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目的:观察STZ鼠肾组织及高糖状态下正常人类系膜细胞(NHMC)中金属基质蛋白酶9(MMP-9)、金属基质蛋白酶组织抑制物1(TIMP-1)表达状况及磷酸肌酸激酶(PKC)抑制剂对其表达的影响,探讨糖尿病肾病(DN)时PKC抑制剂在细胞外基质降解中的作用。方法:Wistar大鼠随机分为正常对照组、DN模型组、PKC抑制剂组。PKC抑制剂组采用根皮素10mg/(kg.d)混悬液灌胃进行干预。第8周处死大鼠(每组6只)。检测24h尿蛋白定量、血肌酐水平。用免疫组化方法检测肾脏组织MMP-9、TIMP-1的表达。用ELISA方法检测肾脏组织PKC活性。体外实验,将NHMC置37℃,5%CO2培养箱中进行培养。并将NHMC分为N组(对照组):糖浓度5mmol/L,H组(高糖组):糖浓度30mmol/L,P组(高糖加PKC抑制剂):糖浓度30mmol/L加chelery thrine chloride 10-5mmol/L,M组(甘露醇组):甘露醇30mmol/L。于培养24、48、72h后用MTT法测定细胞增值。采用ELISA方法检测四组PKC活性。分别用RT-PCR及Western Blot检测各组MMP-9、TIMP-1mRNA及蛋白表达。结果:DN模型组尿蛋白排泄明显增加(P<0.01),血肌酐上升(P<0.05),PKC活性明显增高,MMP-9和TIMP-1出现表达,MMP-9/TIMP-1比值降低。PKC抑制剂干预后其尿蛋白排泄明显减少,血肌酐水平降低,PKC活性下降,MMP-9、TIMP-1表达上调,其MMP-9/TIMP-1比值增高。体外实验中,高糖能促进NHMC增殖,且NHMC的增殖状况随时间的递增而明显增加。高糖(30mmol/L)能增加系膜细胞PKC的活性,MMP-9、TIMP-1较高表达,MMP-9/TIMP-1比值降低。而PKC抑制剂使PKC的活性降低同时,MMP-9、TIMP-1表达上调,MMP-9/TIMP-1比值增高。结论:高糖可诱导PKC活性,PKC抑制剂能使MMP-9、TIMP-1表达上调,MMP-9/TIMP-1表达比值升高,推测PKC的活性状况可影响DN细胞外基质降解过程。
OBJECTIVE: To observe the expression of matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 1 (TIMP-1) in human kidney tissue and normal human mesangial cells (NHMC) (PKC) inhibitor on the expression of PKC inhibitors in diabetic nephropathy (DN) in the role of extracellular matrix degradation. Methods: Wistar rats were randomly divided into normal control group, DN model group and PKC inhibitor group. PKC inhibitor group with phloretin 10mg / (kg.d) suspension intragastric intervention. Rats were sacrificed at Week 8 (6 in each group). 24h urine protein detection, serum creatinine levels. Immunohistochemistry was used to detect the expression of MMP-9 and TIMP-1 in kidney. The PKC activity in kidney tissue was detected by ELISA. In vitro experiments, NHMC home 37 ℃, 5% CO2 incubator for culture. The NHMCs were divided into N groups (control group): sugar concentration 5mmol / L, H group (high glucose group): glucose concentration 30mmol / L, P group (high glucose plus PKC inhibitor) thrine chloride 10-5mmol / L, M group (mannitol group): mannitol 30mmol / L. After cultured for 24,48,72h, the cell proliferation was measured by MTT method. Four groups of PKC activity were detected by ELISA. The mRNA and protein expressions of MMP-9 and TIMP-1 in each group were detected by RT-PCR and Western Blot respectively. Results: Urinary protein excretion was significantly increased (P <0.01), serum creatinine was increased (P <0.05), PKC activity was significantly increased, and MMP-9 and TIMP-1 were decreased and MMP-9 / TIMP- PKC inhibitor intervention significantly decreased urinary protein excretion, decreased serum creatinine, decreased PKC activity, increased MMP-9 and TIMP-1 expression, and increased MMP-9 / TIMP-1 ratio. In vitro experiments, high glucose can promote the proliferation of NHMC, and the proliferation of NHMC significantly increased with time. High glucose (30mmol / L) increased the activity of PKC in mesangial cells, with higher MMP-9 and TIMP-1 expression and lower MMP-9 / TIMP-1 ratio. While PKC inhibitor decreased the activity of PKC, the expression of MMP-9, TIMP-1 was up-regulated and the ratio of MMP-9 / TIMP-1 was increased. CONCLUSION: High glucose can induce the activity of PKC. PKC inhibitor can increase the expression of MMP-9, TIMP-1 and increase the ratio of MMP-9 / TIMP-1.