【作者】 牛洪琳；

【导师】 李英；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:观察高糖培养下肾小球系膜细胞对ILK的表达以及Benazepril对ILK的表达的干预作用。目前糖尿病肾病(diabetic nephropathy, DN)已经成为在美国和欧洲引起终末期肾病(ESRD)最常见的原因,是糖尿病最严重的并发症之一,但是迄今为止DN的发病和早期预防措施并不明确。DN主要的病理改变是:肾小球基底膜增厚、肾小球系膜细胞(Glomerular mesangial cell, GMC)增殖与系膜基质的积聚,最终导致肾小球硬化。GMC是肾小球中最活跃的细胞,对于维持肾脏正常组织结构和生理功能发挥着重要作用,因此积极探讨GMC在生理、病理下的变化机制对于临床DN的防治和早期发现具有重大而深远的的意义。整合素连接激酶(Intergrin-linked Kinase, ILK)是一种存在于细胞胞浆中的丝氨酸/苏氨酸蛋白激酶,通过多种信号传导通路,包括整合素、生长因子及Wnt信号传导通路,来参与调节细胞的黏附、维持细胞存活、抑制细胞凋亡、控制细胞形态和基因的表达。近年来研究发现正常的肾脏组织中ILK在GMC表达较弱或几乎不表达,但ILK表达的增高常伴随GMC细胞增殖及细胞外基质(extracellular matrix, ECM)分泌。α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA )是血管平滑肌特有的细胞骨架蛋白,即血管平滑肌的标志,正常成熟的GMC是不表达的。但在病理情况下GMC发生表型转化可表达α-SMA ,其表达情况和GMC增殖改变,细胞外基质的积聚,肾功能持续下降密切联系。而DN早期病理改变主要就是GMC增殖肥大和ECM积聚,因此探讨ILK、α-SMA与GMC增殖及ECM分泌的关系,可以更加深入阐明DN的发病机制。Benazepril(贝那普利)是新一代血管紧张素转换酶抑制剂(ACEI),它通过抑制血管紧张素转换酶,使循环和组织中血管紧张素Ⅱ(AngⅡ)减少,并同时减少AngⅡ的刺激,阻止其与受体AT1结合。有实验证明高糖刺激下的GMC可自分泌AngⅡ,后者和GMC膜上AT1结合,进而导致了GMC内PKC等通路的活化,引起一些其他细胞生长因子基因和蛋白的异常表达,致使GMC增殖和分化。本实验利用贝那普利进行干预,观察ILK、α-SMA在高糖培养GMC的表达及药物的保护作用,为临床DN防治提供实验性理论依据。方法:肾小球系膜细胞,常规培养消化传代,将其随机分为四组:即正常糖对照组(D-葡萄糖5.5mmol/L,NG组)、甘露醇组(甘露醇20mmol/L,MG组)、高糖组(D-葡萄糖30 mmol/L,HG组)、高糖+Benazepril干预组(D-葡萄糖30mmol/L+贝那普利10μmol/L,HG+Benazepril)分别培养。四组于实验开始后3、6、12、24、48、72h收集细胞,用逆转录-聚合酶链式反应(RT-PCR)法检测GMC中ILK、α-SMA mRNA的表达,用western blot法检测各实验组GMC中ILK、α-SMA蛋白的表达。结果:1.细胞培养结果:倒置相差显微镜下可见MC呈梭形、不规则星形或三角形的、树枝形,当细胞密集时可重叠生长,胞浆多突起,有大量微丝样结构,核居中央,多呈圆形或椭圆形; 2.高糖环境中GMC ILK mRNA和蛋白水平3h时较正常糖组明显升高(P<0.05),且随时间延长有增高趋势(P<0.05),表达量在48h达到最高(P<0.05),但在72h开始下降(P<0.05)。药物干预后上述指标较高糖组表达水平下降,但未能恢复至对正常照组水平(P<0.01);同时间点甘露醇组ILK mRNA和蛋白水平与正常糖组无明显差别(P>0.05)。3.高糖环境中GMCα-SMA mRNA和蛋白水平较正常糖组升高(P<0.01),随时间延长逐渐增高(P<0.05),表达量在72h达到最高(P<0.01),药物干预后上述指标较高糖组表达水平下降(P<0.01),但未能恢复至对照组水平(P<0.05);同时间点甘露醇组α-SMA mRNA和蛋白水平与较正常糖组升高(P<0.05),不能排除高糖组渗透压依赖性引起的α-SMA升高。结论:上述结果提示1.高糖能以时间依赖的方式诱导ILK在GMC高表达。2.高糖能以时间依赖的方式使α-SMA在GMC表达升高,介导GMC发生表型转化;3.贝那普利可抑制上述作用。4.甘露醇作用下α-SMA异常表达,提示高糖环境下GMC发生明显的表型转化可能同时存在渗透压依赖性。更多还原

【Abstract】 Objective: To investigate the expression of ILK andα-SMA in the glomerular mesangial cells induced by high-glucose and observe the effect of Benazepril on the the expression of ILK andα-SMA. Recently diabetic nephropathy (DN) has become the most common cause of end-stage renal disease (ESRD), which is one of the most serious complications of diabetes mellitus in the United States and Europe. But, up to now, the mechanisms of the onset of DN and its preventive measures in early stage are not clear. The thickening of glomerular basement membrane(GBM), the proliferation of the glomerular mesangial cells (GMC) and the accumulation of the mesangial matrix are the main pathological changes in DN which led to glomerulosclerosis. Glomerular mesangial cells are the most active cells in the glomerular, which play an important role for the normal histio-structure and physiologic function in the kidney. So it is very important to explore and clarify the mechanisms of physiological and pathological changes in Glomerular mesangial cells for the clinical prevention and finding DN in early time.Intergrin-linked Kinase is a kind of serine / threonine protein kinase in the cytoplasm, which plays a role in regulating cell adhesion, maintaining cell survival, morphology and regulating gene expression through many kinds of signal transduction pathways including integrin, growth factor and Wnt. Recently, some researches show that there is nothing or little ILK in the GMC of normal kidney tissues. But the increasing expression of ILK induce the cell proliferation and the secretion of extracellular matrix (ECM). A-smooth muscle actin (α-SMA) is a special cytoskeleton protein of vascular smooth muscle. It is the symbol of the vascular smooth muscle, which has no expression in normal maturation GMC. Butα-SMA can be expressed in the pathological GMC because of its transformed phenotype. The increasing expression ofα-SMA in GMC are related to the proliferation of GMC and the accumulation of extracellular matrix,which are also lead to the continuous decline of renal function. Because the proliferation of GMC and the accumulation of extracellular matrix are the early pathological changes in DN, the relations among ILK,α-SMA, the GMC proliferation and the secretion of ECM will help us to clarify the mechanism of the the pathogenesis of DN more deeply.Benazepril is one of the new generation of angiotensin -converting enzyme inhibitors(ACEIs), which can decrease angiotensinⅡ(AngⅡ) in the circulation and organisms by inhibiting the angiotensin-converting enzyme. It can also weaken the stimulus of AngⅡand prevent AngⅡfrom combineing with its receptors. Some experiments have proved that the GMC can autocrine AngⅡby the stimulus of high glucose. And then it will combine with AT1 receptors in GMC, which led to activate the signal transduction pathyways PKC. So some other cell growth factor genes and proteins will be expressed, which lead to the proliferation and differentiation of GMC. In this experiment we use the effects of benazepril on ILK andα-SMA to observe their expressions in the cultured GMC exposed by high glucose and the protective effects of the drug, which may provide us experimental theory in clinical DN.Methods: The mesangial cells of SD rat (HBZY-1) were cultured conventionally and randomly divided into four groups: normal glucose( D-glucose 5.5mmol/L, group NG ) ,Mannitol-treated normal glucose group (D-glucose 5.5mmol/L+ mannitol 20mmol/L,group MG), high glucose (D-glucose 30mmol/L,group HG), Benazepril-treated high glucose group(D-glucose 30mmol/L+ Benazepril 10μmol/L, group HG+Benazepril). NG, MG, HG ,HG+Benazepril were all collected on 3hour, 6hour, 12hour, 24 hour, 48 hour and 72 hour respectively. The mRNA of ILK andα-SMA expressions of the four groups were detected by reverse transcriptase- polymerase chain reaction (RT-PCR) . The protein of ILK andα-SMA were detected by western blot.Results:1.The shapes of the cultured cells showed as fusiform, irregular stellar, trianglar or branch-shaped by phase contrast microscopy. When the cells growed overlap-intensively, their endochylemas had some ecphymas and there are a lot of microfilaments in it. Their nucleus were in the center, which were round or oval. 2.The expressions of ILK mRNA and protein in HG group were significantly increased on 3h compared to those in NG group (P <0.05). The increasing expression of ILK in HG group were time-dependent and the expression reached the peak on 48h (P <0.05), while decreased on 72h(P <0.05). The expression of ILK in HG+Benazepril group were lower than that in HG group (P <0.01), but failed to drop to the same level as that in NG(P<0.01). There were no significant differences of ILK expressions between MG group and NG group at the same time(P> 0.05). 3.The expressions ofα-SMA mRNA and protein in HG were higher than that in NG at anytime(P <0.05). The increasing expression ofα-SMA was also time-dependent and it reached the peak on 72h(P<0.05). The expression ofα-SMA in HG+Benazepril was lower than that in HG(P<0.01), while failed to drop to the same level as that in NG(P <0.01). The expressions ofα-SMA mRNA and protein in MG were higher than that in NG (P <0.05),so we couldn’t eliminate the factor of high osmotic pressure which may cause the increasing ofα-SMA.Conclusion: 1.High glucose induced ILK expression in the GMC in a time-dependent manner. 2.High glucose could also inducedα-SMA expression in the GMC in a time-dependent manner and mediated the phenotypic transformation of GMC. 3.Benazepril could inhibit the processes that were mentioned above in GMC. 4.The abnormal expressions ofα-SMA suggested that there was phenotypic transformation of GMC in glucose. The increasing ofα-SMA may be dependent on high osmotic pressure.更多还原