人脐带间充质干细胞对低氧环境下的肾小管上皮细胞中肝细胞生长因子(HGF)表达的影响  

Effects of Human Wharton jelly-derived Mesenchymal Stromal Cells on Hepatocyte Growth Factor (HGF) Expression in Hypoxic Kidney Proximal Tubular Epithelial Cells

作　　者：邹翔宇[1] 杜涛[1] 程中良[1] 琚官群[1] 苗帅[1] 朱英坚[1] 刘国华[1] 

机构地区：[1]上海交通大学附属第一人民医院泌尿外科,上海201600

出　　处：《现代生物医学进展》2013年第32期6219-6222,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81170642);上海市科学技术委员会医学引导项目(10411967200)

摘　　要：目的:肾脏的急性缺血缺氧性损伤是泌尿外科常见病,以肾小管间质纤维化为主要病理特点,成体干细胞在急性肾脏损伤动物模型中可以促进肾脏结构修复、改善肾脏功能,肝细胞生长因子作为抗纤维化的主要生长因子,在成体干细胞干预的急性肾脏损伤动物模型实验中起重要作用,然而以往的研究对象主要以动物模型为主,成体干细胞对肝细胞生长因子的具体调节机制尚不清。本实验通过体外分离、培养人的脐带间充质干细胞,来干预离体低氧预处理后的大鼠近端肾小管上皮细胞,探讨在体外培养条件下人脐带间充质干细胞对低氧预处理大鼠近端肾小管上皮细胞肝细胞生长因子表达的影响,为今后研究间充质干细胞治疗肾功能损害提供可靠的理论依据。方法:采用贴壁培养的方法无菌条件下分离、培养、传代人脐带间充质干细胞,细胞融合达90%时更换无血清培养基(serum-free medium,SFM)培养24 h,收集细胞上清液即为人脐带间充质干细胞条件培养基(condition medium,CM);大鼠近端肾小管上皮细胞(tubular epithelial cells,TECs)于低氧环境处理1 h后,随机分为对照组与CM干预组,分别培养24 h和48 h后检测TECs中大鼠肝细胞生长因子(hepatocyte growth factor,HGF)的mRNA水平、收集上清液测定大鼠HGF蛋白的含量,同时收集CM干预组中0h,12h、24h、48h的上清液,测定其中人来源HGF蛋白的含量,并对TECs在CM干预24 h和48 h后行免疫组化定性人的HGF蛋白的表达。结果:在低氧预处理的培养环境下,CM干预组中,TECs中大鼠来源的HGF mRNA表达水平在24 h和48 h时明显高于对照组(P<0.05);上清液中大鼠HGF蛋白的含量在24 h和48 h,CM干预组显著高于对照组(P<0.05);上清液中人的HGF蛋白的含量随时间进行性增高,免疫组化染色显示在24 h和48 h大鼠TECs中能检测人的HGF蛋白的表达。结论:在低氧预处理的体外培养环境下,脐带间充质�Objective： Acute renal ischemia and hypoxia injury is a common disease in urology, and the main pathological feature is tubulointerstitial fibrosis. The adult stromal stem cells can repair kidney and improve renal functions in animal models. Hepatocyte growth factor as a major antifibrotic factor has an significantly alterations in these experiments. However previous studies mainly targeted in animal models, and the correct mechanisms is not clear. In our experiment, we isolated human Wharton jelly-derived mesenchymal stromal cells （WJMSCs） to intervene the hypoxic pretreated kidney proximal tubular epithelial cells in vitro culture conditions, so as to investigate the effects of WJMSCs on hepatocyte growth factor expression in hypoxic pretreated kidney proximal tubular epithelial cells, and provide a theoretical basis for revealing the repair mechanisms of stromal stem cells in the kidney tissue after renal ischemia and hypoxia injury. Methods： The WJMSCs were separated, cultured and expanded sterilely, and the culture medium was changed when the cells grew to 90%, the cells were cultivated in senlm-free medium （SFM） for 24 hours, the cell culture medium was collected as the condition medium （CM） of WJMSCs. Rat tubular epithelial cells （TECs）were cultured in hypoxia injury for 1 h, CM or without CM conducted for 12 h, 24 h and 48 h,collect the TECs to assess the level of Rat-HGF m-RNA use the Real time-PCR method, and collect the supernatants to assess the level of Human-HGF protein and Rat-HGF protein using the ELISA method. Immunochemistry staining method was used to explain whether TECs can synthesize human HGF proteins. Results： In hypoxic conditions, the CM of WJMSCs can significantly improve the level Rat-HGF m-RNA of TECs in 24 h （P〈0.05） and 48 h （P〈0.05）, and the Rat-HGF protein in the supematants also significantly rised in 24 h （P〈0.05） and 48 h （P〈0.05）compared to the control. The Human-HGF protein in the supematants increases over time within 0 h, 12 h,

关 键 词：人脐带间充质干细胞 近端肾小管上皮细胞低氧培养 肝细胞生长因子 

分 类 号：R692[医药卫生—泌尿科学]