KLF-4靶向调控E-cadherin表达对高糖腹膜透析液诱导的大鼠腹膜纤维化的影响  

Effect of KLF-4 on peritoneal fibrosis induced by high glucose peritoneal dialysate via targeting regulation of the expression of E-cadherin

作　　者：车明文 徐国顺 龚杜景子 赵建乐 李晶 陈亚蓉 蒋希 马兴杰 何丽洁 Che Ming-Wen;Xu Guo-Shun;Gong Du-Jing-Zi;Zhao Jian-Le;Li Jing;Chen Ya-Rong;Jiang Xi;Ma Xing-Jie;He Li-Jie(Department of Internal Medicine,the 951 Hospital of PLA,Korla,Xinjiang 841000,China;Department of General Surgery,69220 Regiment Hospital of PLA,Kuqa,Xinjiang 842000,China;Department of Nephrology,Xijing Hospital,Air Force Military Medical University,Xi’an 710032,China)

机构地区：[1]解放军第951医院内科,新疆库尔勒841000 [2]解放军第69220部队医院普通外科,新疆库车842000 [3]空军军医大学西京医院肾脏内科,西安710032

出　　处：《解放军医学杂志》2020年第9期904-912,共9页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81270768)。

摘　　要：目的探索Kruppel样因子4(KLF-4)与E-钙黏素(E-cadherin)在人腹膜间皮细胞(HPMCs)中的关系,以及二者在高糖腹膜透析液诱导的腹膜纤维化动物模型中的表达及作用。方法在HPMCs中共转染KLF-4及E-cadherin启动子区域结合位点或其突变体质粒,利用试剂盒检测各结合位点及其匹配的突变体的荧光素酶活性,判断KLF-4是否可与E-cadherin启动子区域结合位点相结合;采用染色质免疫共沉淀实验(CHIP)检测KLF-4是否可与E-cadherin启动子区域结合位点相结合;采用Real-time PCR及Western blotting检测b、d、f、g结合位点及其匹配突变体E-cadherin的表达。将30只SD大鼠随机均分为盐水组、腹膜透析液组及实验组,每组10只。盐水组腹腔内注射0.9%NaCl溶液,腹膜透析液组腹腔内注射4.25%高糖腹膜透析液,实验组腹腔内注射4.25%高糖腹膜透析液并经尾静脉注射含有超声微泡的KLF-4质粒混悬液。采用HE染色观察3组大鼠腹膜组织的厚度,Masson染色观察3组大鼠腹膜组织中胶原纤维的沉积情况,免疫组织化学染色观察3组大鼠腹膜组织中KLF-4、E-cadherin、α-平滑肌肌动蛋白(α-SMA)及纤维连接蛋白(FN)的表达水平。结果启动子荧光素酶报告基因及CHIP检测结果显示,KLF-4可以与HPMCs中E-cadherin启动子区域的结合位点相结合。Real-time PCR及Western blotting结果显示,KLF-4可正向调控E-cadherin的表达。HE染色结果显示,腹膜透析液组大鼠的腹膜组织厚度[(105.91±12.0)μm]明显大于盐水组[(20.89±5.39)μm]及实验组[(23.05±6.07)μm],差异有统计学意义(P<0.05),而盐水组与实验组差异无统计学意义(P>0.05)。Masson染色结果显示,腹膜透析液组的胶原纤维沉积(0.89±0.09)明显高于盐水组(0.19±0.03)及实验组(0.15±0.06),差异有统计学意义(P<0.05),而盐水组与实验组差异无统计学意义(P>0.05)。免疫组织化学染色结果显示,腹膜透析液组的KLF-4(0.27±0.09)及E-cadherin(0.31±0.03)�Objective To explore the relationship between Kruppel-like factor 4(KLF-4)and E-cadherin in human peritoneal mesothelial cells(HPMCs),and the expression and function of KLF-4 in the animal model of peritoneal fibrosis induced by high glucose peritoneal dialysate.Methods Co-transfection in HPMCs with the plasmid of KLF-4 and the bind site or mutant in the promoter region of E-cadherin,and then the luciferase activity was measured of the each bind site and its matched mutants to estimate whether KLF-4 can combine with the bind site in the promoter region of E-cadherin;Chromatin immunocoprecipitation(CHIP)was exploited to verify if KLF-4 can combine with the bind site in the promoter region of E-cadherin;Real-time PCR and Western blotting were performed to detect the expression of E-cadherin at the bind site and matched mutants of b,d,f and g.Thirty SD rats were randomly divided into saline group,peritoneal dialysate group and experimental group(10 each).Rats in saline group were given intraperitoneal injection with 0.9%NaCl,in peritoneal dialysate group were given with 4.25%high glucose peritoneal dialysate,and in experimental group were given via tail vein with 4.25%high glucose peritoneal dialysate and the mixture of KLF-4 plasmid suspension containing ultrasound microbubble.To observe the peritoneal tissue thickness of the 3 groups of rats by Hematoxylin and Eosin staining.Masson trichrome staining was performed to detect the deposition of collagen fibers in peritoneal tissue of the 3 groups of rats.Immunohistochemistry was used to detect the expression level of KLF-4,E-cadherin,α-SMA and fibronectin(FN)in peritoneal tissue of the 3 groups of rats.Results Promoter luciferase reporter gene and CHIP results showed that KLF-4 can combine with the bind site in the promoter region of E-cadherin in HPMCs.Real-time PCR and Western blotting showed that KLF-4 can positively regulate the expression of E-cadherin.HE staining showed that the peritoneal tissue was obviously thickened in rats of peritoneal dialysate group[(1

关 键 词：Kruppel样因子4 人腹膜间皮细胞 E-钙黏素 腹膜纤维化 

分 类 号：R459.5[医药卫生—治疗学]