高糖环境下Trx1过表达对HBZY-1细胞中MMP9表达的影响  

Effect of Trx1 Overexpression on Expression Level of MMP9 under High Glucose Condition in HBZY-1

作　　者：房绍红[1,2] 刘心平[1] 靳玉宏[1] 贾慧婕[1] 那丽颖[1] 郑海霞[1] 周宏博[1,3] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,哈尔滨150081 [2]哈尔滨医科大学附属二院科研实验中心,哈尔滨150086 [3]哈尔滨医科大学生物技术实验教学中心,哈尔滨150081

出　　处：《中国生物化学与分子生物学报》2009年第5期465-472,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：黑龙江省卫生厅基金(No.2006-273);哈尔滨医科大学青年基金(No.060024)资助项目~~

摘　　要：硫氧还蛋白1(thioredoxin1,Trx1)是细胞内一种重要的巯基-二硫键氧化还原酶,在细胞内氧化还原状态的调控及抵抗氧化应激损伤过程中发挥重要的作用.为了探讨高糖环境下Trx1过表达对肾小球系膜细胞(glomerular mesangial cells)HBZY-1中基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)表达水平的影响,本实验采用脂质体介导的瞬时转染实现Trx1蛋白过表达;采用RT-PCR和明胶酶谱法检测HBZY-1中MMP9 mRNA及酶活性的变化;通过流式细胞仪检测细胞内活性氧的含量.实验结果显示,高糖状态下,细胞中MMP9的mRNA和酶活性分别在12 h、24 h、48 h时表达增加(P<0.05);HBZY-1细胞中转染正义Trx1组,MMP9 mRNA水平及MMP9酶活性,高糖组与正常糖组无明显差异(P>0.05),转染反义Trx1组和未转染组中,高糖组均比正常糖组表达增加,差异有统计学意义(P<0.01);细胞中活性氧含量,高糖作用12 h、24 h、48 h均较正常糖组明显增多(P<0.01),高糖环境下转染正义Trx1质粒较转染反义Trx1质粒,细胞中活性氧含量明显减少,差异有统计学意义(P<0.05).实验提示,高糖环境下,Trx1过表达对MMP9的抑制作用是通过减少细胞内活性氧含量来实现的.本实验为Trx1的抗氧化作用提供新的证据,也为继续探讨Trx1在糖尿病肾病的预防和治疗提供新的思路.Thioredoxinl （Trx1）is an important thiol disulfide oxidoreductase in the cell, which plays a crucial role in the maintenance of cellular redox homeostasis against oxidative stress. To study the effects of Trxl overexpression on matrix metalloproteinase 9 （MMP9） expression, HBZY-1 cells were transfected with either PIRESpuro3-sense-Trxl and PIRESpuro3-antisense-Trxl. The Trxl expression was assayed by RT-PCR and Western blotting. The mRNA level of MMP9 and its enzyme activity were determined by RT-PCR and gelatin zymography. The ROS level was detected by flow cytometry. The results showed that the MMP9 mRNA and enzyme activity were significantly increased at 12, 24 and 48 hours under high glucose condition （P 〈 0.05）.A similar response was showed in antisense-Trxl transfected cells （ P 〈 0.01）, but for sense-Trxl transfected cells, no such increase was observed （ P 〉 0.05）. The ROS level under high glucose was significantly higher than that under normal glucose at 12, 24, and 48 hours （ P 〈 0.01 ）. In sense-Trxl transfected cells, the ROS level was significantly lower than that in antisense-Trxl transfected cells （P 〈 0.05）. Taken together, the inhibition of MMP9 expression and activity by Trxl over-expression was regulated by the ROS level under high glucose conditions in HBZY-1 cells. This suggests that Trxl may be applied for the treatment of diabetic nephropathy.

关 键 词：糖尿病肾病 硫氧还蛋白1 基质金属蛋白酶9 活性氧 

分 类 号：Q51[生物学—生物化学]