【摘要】：目的：膜性腎病是成人原發(fā)性腎病綜合征的常見病因，病理主要表現(xiàn)為腎小球臟層上皮細(xì)胞下免疫復(fù)合物彌漫性沉積、腎小球基底膜增厚伴“釘突”形成，臨床主要表現(xiàn)為大量蛋白尿、低蛋白血癥、高脂血癥。大量蛋白尿已成為腎臟病進(jìn)展的獨(dú)立危險因素，有效控制蛋白尿可以延緩腎臟病進(jìn)展。近年國外研究發(fā)現(xiàn)硫酸乙酰肝素蛋白聚糖（Heparan sulfateproteoglycans，HSPG）在腎臟過濾裝置中大量表達(dá)，尤其是腎小球基底膜,生化學(xué)研究顯示，HSPG是GBM上不可或缺的有機(jī)組成成分。在很多腎病中，包括糖尿病腎病、微小病變和膜性腎病，HSPG表達(dá)下降，一般HSPG表達(dá)下降與尿蛋白水平升高有關(guān)。且研究發(fā)現(xiàn)在被動性Heyemann腎炎（給大鼠直接注射抗刷狀緣抗體造成類似于人類膜性腎病的腎炎表現(xiàn)）中類肝素酶（Heparanase HPA）在內(nèi)皮和腎臟上皮細(xì)胞表達(dá)增加對尿蛋白形成是有作用的。HPA可以有選擇性的作用于基底膜上HSPG帶有負(fù)電荷的側(cè)鏈，帶負(fù)電荷的HSPG的缺失可改變腎小球基底膜的通透性，導(dǎo)致蛋白漏出。舒洛地特可以抑制類肝素酶，修復(fù)內(nèi)皮細(xì)胞負(fù)電荷，減少帶負(fù)電荷蛋白的漏出。應(yīng)用舒洛地特是否可以通過降低膜性腎病腎臟內(nèi)皮及上皮細(xì)胞類肝素酶的表達(dá)，進(jìn)而使HSPG的表達(dá)上調(diào)而發(fā)揮降低尿蛋白作用?目前國內(nèi)外尚缺乏該方面的研究。本實(shí)驗(yàn)通過檢測HSPG、HPA在膜性腎病大鼠腎臟中的表達(dá)，并應(yīng)用舒洛地特給予干預(yù)，研究HSPG與HPA之間的關(guān)系，探討舒洛地特降低膜性腎病蛋白尿的相關(guān)機(jī)制，從而為延緩膜性腎病的腎臟病變進(jìn)展提供新的思路。 方法：選擇清潔級健康雄性SD大鼠40只，體重160±20g，適應(yīng)性飼養(yǎng)1周后，檢測尿蛋白均為陰性，隨機(jī)將其分為4組，正常對照組、模型組、高劑量舒洛地特組、低劑量舒洛地特組，每組10只。模型組、高劑量舒洛地特組、低劑量舒洛地特組應(yīng)用自備陽離子化牛血清白蛋白（C-BSA）預(yù)免疫1周后，隔日尾靜脈注射C-BSA，正常對照組尾靜脈注射等體積生理鹽水，連續(xù)4周后代謝籠中留取各組大鼠24小時尿液，檢測24小時尿蛋白定量。造模成功后高劑量舒洛地特組給予舒洛地特20mg/kg灌胃，低劑量舒洛地特組給予舒洛地特10mg/kg灌胃。模型組和正常對照組灌服等體積生理鹽水，各組大鼠自由進(jìn)食、飲水。連續(xù)4周后再次代謝籠中留取各組大鼠24小時尿液，檢測24小時尿蛋白定量，之后處死大鼠，留取腎組織，分別應(yīng)用HE、Masson和PAS染色在光鏡下觀察腎小球基底膜病變情況，并通過電鏡觀察腎小球基底膜及足突病理變化，應(yīng)用免疫組織化學(xué)方法檢測HSPG、HPA的表達(dá)情況，結(jié)果應(yīng)用圖像分析系統(tǒng)IPP(Image-Pro Plus)進(jìn)行半定量分析。實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(x±S)表示，各組間比較采用單因素方差分析（滿足正態(tài)性和方差齊性），兩組間比較采用兩獨(dú)立樣本t檢驗(yàn)，不滿足正態(tài)性和方差齊性的數(shù)據(jù)資料采用非參數(shù)檢驗(yàn)，應(yīng)用SPSS13.0統(tǒng)計分析軟件處理實(shí)驗(yàn)數(shù)據(jù)，以P＜0.05為具有統(tǒng)計學(xué)意義。 結(jié)果： 1.24小時尿蛋白定量 隔日尾靜脈注射C-BSA，連續(xù)4周后，模型組、高劑量舒洛地特組、低劑量舒洛地特組均出現(xiàn)大量蛋白尿，與正常對照組比較有顯著性差異（P均0.05），，連續(xù)舒洛地特灌胃4周后，高劑量舒洛地特組與低劑量舒洛地特組24小時尿蛋白較前均降低，與模型組相比有顯著性差異（P均0.05），與低劑量舒洛地特組相比，高劑量舒洛地特組24小時尿蛋白降低更明顯（P0.05）。 2.腎組織病理形態(tài)學(xué)觀察 光鏡及電鏡下正常對照組腎小球結(jié)構(gòu)無明顯異常。模型組光鏡下腎小球基底膜明顯增厚，電鏡下腎小球基底膜內(nèi)大量電子致密物沉積，足細(xì)胞足突彌漫融合。與模型組相比，低劑量舒洛地特組與高劑量舒洛地特組光鏡下腎小球基底膜輕度增厚，電鏡下腎小球基底膜電子致密物沉積減少，足細(xì)胞足突融合減輕。 3.腎組織中HSPG、HPA免疫組化結(jié)果 HSPG在正常對照組腎小球基底膜上大量表達(dá)，模型組、低劑量舒洛地特組及高劑量舒洛地特組腎小球基底膜上有表達(dá)，較正常對照組表達(dá)降低，有統(tǒng)計學(xué)差異（P均0.05），模型組較低劑量舒洛地特組及高劑量舒洛地特組表達(dá)均降低，有統(tǒng)計學(xué)差異（P均0.05），低劑量舒洛地特組較高劑量舒洛地特組表達(dá)降低，有統(tǒng)計學(xué)差異（P0.05）。 HPA在模型組腎小球內(nèi)皮及上皮細(xì)胞大量表達(dá)，正常對照組微量表達(dá)，低劑量舒洛地特及高劑量舒洛地特組有表達(dá)，較模型組表達(dá)均降低，有統(tǒng)計學(xué)差異（P均0.05），較正常對照組表達(dá)增加，有統(tǒng)計學(xué)差異（P均0.05），模型組與正常對照組相比表達(dá)明顯增強(qiáng)，有顯著性差異（P0.05）。 4.相關(guān)性分析 對HSPG的表達(dá)水平與HPA的表達(dá)情況進(jìn)行了Pearson相關(guān)分析，結(jié)果表明：HSPG與HPA的表達(dá)水平呈負(fù)相關(guān)（r=㧟0.932，P0.05）。 結(jié)論： 1.舒洛地特可以降低膜性腎病大鼠尿蛋白程度。 2.舒洛地特可能通過抑制HPA在腎組織的表達(dá)，上調(diào)腎小球基底膜上的HSPG表達(dá)，從而具有改善腎小球基底膜的通透性，減輕腎臟損傷，降低蛋白尿的作用。 3.高劑量舒洛地特較低劑量舒洛地特治療效果明顯。
[Abstract]:Objective: The membranous nephropathy is a common cause of primary nephrotic syndrome in adults. The pathological changes are mainly characterized by the diffuse deposition of the immune complex in the epithelial cells of the mesangial cells, the thickening of the glomerular basement membrane and the formation of the "spike", and the clinical manifestation is a large amount of proteinuria and hypoproteinemia. Hyperlipemia. A large amount of proteinuria has become an independent risk factor for the progression of kidney disease, and the effective control of proteinuria can delay the progression of kidney disease. In recent years, it has been found that heparin proteoglycan (HSPG) has been expressed in the renal filtration device, especially the glomerular basement membrane, and the chemical research shows that the HSPG is an integral component of the GBM. In many cases of kidney disease, including diabetic nephropathy, microlesion and membranous nephropathy, the expression of HSPG decreased, and the expression of HSPG in general was related to the increase of the level of urinary protein. It was found that the increase of the expression of heparanase in the endothelial and renal epithelial cells was effective in the expression of the endothelial and renal epithelial cells in the passive Heyemann nephritis (which caused the direct injection of the anti-brush-like edge antibody to the rat). The HPA can selectively act on the side chain with negative charge of the HSPG on the basement membrane, and the deletion of the negatively charged HSPG can change the permeability of the glomerular basement membrane and lead to the leakage of the protein. Schlosin can inhibit the heparanase, repair the negative charge of the endothelial cell, and reduce the leakage of the negatively charged protein. Whether the expression of the heparanase of the renal endothelium and the epithelial cells of the membranous nephropathy can be reduced by using the Schloway, and then the expression of the HSPG is up-regulated and the effect of the urinary protein can be reduced. At present, there is no research on this aspect at home and abroad. The relationship between HSPG and HPA was studied by detecting the expression of HSPG and HPA in the kidney of rat kidney, and the relationship between HSPG and HPA was studied. So as to provide a new way for delaying the progression of the kidney disease of the membranous nephropathy. Methods:40 healthy male SD rats were randomly divided into 4 groups, the normal control group, the model group, the high-dose sulotrete group and the low-dose sulotrete group, each group of 10 healthy male SD rats were randomly divided into 4 groups. One week after the pre-immunization with self-contained cationic bovine serum albumin (C-BSA) in the model group, the high-dose sulotreotide group and the low-dose sulotreotide group, the volume physiological salts such as C-BSA and the tail vein of the normal control group were injected intravenously. The 24-hour urine of each group was retained in the cage for 24 hours after 4 weeks of continuous water, and the urine protein was determined for 24 hours. In this study, the high dose of the high-dose sulotreotide group was given in the high dose group with the dose of 20 mg/ kg and the dosage of the low-dose sulotreotide group was 10 mg/ kg in the low-dose group. The rats were divided into three groups: the stomach, the model group and the normal control group, and the rats were free to eat, drink and drink. Water was collected for 24 hours after 4 weeks of continuous metabolism,24 hours of urine was collected in each group of rats,24 hours of urine protein was measured, and the rats were sacrificed, and the kidney tissues were sacrificed. HE, Masson and PAS staining were used to observe the pathological conditions of the glomerular basement membrane under the light microscope. The expression of HSPG and HPA was detected by the immunohistochemical method, and the image analysis system IPP (Image-Pro Plus) was used for semi-quantitative analysis. The mean square standard deviation (x-S) of the experimental data was expressed by the standard deviation (x-S) of the average number of the experimental data. The single-factor analysis of variance was used for the comparison between the two groups. The two independent samples (t) were used for the comparison between the two groups, and the non-parametric test was used for the data which did not meet the positive and the standard deviation. The software of SPSS13.0 statistical analysis software was applied to deal with the experimental data, which was statistically significant with P <0.05. Righteousness. Result: 1.24 small There was a significant difference between the normal control group and the normal control group (P <0.05). The 24-hour urine protein of the high-dose sulotreotide group and the low-dose sulotreotide group was lower than that of the low-dose sulotreotide group after 4 weeks, and there was a significant difference compared with the model group (P <0.05), and the low-dose group was significantly different from the model group (P <0.05). The 24-hour urinary protein decrease of the high-dose sulotreotide group is more obvious than that of the Schlosin group. (P0.05). 2. The pathological and morphological observation of the renal tissue and the normal control under the electron microscope The glomerular basement membrane was significantly thickened under the light microscope of the model group, and a large number of electron dense deposits were found in the glomerular basement membrane under the electron microscope. In comparison with the model group, the low dose of the sulosin group and the high dose of the Schlosin group had mild thickening of the glomerular basement membrane under the light microscope, and the electron dense deposit of the glomerular basement membrane was reduced under the electron microscope. Small, foot-cell-apophysis fusion-reduced.3. Renal tissue The expression of HSPG and HPA in the Glomerular basement membrane of the normal control group was expressed, and the expression of HSPG on the glomerular basement membrane of the low-dose and high-dose sulosin group was reduced, and the expression of HSPG in the normal control group was lower. There was a statistical difference (P <0.05), and the expression of the low-dose and the high-dose sulotreotide group was lower in the model group than in the low-dose and the low-dose group. There was a significant difference in the expression of HPA in the glomerular endothelial and epithelial cells of the model group. (0.05), the expression of the normal control group was increased, there was a statistical difference (P <0.05), and the model group was expressed as compared with the normal control group. Significant enhancement, significant 4. The expression level of HSPG and the expression of HPA were analyzed by Pearson correlation analysis. The results showed that the expression of HSPG and HPA was higher than that of HPA. flat negative Correlation (r =? 0.932, P0.05). The expression of HspG in the glomerular basement membrane can be up-regulated by inhibiting the expression of the HPA in the renal tissue, thus having the effect of modifying the expression of the HPA in the renal tissue, Good glomerular basement membrane permeability, reduced renal damage, and reduced proteinuria
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692

【參考文獻(xiàn)】

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1 陳珊;方展;朱忠華;鄧安國;劉建社;張春;;Protective Effect of Sulodexide on Podocyte Injury in Adriamycin Nephropathy Rats[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2009年06期

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