MMP-9/TIMP-1在HHcy致ApoE^(-/-)小鼠肾脏损伤中的作用机制  

Effects and Mechanisms of MMP-9 / TIMP-1 in HHcy Induced Apo E^(- /-) Mice Kidney Damage

作　　者：孔繁琪[1] 马胜超[2] 张辉[3] 和杨杨 赵丽[1] 孙炜炜[3] 曹成建[3] 王艳华[3] 田珏[1] 杨晓玲[1] 姜怡邓[1] 

机构地区：[1]宁夏医科大学基础医学院,宁夏银川市750004 [2]四川大学基础医学与法医学院,四川省成都市610041 [3]宁夏医科大学检验学院,宁夏银川市750004

出　　处：《中国动脉硬化杂志》2015年第6期541-548,共8页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金资助(81260105;81160044;81260063);宁夏自然科学基金重点项目(NZ13054);宁夏回族自治区科技厅科技攻关项目(20100820;[2012]17号)

摘　　要：目的探讨高同型半胱氨酸血症(HHcy)导致Apo E-/-小鼠肾脏损伤的作用机制,为HHcy所致肾脏疾病的预防和治疗提供理论和实验依据,为肾病患者的疾病监测提供新的指标。方法将5周龄雄性纯合子Apo E-/-小鼠随机分组:Apo E-/-对照组饲以普通饮食,Apo E-/-高蛋氨酸组饲以高蛋氨酸饮食,Apo E-/-干预组饲以加叶酸和维生素B12的高蛋氨酸饮食,5周龄SPF级C57BL/6J雄鼠饲以普通饮食作为正常对照组,喂养14周后,生化分析仪测定血清同型半胱氨酸(Hcy)、肌酐及尿素浓度,透射电镜和PAS染色检测小鼠肾脏损伤情况,荧光定量PCR检测肾脏基质金属蛋白酶9(MMP-9)和组织型基质金属蛋白酶抑制剂1(TIMP-1)m RNA表达水平,并以免疫组织化学染色法分析肾脏MMP-9蛋白表达,ELISA检测TIMP-1蛋白表达。结果与正常对照组相比,Apo E-/-高蛋氨酸组血清Hcy、肌酐及尿素浓度分别升高了3.66倍、1.1倍和1.6倍(P<0.01);透射电镜和PAS染色结果显示,Apo E-/-高蛋氨酸组较正常对照组肾脏损伤明显,荧光定量PCR结果显示MMP-9及TIMP-1 m RNA表达则分别升高了5.25倍和1.38倍(P<0.01);免疫组织化学结果显示Apo E-/-高蛋氨酸组MMP-9表达较正常对照组明显升高(P<0.01);ELISA结果显示Apo E-/-高蛋氨酸组TIMP-1蛋白表达比正常对照组明显升高(P<0.01)。与Apo E-/-高蛋氨酸组相比,Apo E-/-干预组血清Hcy浓度下降,且肌酐和尿素浓度均降低(P<0.01);透射电镜和PAS染色结果显示,Apo E-/-干预组肾脏损伤较Apo E-/-高蛋氨酸组减轻。血清Hcy浓度与其肌酐及尿素浓度呈正相关(r2=0.4344,P<0.0001;r2=0.4478,P<0.0001),与MMP-9/TIMP-1比值也呈正相关(r2=0.39309,P<0.001),且小鼠肾脏MMP-9/TIMP-1比值与肌酐及尿素浓度也成正相关(r2=0.1464,P<0.05;r2=0.3027,P<0.01)。结论 HHcy可能是经MMP-9/TIMP-1比例上调致细胞外基质降解,继而导致肾脏损伤。Aim To explore the effect and mechanisms of matrix metalloproteinase-9( MMP-9) /tissue inhibitor of metalloproteinase-1( TIMP-1) in Apo E- /-mice kidney damage induced by hyperhomocysteinemia( HHcy),provide theoretical and experimental basis for the prevention and treatment of kidney disease,and supply a new indicator for kidney disease surveillance. Methods 5 week old male Apo E- /-mice were divided into three groups: Apo E- /-control group,Apo E- /-high methionine diet group and Apo E- /-intervention group,and 5 week old SPF male C57 BL /6J mice were chosen as normal control group. After fed for 14 weeks,the serum concentration of homocysteine( Hcy),creatinine( Cr) and urea were detected by biochemical analyzer,transmission electron microscopy and PAS staining were used to show the damage of kidney. MMP-9 and TIMP-1 m RNA expression levels of kidney were detected by real-time PCR,MMP-9 protein expression of kidney was assayed by immunohistochemical staining and TIMP-1 protein expression was detected by ELISA. Results Compared with normal control group,the serum concentration of Hcy,Cr and urea of Apo E- /-high methionine diet group were significantly increased by 3. 66-,1. 1-and 1. 6-folds( P < 0. 01). Transmission electron microscopy and PAS staining showed that kidney damage of Apo E- /-high methionine diet group were more serious than normal control group. Real-time PCR results showed that m RNA expressions of MMP-9 and TIMP-1 were evidently increased by 5. 25-and 1. 38-folds respectively( P < 0. 01). The results of immunohistochemistry assay showed that protein expression of MMP-9 in Apo E- /-high methionine diet group was significantly higher than that in the normal control group( P < 0. 01),ELISA assay showed TIMP-1 protein expression in the Apo E- /-high methionine diet group was significantly higher than that in the normal control group( P < 0. 01). Compared with Apo E- /-high methionine diet group,Hcy levels decreased in Apo E- /-intervention group( P < 0. 01),and Cr and urea levels were significantly

关 键 词：高同型半胱氨酸血症 基质金属蛋白酶9 组织型基质金属蛋白酶抑制剂1 肾脏损伤 

分 类 号：R692[医药卫生—泌尿科学]