高糖对人类肾小球系膜细胞PKC及MMPs/TIMPs的影响

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，5 mmol/L葡萄糖）；H组（高糖组，30 mmol/L葡萄糖）；P组（抑制剂组，30 mmol/L葡萄糖+10-5mol/L白屈菜红碱）；M组(甘露醇组，5 mmol/L葡萄糖+25 mmol/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24,48,72 h用MTT法测定NHMC增殖；并于培养后第24,48 h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， TIMP1，2 mRNA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， TIMP1，2 mRNA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, TIMP1 mRNA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。

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

ObjectiveTo 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).MethodsNormal 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.ResultsHigh 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 TIMP1 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).ConclusionHigh glucose can activate PKC in mesangial cells. The activity of PKC influences the expression of MMPs/TIMPs in the progressing of DN.