本文選題：阿霉素 + 蛋白尿��； 參考：《石河子大學(xué)》2010年碩士論文

【摘要】：目的：肝細(xì)胞生長因子(hepatocyte growth factor, HGF)是一種多效性多肽生長因子,它能刺激多種上皮和內(nèi)皮細(xì)胞進(jìn)行有絲分裂、運(yùn)動,在腎小管形態(tài)發(fā)生、腎臟胚胎的形成、腎組織的損傷修復(fù)和再生中起著非常重要的營養(yǎng)作用。而整合素連接激酶(integrin-linked kinase, ILK)是一種新發(fā)現(xiàn)的粘附蛋白,與整合素p的胞漿區(qū)域相連,是細(xì)胞內(nèi)絲氨酸／蘇氨酸蛋白激酶。它在調(diào)節(jié)細(xì)胞周期、基質(zhì)積聚、腫瘤發(fā)生等中起重要作用,也參與了腎臟疾病的發(fā)生發(fā)展過程。新近的研究表明ILK在足細(xì)胞中表達(dá)水平較高,對維持腎小球基底膜(glomerular basement membrane, GBM)的完整性、保持腎小球的濾過功能和防止蛋白質(zhì)的漏出有重要意義。本研究主要通過組織病理學(xué)觀察、免疫組化、免疫熒光方法,觀察在微小病變腎病(Minimal Change Disease, MCD)大鼠體內(nèi)不同時間點(diǎn)的ILK蛋白水平變化,探討ILK在足細(xì)胞損傷中的意義及其可能機(jī)制,同時進(jìn)一步探討了HGF干預(yù)時,微小病變腎病模型中ILK的表達(dá)變化。 方法：1.微小病變腎病(MCD)動物模型的建立：選取清潔級雄性SD大鼠72只,隨機(jī)分為三組：對照組、模型組、肝細(xì)胞生長因子干預(yù)組,模型組和肝細(xì)胞生長因子干預(yù)組的大鼠經(jīng)尾靜脈一次性注射阿霉素注射液(濃度2mg/ml, 7.5mg/kg劑量),復(fù)制MCD模型,肝細(xì)胞生長因子干預(yù)組從注射阿霉素后的第三周開始給予腹腔注射肝細(xì)胞生長因子(30ug·kg-1·d-1),直至試驗(yàn)結(jié)束；正常對照組尾靜脈一次性注射等體積的生理鹽水。各組大鼠分別于建模后第7、14、21、28天宰殺,每組各6只,留取腎臟標(biāo)本,處死前于大鼠內(nèi)眥靜脈采血約3m1,離心分離血清測血漿白蛋白、尿素氮、肌酐、膽固醇。大鼠處死前1天代謝籠收集24小時尿液,測24小時尿蛋白定量。2.進(jìn)行HE染色,光鏡、電鏡下觀察腎臟病理改變情況。3.用免疫熒光技術(shù)檢測ILK蛋白在腎小球的定位分布,分析各組間ILK在不同時間點(diǎn)的表達(dá)情況,及其與腎小球足細(xì)胞損傷程度間的關(guān)系。4.采用電泳法測定血漿白蛋白,采用磺基水楊酸法測24小時尿蛋白定量,分析各組間血漿白蛋白、24小時尿蛋白的變化,以及與ILK的關(guān)系。 結(jié)果：1.采用尾靜脈一次性注射阿霉素注射液復(fù)制MCD模型成功。2.模型組大鼠腎小球中ILK蛋白的表達(dá)7天時減少,但與對照組相比,差異無統(tǒng)計(jì)學(xué)意義(p0.05)；14天明顯增加,21天達(dá)到最大值,與對照組相比差異有統(tǒng)計(jì)學(xué)意義(p0.05)。與模型組相比,HGF干預(yù)組大鼠腎小球中ILK蛋白的表達(dá)21天減少,28天明顯減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05)；3.與對照組相比,模型組大鼠24h尿蛋白的排泄量7天增加,21天達(dá)高峰,差異有統(tǒng)計(jì)學(xué)意義(p0.01)；與模型組相比,HGF干預(yù)組24h尿蛋白的排泄量21天及28天均明顯降低,差異有統(tǒng)計(jì)學(xué)意義(p0.05)；4.與對照組相比,模型組血清白蛋白7天開始下降,21天降至最低點(diǎn),差異有統(tǒng)計(jì)學(xué)意義(p0.01)；與模型組相比,HGF干預(yù)組血清白蛋白在21天及28天均明顯升高,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。5.腎組織形態(tài)學(xué)與病理學(xué)改變：肉眼觀對照組、干預(yù)組腎臟體積大小正常,顏色深紅,切面皮髓質(zhì)分界清楚。模型組腎臟體積稍大,顏色深紅,切面皮髓質(zhì)分界也清楚。光鏡下可見模型組少數(shù)腎小球體積稍增大,腎小球毛細(xì)血管可見紅細(xì)胞滲出,近端腎小管可見脂肪變性、脫落,腎小管管腔內(nèi)蛋白管型形成,隨著時間延長,病變程度加重,21天時最明顯。與模型組比,HGF干預(yù)組腎小球體積稍減小,腎小球毛細(xì)血管紅細(xì)胞滲出減少,近端腎小管上皮細(xì)胞脂肪變性減輕,腎小管蛋白管型減少。電鏡下：模型組可見彌漫性腎小球足細(xì)胞的足突大部分融合或消失,部分足細(xì)胞的足突斷裂；HGF干預(yù)組足突融合較模型組有所減輕。6.相關(guān)分析結(jié)果顯示,模型組ILK表達(dá)的變化與24h尿蛋白定量的變化呈正相關(guān)(r=0.743,P0.01)。 結(jié)論：1.ILK表達(dá)增加及分布異常,與足細(xì)胞病變密切相關(guān),可能是判斷腎小球足細(xì)胞損傷及導(dǎo)致蛋白尿原因的一個早期重要指標(biāo)。2.ILK與蛋白尿呈正相關(guān)關(guān)系,其在腎組織中表達(dá)的高低對腎臟疾預(yù)后的判斷具有一定的意義。3.HGF能抑制微小病變腎病大鼠腎小球ILK蛋白的表達(dá),對MCD在腎臟結(jié)構(gòu)及功能上既有保護(hù)作用又有治療作用。
[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.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R692;R-332

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