HGF对大鼠微小病变肾病模型中ILK表达的影响

发布时间：2018-05-12 13:10

本文选题：阿霉素 + 蛋白尿　；参考：《石河子大学》2010年硕士论文

【摘要】：目的：肝细胞生长因子(hepatocyte growth factor, HGF)是一种多效性多肽生长因子,它能刺激多种上皮和内皮细胞进行有丝分裂、运动,在肾小管形态发生、肾脏胚胎的形成、肾组织的损伤修复和再生中起着非常重要的营养作用。而整合素连接激酶(integrin-linked kinase, ILK)是一种新发现的粘附蛋白,与整合素p的胞浆区域相连,是细胞内丝氨酸／苏氨酸蛋白激酶。它在调节细胞周期、基质积聚、肿瘤发生等中起重要作用,也参与了肾脏疾病的发生发展过程。新近的研究表明ILK在足细胞中表达水平较高,对维持肾小球基底膜(glomerular basement membrane, GBM)的完整性、保持肾小球的滤过功能和防止蛋白质的漏出有重要意义。本研究主要通过组织病理学观察、免疫组化、免疫荧光方法,观察在微小病变肾病(Minimal Change Disease, MCD)大鼠体内不同时间点的ILK蛋白水平变化,探讨ILK在足细胞损伤中的意义及其可能机制,同时进一步探讨了HGF干预时,微小病变肾病模型中ILK的表达变化。方法：1.微小病变肾病(MCD)动物模型的建立：选取清洁级雄性SD大鼠72只,随机分为三组：对照组、模型组、肝细胞生长因子干预组,模型组和肝细胞生长因子干预组的大鼠经尾静脉一次性注射阿霉素注射液(浓度2mg/ml, 7.5mg/kg剂量),复制MCD模型,肝细胞生长因子干预组从注射阿霉素后的第三周开始给予腹腔注射肝细胞生长因子(30ug·kg-1·d-1),直至试验结束；正常对照组尾静脉一次性注射等体积的生理盐水。各组大鼠分别于建模后第7、14、21、28天宰杀,每组各6只,留取肾脏标本,处死前于大鼠内眦静脉采血约3m1,离心分离血清测血浆白蛋白、尿素氮、肌酐、胆固醇。大鼠处死前1天代谢笼收集24小时尿液,测24小时尿蛋白定量。2.进行HE染色,光镜、电镜下观察肾脏病理改变情况。3.用免疫荧光技术检测ILK蛋白在肾小球的定位分布,分析各组间ILK在不同时间点的表达情况,及其与肾小球足细胞损伤程度间的关系。4.采用电泳法测定血浆白蛋白,采用磺基水杨酸法测24小时尿蛋白定量,分析各组间血浆白蛋白、24小时尿蛋白的变化,以及与ILK的关系。结果：1.采用尾静脉一次性注射阿霉素注射液复制MCD模型成功。2.模型组大鼠肾小球中ILK蛋白的表达7天时减少,但与对照组相比,差异无统计学意义(p0.05)；14天明显增加,21天达到最大值,与对照组相比差异有统计学意义(p0.05)。与模型组相比,HGF干预组大鼠肾小球中ILK蛋白的表达21天减少,28天明显减少,差异有统计学意义(p0.05)；3.与对照组相比,模型组大鼠24h尿蛋白的排泄量7天增加,21天达高峰,差异有统计学意义(p0.01)；与模型组相比,HGF干预组24h尿蛋白的排泄量21天及28天均明显降低,差异有统计学意义(p0.05)；4.与对照组相比,模型组血清白蛋白7天开始下降,21天降至最低点,差异有统计学意义(p0.01)；与模型组相比,HGF干预组血清白蛋白在21天及28天均明显升高,差异有统计学意义(p0.05)。5.肾组织形态学与病理学改变：肉眼观对照组、干预组肾脏体积大小正常,颜色深红,切面皮髓质分界清楚。模型组肾脏体积稍大,颜色深红,切面皮髓质分界也清楚。光镜下可见模型组少数肾小球体积稍增大,肾小球毛细血管可见红细胞渗出,近端肾小管可见脂肪变性、脱落,肾小管管腔内蛋白管型形成,随着时间延长,病变程度加重,21天时最明显。与模型组比,HGF干预组肾小球体积稍减小,肾小球毛细血管红细胞渗出减少,近端肾小管上皮细胞脂肪变性减轻,肾小管蛋白管型减少。电镜下：模型组可见弥漫性肾小球足细胞的足突大部分融合或消失,部分足细胞的足突断裂；HGF干预组足突融合较模型组有所减轻。6.相关分析结果显示,模型组ILK表达的变化与24h尿蛋白定量的变化呈正相关(r=0.743,P0.01)。结论：1.ILK表达增加及分布异常,与足细胞病变密切相关,可能是判断肾小球足细胞损伤及导致蛋白尿原因的一个早期重要指标。2.ILK与蛋白尿呈正相关关系,其在肾组织中表达的高低对肾脏疾预后的判断具有一定的意义。3.HGF能抑制微小病变肾病大鼠肾小球ILK蛋白的表达,对MCD在肾脏结构及功能上既有保护作用又有治疗作用。
[Abstract]:Objective: hepatocyte growth factor (HGF) is a pleiotropic polypeptide growth factor, which stimulates mitosis and movement of various epithelial and endothelial cells. It plays a very important role in renal tubule morphogenesis, kidney embryo formation, renal tissue injury repair and regeneration. Integrin-linked kinase (ILK) is a newly discovered adhesion protein, which is linked to the cytoplasmic region of integrin P and is an intracellular serine / threonine protein kinase. It plays an important role in regulating cell cycle, matrix accumulation, tumor occurrence and so on. It also participates in the development of renal disease. Recent studies have shown that ILK is in the foot. High levels of expression in cells are important for maintaining the integrity of the glomerular basement membrane (glomerular basement membrane, GBM), maintaining the glomerular filtration function and preventing the leakage of protein. This study was mainly observed by histopathological observation, immunohistochemistry, immunofluorescence, and the observation of Minimal Change Disea (Minimal Change Disea). Se, MCD) changes in the level of ILK protein at different time points in rats, explore the significance and possible mechanism of ILK in the injury of Poda, and further explore the changes of ILK expression in the model of HGF intervention in the model of small lesion nephropathy.
Methods: the establishment of the animal model of 1. small lesion nephropathy (MCD): 72 male clean grade male SD rats were randomly divided into three groups: control group, model group, hepatocyte growth factor intervention group, model group and rat hepatocyte growth factor intervention group with doxorubicin injection (concentration of 2mg/ml, 7.5mg/kg dose) in the tail vein of hepatocyte growth factor intervention group and M CD model, the hepatocyte growth factor intervention group was given the hepatocyte growth factor (30ug. Kg-1. D-1) from the third week after injection of adriamycin, until the end of the test. The normal control group was injected with the same volume of normal saline in the tail vein. The rats in each group were slaughtered on 7,14,21,28 day after Yu Jianmo, and 6 rats in each group were left to take the kidney. Before death, the specimens were collected from the canthus vein of the rat for about 3M1. The serum albumin, urea nitrogen, creatinine and cholesterol were measured by centrifugation serum. The urine was collected for 24 hours by the metabolic cage 1 days before the death of the rat. The urine protein quantitative.2. was measured by HE staining, and the changes of kidney disease were observed under light microscope and electron microscope, and.3. was used to detect the ILK protein in the kidney by immunofluorescence technique. The location and distribution of small balls, the expression of ILK at different time points, and the relationship with the degree of glomerular podocyte injury, the plasma albumin was measured by electrophoretic method and 24 hours urine protein was measured by sulfonylic salicylic acid method. The changes of plasma albumin, 24 hour urine protein and the relationship with ILK were analyzed in each group. The changes of plasma albumin, 24 hour urine protein and ILK were analyzed.
Results: 1. the expression of ILK protein in the glomeruli of the.2. model group was reduced at 7 days, but the difference was not statistically significant (P0.05) compared with the control group (P0.05), the difference between the 14 days and the control group was statistically significant (P0.05). Compared with the model group, the difference was statistically significant (P0.05). Compared with the HGF intervention group, the expression of ILK protein in the glomeruli decreased for 21 days, and the 28 day decreased significantly (P0.05). 3. compared with the control group, the excretion of 24h urine protein increased for 7 days, 21 Tianda peak, and the difference was statistically significant (P0.01). Compared with the model group, the excretion of 24h urine protein in the HGF intervention group was 21 days. The difference was statistically significant (P0.05) and the difference was statistically significant (P0.05). 4. compared with the control group, the serum albumin in the model group began to decline for 7 days and to the lowest point at 21 days, and the difference was statistically significant (P0.01). Compared with the model group, the serum albumin in the HGF intervention group increased significantly at 21 days and 28 days, and the difference was statistically significant (P0.05).5. renal tissue shape. In the control group, the size of kidney in the control group was normal, the color was dark red, and the cut dough medulla demarcated clearly. The kidney volume of the model group was slightly larger, the color was dark red, and the demarcation of the tangent skin was clear. The small size of glomeruli in the model group showed a slight increase in the model group, the glomerular capillary was visible red cell exudation and the proximal end. The renal tubules showed fat degeneration, abscission, and the formation of protein tube in the renal tubule cavity. With time prolonging, the degree of pathological changes was more obvious. Compared with the model group, the glomerular volume of the HGF intervention group decreased slightly, the glomerular capillary red cell exudation decreased, the proximal renal tubular epithelial cell adipose degeneration lightened, and the renal tubule protein tube type decreased. Under the electron microscope, the model group showed that most of the podocytes of diffuse glomerular podocytes were fused or disappeared, and the foot process of partial podonus was broken. The HGF intervention group reduced the.6. correlation analysis of the foot process fusion compared with the model group. The changes of ILK expression in the model group were positively correlated with the quantitative alteration of 24h urine protein (r=0.743, P0.01).
Conclusion: the expression of 1.ILK and abnormal distribution are closely related to the disease of podonus. It may be an important early marker to judge the damage of the glomerular podocyte and the cause of proteinuria..2.ILK is positively correlated with proteinuria. Its expression in the renal tissue is of certain significance to the prognosis of renal disease..3.HGF can inhibit the microorganism. The expression of ILK protein in glomeruli of rats with small lesion nephropathy has both protective and therapeutic effects on the structure and function of MCD.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R692;R-332

【参考文献】