高糖对人类肾小球系膜细胞PKC及MMPs/TIMPs的影响  

Effect of high glucose on PKC and MMPs/TIMPs in human mesangial cells

作　　者：杨敬华[1] 周巧玲[1] 王衍慧[1] 刘抗寒[1] 张军[1] 

机构地区：[1]中南大学湘雅医院肾内科,长沙410008

出　　处：《中南大学学报（医学版）》2009年第5期425-431,共7页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(30370663)~~

摘　　要：目的:研究体外高糖环境下人类肾小球系膜细胞(NHMC)中PKC激活或使用PKC抑制剂干预后MMP2,9/TIMP1,2的表达,探讨PKC与MMPs/TIMPs信号转导在糖尿病肾病发生发展中的作用。方法:将NHMC分4组:N组(对照组,5mmol/L葡萄糖);H组(高糖组,30mmol/L葡萄糖);P组(抑制剂组,30mmol/L葡萄糖+10-5mol/L白屈菜红碱);M组(甘露醇组,5mmol/L葡萄糖+25mmol/L甘露醇)。分别于上述4种不同成分培养基中进行细胞培养,第24,48,72h用MTT法测定NHMC增殖;并于培养后第24,48h收集细胞,抽提mRNA及蛋白质,用ELISA方法测定胞膜、胞核蛋白质PKC活性。用RT-PCR和Western印迹方法检测MMP2,9,TIMP1,2mRNA和蛋白质的表达。结果:高糖组细胞膜和胞核部分的PKC活性较对照组明显升高(P<0.01),MMP2,9,TIMP1,2mRNA及蛋白质的表达较对照组明显上升(P<0.01);而MMP-9/TIMP-1,MMP-2/TIMP-2比值较对照组明显下降(P<0.05)。高糖加PKC抑制剂白屈菜红碱后,MMP2,9,TIMP1mRNA及蛋白质表达较对照组明显升高(P<0.01),但MMP9/TIMP1,MMP2/TIMP2比值较高糖组明显升高(分别P<0.05,P<0.01)。PKC活性与MMP-2/TIMP-2及MMP-9/TIMP-1的蛋白质比值均呈负相关(分别r=-0.651,r=-0.702,均P<0.05)。结论:高糖可刺激人类肾小球系膜细胞PKC活化,在DN中PKC活化与MMP2,9/TIMP1,2的表达水平有密切关系。Objective To explore the relationship between protien kinase C （PKC） and matrix metalloproteinase （ MMPs ）/tissue inhibitor of metalloproteinase （ TIMPs ） in human mesangial cells under the high glucose medium, and to analyze the effect of PKC and MMPs/TIMPs in diabetes nephropathy （ DN ）. Methods Normal human mesangial cells （ NHMC ） were divided into 4 groups ： a control group（N, 5 mmol/L glucose） , a high glucose group （H, 30 mmol/L glucose） , a PKC inhibition group （P, 30 mmol/L glucose plus 10^-5mol/L chelerythrine chloride） , and an mannitol group （M, 5 mmol/L glucose plus 25 mmol/L mannitol）. Cell proliferation was measured by MTT at 24,48 or 72 hours. The activity of PKC was measured by ELISA and the mRNA and protein expressions of MMP2, 9 and TIMP1 , 2 were examined by RT-PCR and Western blot. Results High glucose increased the activity of PKC as well as the expressions of mRNA and protein of MMP2, 9 and TIMP1 , 2. The ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1 was significantly decreased in the high glucose group compared with that of the control group （ P 〈 0.05 ）. The mRNA and protein expressions of MMP2, 9 and TIMP 1 were significantly increased in the PKC inhibition group compared with the control group （ P 〈 0.01 ）. The ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1 increased in the inhibition group compared with that of the high glucose group （ P 〈 0.05 or P 〈 0.01 ）. The activity of PKC was negatively correlated with the protein ratio of MMP-2/TIMP-2 and MMP-9/ TIMP-1 （ r = - 0. 651 , r = - 0. 702, both P 〈 0.05 ）. Conclusion High glucose can activate PKC in mesangial cells. The activity of PKC influences the expression of MMPs/TIMPs in the progressing of DN.

关 键 词：蛋白激酶C 基质金属蛋白酶 金属蛋白酶组织抑制物 糖尿病肾病 系膜细胞 

分 类 号：R692[医药卫生—泌尿科学]