CD2AP分子mRNA和蛋白的表达在肾组织及细胞损伤中的作用  

The roles of CD2AP molecule mRNA and protein in renal tissues and cell injury

作　　者：李晓艳[1] 于力[1] 于生友 温捷 

机构地区：[1]广州医学院附属广州市第一人民医院儿科,510180 [2]广东省临床分子医学及分子诊断重点实验室,广州510180

出　　处：《广东医学》2013年第6期821-826,共6页Guangdong Medical Journal

基　　金：国家自然科学基金面上项目(编号:81273205);广东省科技计划项目(编号:2011B020800002);广州市科技计划项目(编号:2010Y1-C521)

摘　　要：目的观察CD2AP分子mRNA和蛋白的表达在嘌呤霉素(PAN)肾病模型及肾小球足细胞系损伤中的作用。方法将雄性SD大鼠64只随机分为肾病模型组和对照组,每组32只。肾病模型组大鼠尾静脉注射PAN 5 mg/kg,建立PAN肾病模型;对照组注射8 mg/kg生理盐水。分别于第2、4、6和8周每组各处死8只大鼠用于实验。体外培养小鼠肾小球足细胞系(MPC5),分为细胞刺激组和对照组,细胞刺激组给予PAN刺激足细胞,于刺激后12、24和48 h收集足细胞用于实验;对照组用RPMI 1640培养液培养足细胞。光镜下观察肾病模型组肾组织的病理学改变;倒置显微镜下观察细胞刺激组足细胞的形态变化;应用荧光定量PCR、Western blot和免疫荧光染色检测肾病模型组、细胞刺激组和对照组不同时间点CD2AP分子mRNA、蛋白表达及分布的变化。结果(1)肾病模型组病理学变化为系膜和细胞外基质增生,部分或广泛肾小球呈局灶节段性硬化;肾病模型组注射PAN后第2周CD2AP分子mRNA和蛋白的表达量明显下降,一直持续至第8周。(2)细胞刺激组形态变化为刺激后12、24及48 h足细胞体积明显缩小,主要表现为足突回缩、消失及失去细胞间连接;细胞刺激组在给予PAN刺激后12 h CD2AP分子mRNA表达明显下降,mRNA表达下降持续至PAN刺激后48 h;CD2AP的蛋白表达在刺激足细胞12 h时无显著改变,在24和48 h时CD2AP的蛋白表达明显下降。(3)免疫荧光染色显示,正常情况下CD2AP均匀分布于足细胞的细胞核、细胞浆及细胞膜;细胞刺激组12和24 h CD2AP分布出现异常,在细胞核和细胞浆呈粗颗粒状分布,刺激48 h后在细胞膜和细胞浆出现分布缺失。结论 CD2AP在大鼠PAN肾病模型及体外培养肾小球足细胞系中mRNA和蛋白的表达明显下调,提示CD2AP分子是肾小球足细胞损伤的重要标志,CD2AP表达量及分布变化可能是临床上蛋白尿发生的分子机制。Objective To investigate the roles of CD2AP molecule mRNA and protein in puromycin - induced nephropathy and glomerular podocyte injury in vitro. Methods 64 male SD rats were randomly divided into nephropathy model group and control group （n = 32）, in which puromycin （PAN, 5 mg/kg） and saline were given respectively. 8 subjects from each groups were sacrificed at the 2nd, 4th, 6th and 8th week. Mouse podocyte cell fine （ MPC5 ） in vitro were divided into stimulate group, in which PAN treatment were given, and control group. The cells were collected 12 h, 24 h and 48 h after culture. Light microscopy and inverted microscopy were applied for observation of renal histopathological and cellular pathological changes. Meanwhile, RT - PCR, Western blot and immunofluorescence staining were used for assessment of the expression and distribution of CD2AP mRNA and protein. Results Glomerular mesangial and extracel- lular matrix proliferation, and partial or extensive focal segmental sclerosis were observed in nephropathy model group, so was the continuous down - regulation of CD2AP mRNA and protein since the 2nd week. Significant shrinkage of podocytes, including pod retraction or disappeared absence and intercellular junction loss, were observed in simulated group ; so was the down - regulation of CD2AP mRNA and protein. Even nuclear, cytoplasm and membrane CD2AP distribution was revealed in control group, while coarse granular CD 2 AP distribution in nucleus and cytoplasm was revealed in stimulated group 12 h and 24 h after stimulation, and even cytoplasm and membrane absence of CD2AP was found after 48 h. Conclusion CD2AP mRNA and protein are both significantly down -regulated in rat puromycin nephropathy model and cultured mouse podocyte cells, suggesting an important indicator of podocyte injury. These changes of CD2AP expression and distribution may be involved in the molecular mechanism of proteinuria.

关 键 词：嘌呤霉素 CD2AP 肾组织 足细胞 

分 类 号：R692[医药卫生—泌尿科学]