重组腺相关病毒介导克老素基因表达对2型糖尿病大鼠肾脏纤维化的作用及其机制  

Effect of recombinant adeno-associated virus-mediated klotho gene expression on kidney fibrosis in rats with type 2 diabetes mellitus and relevant mechanism

作　　者：邓明洪 马厚勋[1] 罗玉梅[1] 李运奎[1] 吴平[1] 王艳娇[1] 李宝善 

机构地区：[1]重庆医科大学附属第一医院老年病科,重庆400016 [2]重庆市急救中心老年病科,重庆400014

出　　处：《中国生物制品学杂志》2013年第11期1593-1598,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30672212);重庆市卫生局科研基金(2010-2-079)

摘　　要：目的观察重组腺相关病毒(recombinant adeno-associated virus,rAAV)介导的克老素(klotho,KL)基因表达对2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠肾脏纤维化的影响及其机制。方法将SD大鼠随机分为4组,随机选3组建立T2DM大鼠模型,分别经尾静脉注射rAAV.mKL(T2DM-mKL组)、rAAV.GFP(T2DM-GFP组)和PBS(T2DM-PBS组),正常对照(SD-PBS组)大鼠经尾静脉注射PBS,12周后,处死动物,收集肾脏组织标本,冰冻切片观察GFP,Masson染色观察肾脏组织病理变化及胶原纤维表达;免疫组化法检测各组大鼠肾脏组织中KL和波形蛋白(vimentin,VIM)的表达;RT-PCR法检测各组大鼠肾脏组织中Rho激酶(Rho associated coiled-coilforming protein kinase,ROCK)基因mRNA转录水平;Western blot法检测各组大鼠肾脏组织中ROCKⅠ蛋白活性。结果 T2DM-GFP组、T2DM-mKL组大鼠肾脏组织中GFP表达较强;T2DM-mKL组大鼠肾小球基底膜结构较清晰完整,肾小球系膜区及肾小管间质区胶原纤维明显较T2DM-PBS组和T2DM-GFP组减少;T2DM-mKL组KL蛋白的表达明显高于其他各组(P<0.01),VIM蛋白的表达明显低于其他各组,与T2DM-PBS组和T2DM-GFP组VIM蛋白的表达相比,差异有统计学意义(P<0.01);T2DM-mKL组ROCKⅠmRNA转录水平及其蛋白活性均明显低于T2DM-PBS组(P均<0.05)。结论 rAAV.mKL转染可明显增加T2DM大鼠肾脏中KL基因的表达,延缓糖尿病肾纤维化进程,其机制可能与KL抑制ROCK信号通路活性相关。Objective To observe the effect of recombinant adeuo-associated virus （rAAV）-mediated klotho （KL） gene expression on kidney fibrosis in rats with type 2 diabetes mellitus （T2DM） as well as the relevant mechanism. Methods Male SD rats were randomly divided into four groups, of which three groups （T2DM-mKL, T2DM-GFP and T2DM-PBS） were selected for establishment of T2DM model. The rats in T2DM-mKL, T2DM-GFP and T2DM-PBS groups were inject-ed i. v. with rAAV. mKL, rAAV. GFP and PBS respectively, while those in normal control（SD-PBS） group with PBS. The rats were killed 12 weeks later, of which the kidney tissue specimens were collected, prepared into frozen section and ob-served for expression of GFP by fluorescent microscopy, for pathological change and expression of collagen fiber by Mas-son staining, for expressions of KL and vimentin（VIM） by immunohistochemistry, for transcription level of mRNA of Rho associated coiled-coilforming protein kinase （ROCK） gene by RT-PCR, and for ROCK I protein activity by Western blot. Results GFP was highly expressed in kidney tissues of rats in T2DM-GFP and T2DM-mKL groups. The structure of glomerular basement membrane of rats in T2DM-mKL group was clear and intact, while the numbers of collagen fibers in mesangial and tubulointerstitial domains decreased significantly as compared with those in T2DM-PBS and T2DM-GFP groups. The expression level of KL in T2DM-mKL group was significantly higher than those in other groups （P 〈 0. 01 ）,while that of VIM was lower than those of other groups and showed significant difference with those in T2DM-PBS and T2DM-GFP groups （P 〈 0. 01）. Both the ROCKI mRNA transcription level and ROCK I activity in T2DM-mKL group were significantly lower than those in T2DM-PBS group （both P 〈 0. 05）. Conclusion The rAAV. mKL transfection sig-nificantly increased the KL expression in kidneys of rats with T2DM, delayed the progression of renal fibrosis in diabetes, of which hthe mechanism might be associated with the

关 键 词：克老素 腺相关病毒 糖尿病 2型 肾脏纤维化 波形蛋白 蛋白激酶 

分 类 号：R587.2[医药卫生—内分泌]