目的 研究肾小管上皮细胞-肌成纤维细胞转分化(TEMT)过程中间隙连接蛋白43(connexin 43)表达和细胞间通讯功能的变化,以及上调connexin 43水平对TEMT的影响。方法 用转化生长因子(TGF)β1刺激人肾小管上皮细胞系(HKC),检测connexin 43、上皮细胞标志物E-钙黏蛋白(E-cadherin)和肌成纤维细胞标志物α-SMA、vimentin的表达。用激光共聚焦显微镜荧光漂白恢复(FRAP)技术检测HKC细胞间通讯功能。通过pLNCX2- connexin 43病毒质粒转染HKC上调connexin 43的表达,检测以上各指标的变化,观察对TEMT的影响。结果 HKC经TGF-β1刺激后.其α-SMA和vimentin mRNA和蛋白质表达增强,而E-cadherin和connexin 43表达下降(P<0.05),细胞间通讯功能降低(P<0.05)。connexin 43高表达后,TEMT程度明显减轻。结论 上调肾小管上皮细胞间通讯功能可部分减轻TEMT程度。
Objective To observe the change and possible role of connexin 43 as well as gap junction intercellular communication function in TGF-~I triggered renal tubular epithelial- myofibroblast transdifferentiation (TEMT) of cultured human renal tubular epithelial cells (HKC). Methods The normal HKC was stimulated with rhTGF-β1 of 10 μg/L. Simuhaneously,pLNCX2- connexin 43 vector was transferred in TGF-β1-stimulated HKC. The morphological alterations were observed under an inverted microscope. The cells were collected at 48th hours after rhTGF-β1 stimulation. The expression levels of connexin 43,E-cadherin, α-smooth muscle actin (α-SMA), vimentin mRNA and protein were detected by RT-PCR and Western blotting. The gap junction intercellular communication function was measured by fluorescence recovery after photobleaching assay (FRAP). Results rhTGF-β1 stimulation altered the HKC's shape from oval to fusiform, down-regulated the connexin 43 and E-cadherin expression and up-regulated α-SMA and vimentin expression (P〈0.05), which resulted in down-regulation of intercellular communication function. The fluorescence recovery rate and recovery period of TGF-β1-stimulation group were significantly decreased compared to control (P〈0.05). In the pLNCX2-connexin 43 vector transferred group, the fluorescence recovery rate and recovery period were significantly increased compared to TGF-β1 stimulated group (P〈0.05). Conclusion Up-regulating connexin 43 expression can delay TEMT exogenous TGF-β1