热休克蛋白47在单侧输尿管梗阻大鼠肾小管上皮细胞转分化中的作用

摘要

目的:研究热休克蛋白47在单侧输尿管梗阻大鼠肾小管上皮细胞转分化(EMT)中的作用,探讨以HSP47为靶点防治肾脏间质纤维化的策略.方法:采用UUO大鼠模型,雄性SD大鼠72只随机分为假手术组(Sham组)、UUO模型组(UUO组)、UUO模型+超声联合脂质微泡+HSP47反义寡核苷酸组(antisenseODNs组)以及UUO模型+超声联合脂质微泡+ HSP47正义寡核苷酸组(senseODNs组),每组18只,于术后14 d处死各组大鼠.HE和Masson染色法观察各组大鼠肾脏病理改变,Western blotting及Real-time PCR方法检测HSP47、波形蛋白(Vimentin)、紧密连接蛋白(Zona occludens-1,ZO-1)及Ⅰ型胶原(Col-Ⅰ)的表达.结果:HE和Masson染色显示,与Sham组相比,UUO组大鼠肾间质宽度明显增加,肾小管扩张及上皮细胞变性,肾小管损伤程度、间质纤维化程度逐渐增加(P＜0.05).与UUO组相比,antisenseODNs组病变程度较UUO组减轻(P＜0.05),senseODNs组无明显差别(P＞0.05).Western blotting及Real-time PCR显示,与Sham组相比,UUO组HSP47蛋白及mRNA表达明显升高(P＜0.05),同时Vimentin、Col-Ⅰ蛋白及mRNA表达上调,而ZO-1蛋白及mRNA表达下调(P＜0.05);与UUO组相比,antisenseODNs组HSP47蛋白及mRNA表达明显降低(P＜0.05),同时Vimentin及Col-Ⅰ蛋白及mRNA表达下调,而ZO-1蛋白及mRNA表达上调(P＜0.05),纤维化程度明显减轻;senseODNs组HSP47、Vimentin、Col-Ⅰ、ZO-1蛋白及mRNA表达无明显变化(P＞0.05).结论:UUO模型大鼠肾小管上皮细胞EMT过程中HSP47表达上调,抑制HSP47表达能部分逆转肾小管上皮细胞EMT.%Objective:To investigate the role of HSP47 in the process of EMT.Methods:Male Sprague Dawley (SD) rats were randomizely divided into sham group (n =18),unilateral ureteral obstructive (UUO) model group (n =18),UUO model with HSP47 antisense oligonucleotides (ODNs)-treat group (antisenseODNs group,n =18) and the sense ODNs-treat group (senseODNs group,n =18).The HSP47 sense or antisense ODNs were transfered into the kidney using ultrasound exposure with liposome microbubble.The obstructed kidneys were harvested 14 days after UUO.Changes in renal morphology were examined by HE and Masson staining.The expressions of HSP47,vimentin,zona occludens-1 (ZO-1) and type Ⅰ collagen (Col-Ⅰ) were examined by Western blotting and Real-time PCR.Results:Compared with the sham group rats,the expressions of HSP47,vimentin,Col-Ⅰ were markedly upregulated in UUO model rats (P ＜ 0.05),but the expression of ZO-1 was downregulated (P ＜ 0.05),more severe tubular injury and tubulointerstitial fibrosis were also noted in UUO model rats (P ＜ 0.05).Treated with antisense ODNs,but not sense ODNs (P ＞ 0.05),abrogated the changes in the expressions of HSP47,vimentin,ZO-1 and Col-Ⅰ (P ＜ 0.05).Conclusion:HSP47 is upregulated in the progress of EMT in the UUO model.Suppression of HSP47 can reverse the EMT of renal tubular epithelial cells.