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目的探讨敲减Runt相关转录因子3(RUNX3)对低氧诱导人心脏微血管内皮细胞(HCMEC)向间质转分化的影响及具体机制。方法低氧条件下培养HCMEC,使用RUNX3干扰慢病毒敲减HCMEC中的RUNX3基因,应用反转录PCR检测内皮细胞间质转分化(EndoMT)相关基因CD31、血管内皮钙黏素(VE-cadherin)、α平滑肌肌动蛋白(α-SMA)、成纤维细胞特异蛋白1(FSP-1)以及RUNX3的mRNA表达,应用Western blot法检测CD31、α-SMA、RUNX3、转化生长因子β2(TGF-β2)、Smad2/3、p-Smad2/3以及Notch-1、Hes1、Hey1蛋白表达,免疫荧光细胞化学染色检测CD31、α-SMA的表达和定位。结果低氧能够诱导HCMEC发生EndoMT;低氧时TGF-β2、Smad2/3、p-Smad2/3以及Notch-1、Hes1、Hey1蛋白均上调;而RUNX3敲减后TGF-β2以及Notch-1蛋白水平增高,然而Smad2/3、p-Smad2/3以及Hes1、Hey1蛋白水平均不同程度下调。结论 RUNX3敲减可减轻低氧诱导的EndoMT,可能是通过部分抑制TGF-β及Notch信号通路。
Objective To investigate the effect of knockdown of Runt-associated transcription factor 3 (RUNX3) on the transdifferentiation of human cardiac microvascular endothelial cells (HCMECs) into mesenchymal cells and its mechanism. Methods HCMEC was cultured under hypoxic conditions, RUNX3 was used to knockdown the RUNX3 gene in HCMEC by lentivirus knockdown, and the expressions of CD31 and VE-cadherin were detected by reverse transcription polymerase chain reaction (RT-PCR) , Α-SMA, FSP-1 and RUNX3 were detected by Western blot. The expressions of CD31, α-SMA, RUNX3 and TGF-β2 ), Smad2 / 3, p-Smad2 / 3 and Notch-1, Hes1, Hey1 protein expression were detected by immunofluorescence cytochemistry staining CD31, α-SMA expression and localization. Results Hypoxia could induce EndoMT in HCMECs. The expression of TGF-β2, Smad2 / 3, p-Smad2 / 3 and Notch-1, Hes1 and Hey1 were all up-regulated in hypoxia. However, Smad2 / 3, p-Smad2 / 3 and Hes1, Hey1 protein levels were down-regulated to some extent. Conclusion RUNX3 knockdown can reduce hypoxia-induced EndoMT, possibly through partial inhibition of TGF-β and Notch signaling.