Klotho基因敲降或过表达对乙醛酸盐诱导的小鼠肾结石模型的影响及机制研究  

Effect of klotho gene knockout/overexpression on calcium oxalate stone in mice and mechansim

作　　者：拜合提亚尔·艾合买提江 刘若天 陈策 贺毅[1] 崔涛[1] 杜恒[1] 木拉提·马合木提[1] Baihetiyaer·Aihemaitijiang;Liu Ruotian;Chen Ce;He Yi;Cui Tao;Du Heng;Mulati·mahemuti(Department of Urology,Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China;Department of Urology,Henan Second People’s Hospital,Zhengzhou 450000,China)

机构地区：[1]新疆医科大学第二附属医院泌尿外科,乌鲁木齐830011 [2]河南省第二人民医院泌尿外科,郑州450000

出　　处：《中华实验外科杂志》2023年第6期1034-1037,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81760128)。

摘　　要：目的调控Klotho基因观察草酸钙晶体对小鼠氧化损伤、细胞凋亡及晶体黏附的影响。方法选取新疆医科大学动物实验室8周龄C57BL/6雄性小鼠30只,构建过表达/沉默Klotho基因小鼠肾结石模型,将模型简单随机抽样法分为分为对照组(Ctrl)组(n=6)、草酸钙结石模型(Model)组(n=6)、模型+生理盐水(Model+Vector)组(n=6)、基因沉默+模型(shKlotho+Model)组(n=6)和基因过表达+模型(Klotho+Model)组(n=6)。应用免疫印迹实验(Western blot)和聚合酶链反应(PCR)检测蛋白级信使RNA(mRNA)的表达;酶联免疫测定(ELISA)检测氧化应激蛋白水平。苏木精-伊红(HE)色观察肾脏病理改变,风库萨(Von Kossa)染色观察肾脏草酸钙晶体黏附情况。脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)法检测肾组织细胞凋亡。两组间统计学差异采用t检验,多组间统计学差异采用单因素方差分析。结果本课题组在体内建立了小鼠草酸钙结石模型,并通过Von Kossa染色验证模型。Model组血清中过氧化氢酶(CAT)[(6.25±0.84)U/ml比(9.55±1.42)U/ml]、谷胱甘肽(GSH)[(4.83±0.64)mg/L比(8.05±0.89)mg/L]、血红素氧化酶(HO-1)[(0.79±0.08)ng/ml比(1.44±0.18)ng/ml]的表达显著下降,乳酸脱氢酶(LDH)[(353.89±51.37)U/L比(179.07±25.87)U/L]的表达显著上升,Ctrl组则相反,差异有统计学意义(F=25.785、39.313、38.321、139.411,P<0.01)。HE和Von Kossa染色发现,Model组部分肾小管变性坏死,Klotho+Model组草酸钙晶体的沉积与黏附面积小于shKlotho+Model组[(9.50±0.54)%比(2.16±0.75)%]。TUNEL染色结果显示,Model组小鼠肾组织中凋亡情况显著增加(9.67±0.81)。shKlotho+Model组细胞凋亡水平明显高于Klotho+Model组[(13.33±0.52)%比(3.83±0.75)%,F=303.615,P<0.01]。Model组细胞凋亡关键蛋白半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3的mRNA及蛋白表达显著上调[(2.15±0.29)ng/ml],B淋巴细胞瘤2(bcl-2)的mRNA表达显著下调[(0.48±0.05)U/L]。shKlothoObjective To observe the effects of calcium oxalate crystals on oxidative damage,apoptosis and crystal adhesion in mice by regulating Klotho gene.Methods A total of 308-week-old C57BL/6 male mice in the Animal Laboratory of Xinjiang Medical University were selected to establish the kidney stone model of overexpressing/silencing Klotho gene.The models were randomly divided into Ctrl,model,model+vector,shKlotho+model and Klotho+model groups.Western blotting and polymerase chain reaction(PCR)were used to detect the expression of protein-grade messenger RNA(mRNA),and enzyme-linked immunosorbent assay(ELISA)was used to detect the level of oxidative stress protein.The pathological changes of kidney were observed by hematoxylin eosin staining and the adhesion of calcium oxalate crystals was observed by Von Kossa staining.Apoptosis in renal tissue was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).The statistical difference between the two groups was tested by t-test,and the statistical difference between multiple groups was analyzed by single factor analysis of variance.Results The model of calcium oxalate stone in mice was established in vivo and verified by Von Kossa staining.In model group,the expression of CAT,GSH and HO-1 decreased significantly,the expression of lactate dehydrogenase(LDH)[(353.89±51.37)U/L vs.(179.07±25.87)U/L]increased significantly,and that of GSH decreased significantly in Ctrl group.The difference was statistically significant(F=25.785,39.313,38.321,139.411,P<0.01).HE and Von Kossa staining showed that part of renal tubules were degenerative and necrotic in model group,and the deposition and adhesion area of calcium oxalate crystals in Klotho+model group was smaller than that in shKlotho+model group[(9.50±0.54)%vs.(2.16±0.75)%].The results of TUNEL staining showed that apoptosis in renal tissue of model group increased significantly.The level of apoptosis in shKlotho+Model group was significantly higher than that in Klotho+Model group[(13.33±0.52)%vs.(

关 键 词：草酸钙肾结石 氧化应激 KLOTHO基因 

分 类 号：R692.4[医药卫生—泌尿科学] R-332[医药卫生—外科学]