【作者】 韩亚莉；

【导师】 傅余芹；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 目的:观察虫草菌丝对治疗前后糖尿病大鼠肾脏组织中基质金属蛋白酶2（MMP-2）及基质金属蛋白酶组织抑制因子2（TIMP-2）表达的变化,分析其与纤维化相关指标Ⅳ型胶原（ColⅣ）等的相关关系,来探讨虫草菌丝对糖尿病大鼠的肾脏保护作用及MMP-2和TIMP-2在糖尿病肾病发生发展中的作用及意义。目前,尚未见相同的研究报道。研究方法:1.动物分组及处理:34只雄性Wistar大鼠行右肾切除术,1周后将存活的33只随机分为甲组（10只）和乙组（23只）。乙组给予一次性腹腔内注射STZ（53mg/kg）诱导糖尿病模型,甲组仅给予等量无菌枸橼酸钠缓冲液。72h后,尾静脉采血测随机血糖≥16.7mmol/L者确定为DM模型,共21只,将其随机分为:糖尿病模型组（DM组,11只）、虫草菌丝干预组（CH组,10只）;另将甲组大鼠设为阴性对照组（NC组）。CH组在糖尿病模型建成后即刻给予虫草菌丝悬浊液(4.95g·kg^-1·d^-1)灌胃,NC组和DM组仅给予等量生理盐水(15ml·kg^-1·d^-1)灌胃。实验期间所有大鼠自由饮水进食,各组大鼠不使用胰岛素及其他降糖药物。所有大鼠每周测一次体重及血糖。2.干预第8周末留取大鼠24h尿液并检测其尿肌酐（Ucr）、24h尿蛋白定量（Upro）等,进行组间差异比较并分析各指标之间的相关性;干预8周后一次性处死大鼠,腹主动脉留取动脉血约4ml,测血糖（Glu）、血肌酐（Scr）、尿素氮（BUN）等,并计算内生肌酐清除率（Ccr）。3.肾脏标本处理:取部分肾组织,剥离被膜,称重,10%福尔马林固定、石蜡包埋、切片。行HE染色及PAS染色镜下观察大鼠肾脏常规病理变化。SABC免疫组化方法检测各组肾脏MMP-2、TIMP-2、Col-Ⅳ蛋白的表达情况。每张切片随机取10个高倍镜视野（×400）,采用Leica Qwin V3图像分析软件,分析各指标阳性着色的灰度,比较三组之间灰度值。其余部分置于液氮保存,以行RT-PCR检测肾组织中MMP-2、TIMP-2、ColⅣ的mRNA水平。收集并整理实验数据,应用单因素方差分析、直线相关分析等进行统计分析,p＜0.05为有显著性差异。结果:1.试验前各组大鼠体重、血糖基础值无显著性差异（p＞0.05）,条件齐同。2.8周后,DM组、CH组大鼠的血糖、Ccr、Upro水平均显著高于NC组（p均＜0.01）。CH组Ccr、Upro水平较DM组显著降低（p均＜0.01）,两组血糖水平无显著差异。3.免疫组化及RT-PCR结果均显示DM组、CH组大鼠肾组织中TIMP-2、ColⅣ水平明显高于NC组（p＜0.01或p＜0.05）,MMP-2水平及MMP-2/TIMP-2比值低于NC组（p＜0.01或p＜0.05）。4.免疫组化及RT-PCR结果显示CH组大鼠肾组织中TIMP-2、ColⅣ水平较DM组降低（p＜0.01或p＜0.05）,MMP-2水平及MMP-2/TIMP-2比值则升高（p均＜0.01）。5.相关分析:将各组大鼠肾脏MMP-2、TIMP-2表达水平及MMP-2/TIMP比值与相应的纤维化指标进行相关分析,结果显示MMP-2和MMP-2/TIMP比值与大鼠ColⅣ表达、24小时尿蛋白定量均存在负相关关系,而TIMP-2与之正相关（p＜0.01或p＜0.05）。相关系数分别为r=-0.394、r=-0.547、r=-0.655、r=-0.747、r=0.576、r=0.571。结论:1.DM大鼠肾脏出现DN病理改变,24小时尿蛋白排泄量增多,肾组织中TIMP-2表达显著升高,MMD-2表达及MMP-2/TIMP比值显著降低,且MMP-2表达及MMP-2/TIMP比值与24小时尿蛋白排泄量呈显著负相关,TIMP-2则与之呈正相关。提示MMP-2和TIMP-2的表达失衡与糖尿病肾病的发生及蛋白尿的形成密切相关。2.DM大鼠肾组织中ColⅣ表达明显升高,并分别与MMP-2及TIMP-2水平呈负相关及正相关,提示DM时,MMP-2表达的减少和TIMP-2表达的增加共同促进了Col-Ⅳ的过度表达,从而促使肾脏ECM积聚,最终导致肾组织纤维化。3.虫草菌丝治疗8周后,DM大鼠肾脏功能明显改善,24小时尿蛋白排泄量减少,肾组织中ColⅣ、TIMP-2水平显著下降,MMP-2则显著上升。提示虫草菌丝可通过纠正MMP-2及TIMP-2的表达异常,促进ColⅣ的降解,抑制肾脏ECM的蓄积,这可能有助于延缓肾小球硬化进程,最终达到减少蛋白尿、保护肾功能的作用。更多还原

【Abstract】 Objective:The objective of this study is to observe the changes of MMP-2 and TIMP-2 expression in the kidneys of diabetic rats with the therapy of cordycepic hypha,to analyze the relationship between MMT-2、TIMP-2 and the fibrous degeneration correlate factor ColⅣ,to study the effects of MMP-2 and TIMP-2 on the progression of diabetic nephropathy and the reno-protective mechanism of cordycepic hypha on Streptozotocin-induced diabetic Wistar rats.Methods:After the rats model with diabetes were induced by right-nephrectomy and peritoneal injection of streptozotocin (STZ,53mg·Kg^-1),the 21 Wistar rats which with fasting blood glucose（FBG） equal to or more than 16.7mmol/L served as diabetic rats were randomly divided into following two groups:diabetes control（n=11,DM group）, diabetic rats treated with cordycepic hypha（n=10,CH group）.At the same time,the rats with right-nephrectomy and peritoneal injection of the equal citric acid buffer were as normal control group（n=10,NC group）.Both DM group and NC group were only given 0.9%Sodium Chloride,while the CH group were treated with cordycepic hypha(4.95g·kg′·d^-1,by gavage).All rats of diabetic groups were not treated with insulin or other anti-diabetes drugs in experimental procedures.After raising rats of the three groups for 8 weeks, Plasma glucose,serum creatinine（Scr）,blood urea nitrogen（BUN）,and 24-hour urinary protein（Upro）were observed.And creatinine clearance（Ccr）was calculated.Then the rats in the three groups were killed.The expressions of MMP-2,TIMP-2 and Col-Ⅳin kidney were identified by immunohistochemistry and the mRNA expression levels of MMP-2,TIMP-2 and ColⅣin kidney were detected by revese transcription polymerase chain reaction（RT-PCR）.The experimental data was statistically analyzed by one-way ANOVA and correlation analysis.Results:1.There were no significant differences among all groups before this experiment（p＞0.05）.2.After 8 weeks,the levels of plasma glucose,Ccr and Upro in DM and CH groups were increased more significantly than NC group（p＜0.01）.The levels of Ccr and Upro in CH group are lower significantly than DM group （p＜0.01）.And the level of plasma glucose in DM and CH groups is not different significantly.3.The results of immunohistochemistry and RT-PCR showed that the expressions of TIMP-2 and ColⅣin DM and CH groups were much higher than NC group（p＜0.01 or p＜0.05）.But the expression of MMP-2 and the relative value of MMP-2 with TIMP-2 were lower than NC group（p＜0.01 or p＜0.05）.4.The results of immunohistochemistry and RT-PCR showed that the expressions of TIMP-2 and ColⅣin CH group were much lower than DM group（p＜0.01 or p＜0.05）.The expression of MMP-2 and the relative value of MMP-2 with TIMP-2 were higher（p＜0.01）5.The correlation analysis showed that there was significant positive correlation of expressions between TIMP-2 and ColⅣor between TIMP-2 and 24-hour urinary protein.There were negative correlation of expressions between MMP-2 or the relative value of MMP-2 with TIMP-2 and ColⅣ.And the same between MMP-2 or the relative value of MMP-2 with TIMP-2 and 24-hour urinary protein.Conclusion:1.There were pathological changes in nephridial tissue of diabetic rat and the 24-hour urinary protein increased significantly.The expression of TIMP-2 was up-regulated in nephridial tissue of diabetic rat while that of MMP-2 and the ratio of MMP-2 with TIMP-2 were down-regulation.The expression of MMP-2 and the ratio of MMP-2 with TIMP-2 has left relationship with 24-hour urinary protein while the expression of TIMP-2 has right relationship with 24-hour urinary protein.Abnor expression of MMP-2 and TIMP-2 has close relationship with diabetic nephropathy and urinary protein.2.Expression of ColⅣhas been demonstrated to increase in nephridial tissue of diabetic rat and has left relationship with MMP-2 while has right relationship with TIMP-2.The over expression of TIMP-2 and low expression of MMP-2 promote over expression of ColⅣ,which promotes to accumulation of extracellular matrix and fibrosis in kidney3.After 8 weeks,the use of cordycepic hypha improve the function of kidney in diabetic rats,24-hour urinary protein decreased significantly. The expression of ColⅣand TIMP-2 were down-regulation,while that of MMP-2 was up-regulated.Cordycepic hypha promote the degradation of ColⅣ,restrain accumulation of extracellular matrix,delay the progression of diabetic nephropathy by correcting the abnor expression of MMP-2及TIMP-2.更多还原