cell patch-clamp technique was used to record the pacing current hyperpolarization-activated inward current(I_f). 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 I_f current in Ad-Prrx1 group rather than in Ad-GFP group. The I_f 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 prrx1 can induce BADSCs to differentiate into sinus node-like cells.更多还原