摘要
目的探讨基质金属蛋白酶-9(MMP-9)DNA甲基化变化在载脂蛋白E基因敲除(ApoE-/-)C57BL/6J小鼠肾脏中的作用及高蛋氨酸饮食的影响。方法将ApoE-/-小鼠随机分成模型组、高蛋氨酸组和叶酸及维生素B12治疗组,并设正常C57BL/6J小鼠为正常对照组,喂养14周后采血,分离肾脏。全自动生化分析仪检测血清同型半胱氨酸(Hcy)、尿素(Urea)和肌酐(Cr)水平;免疫组织化学法分析肾脏MMP-9蛋白表达;荧光定量RT-PCR检测MMP-9 mRNA表达;巢式甲基化特异性PCR(nMS-PCR)和降落式PCR检测MMP-9 DNA甲基化变化程度。结果模型组血清Hcy、Urea、Cr浓度均明显升高,MMP-9甲基化程度降低,mRNA和蛋白表达增强(均P<0.05),与正常对照组比较,差异有统计学意义(P<0.05);高蛋氨酸组各项指标变化较模型组更为明显(均P<0.05);而叶酸及维生素B12治疗组MMP-9 mRNA和蛋白表达减少,甲基化程度升高(P<0.05)。结论 MMP-9 DNA甲基化变化可能是ApoE-/-小鼠高蛋氨酸饮食致肾功能损伤的重要机制之一。
【Objective】 To study the effects of matrix metalloproteinase-9 （MMP-9） DNA methylation changes and high methionine diet on the kidneys of apolipoprotein E -deficient （ApoE -/） C57BL/6J mice. 【Methods】 ApoE -/mice were randomly divided into model control group, high-methionine diet group, folic acid and vitamin B12 treatment group, and set the normal C57BL/6J mice as normal control group. The blood and kidneys were separated after the mice were fed for 14 weeks. The serum homocysteine （Hcy） and renal function markers such as Urea, Cr were detected by automatic biochemical analyzer. Immunohistochemistry was performed to investigate the expression of MMP-9 in kidneys. Fluorescence quantitative RT-PCR was applied to detect the expression of MMP-9 mRNA. MMP-9 DNA methylation changes were detected by nested methylation-specific PCR （nMS-PCR） combined with touch-down PCR. 【Results】 The concentrations of Hcy, Urea and Cr in model control group and high methionine diet group increased significantly, MMP -9 methylation levels decreased, whereas MMP -9 mRNA and protein expression increased. The differences were significant compared with normal control group （P 0.05）. But the expression of MMP-9 decreased in the folate and vitamin B12 treatment group. 【Conclusion】 MMP-9 DNA methylation changes may be an important mechanism of high methionine diet-induced renal function impairment in ApoE -/mice.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2012年第32期7-12,共6页
China Journal of Modern Medicine
基金
国家自然科学基金(No:30960124)
2010年教育部新世纪优秀人才支持计划(No:NCET-10-0916)
国家自然科学基金(No:81160044)
