舒洛地特对膜性肾病大鼠HSPG、Heparanase表达的影响

发布时间：2019-06-02 03:00

【摘要】：目的：膜性肾病是成人原发性肾病综合征的常见病因，病理主要表现为肾小球脏层上皮细胞下免疫复合物弥漫性沉积、肾小球基底膜增厚伴“钉突”形成，临床主要表现为大量蛋白尿、低蛋白血症、高脂血症。大量蛋白尿已成为肾脏病进展的独立危险因素，有效控制蛋白尿可以延缓肾脏病进展。近年国外研究发现硫酸乙酰肝素蛋白聚糖（Heparan sulfateproteoglycans，HSPG）在肾脏过滤装置中大量表达，尤其是肾小球基底膜,生化学研究显示，HSPG是GBM上不可或缺的有机组成成分。在很多肾病中，包括糖尿病肾病、微小病变和膜性肾病，HSPG表达下降，一般HSPG表达下降与尿蛋白水平升高有关。且研究发现在被动性Heyemann肾炎（给大鼠直接注射抗刷状缘抗体造成类似于人类膜性肾病的肾炎表现）中类肝素酶（Heparanase HPA）在内皮和肾脏上皮细胞表达增加对尿蛋白形成是有作用的。HPA可以有选择性的作用于基底膜上HSPG带有负电荷的侧链，带负电荷的HSPG的缺失可改变肾小球基底膜的通透性，导致蛋白漏出。舒洛地特可以抑制类肝素酶，修复内皮细胞负电荷，减少带负电荷蛋白的漏出。应用舒洛地特是否可以通过降低膜性肾病肾脏内皮及上皮细胞类肝素酶的表达，进而使HSPG的表达上调而发挥降低尿蛋白作用?目前国内外尚缺乏该方面的研究。本实验通过检测HSPG、HPA在膜性肾病大鼠肾脏中的表达，并应用舒洛地特给予干预，研究HSPG与HPA之间的关系，探讨舒洛地特降低膜性肾病蛋白尿的相关机制，从而为延缓膜性肾病的肾脏病变进展提供新的思路。方法：选择清洁级健康雄性SD大鼠40只，体重160±20g，适应性饲养1周后，检测尿蛋白均为阴性，随机将其分为4组，正常对照组、模型组、高剂量舒洛地特组、低剂量舒洛地特组，每组10只。模型组、高剂量舒洛地特组、低剂量舒洛地特组应用自备阳离子化牛血清白蛋白（C-BSA）预免疫1周后，隔日尾静脉注射C-BSA，正常对照组尾静脉注射等体积生理盐水，连续4周后代谢笼中留取各组大鼠24小时尿液，检测24小时尿蛋白定量。造模成功后高剂量舒洛地特组给予舒洛地特20mg/kg灌胃，低剂量舒洛地特组给予舒洛地特10mg/kg灌胃。模型组和正常对照组灌服等体积生理盐水，各组大鼠自由进食、饮水。连续4周后再次代谢笼中留取各组大鼠24小时尿液，检测24小时尿蛋白定量，之后处死大鼠，留取肾组织，分别应用HE、Masson和PAS染色在光镜下观察肾小球基底膜病变情况，并通过电镜观察肾小球基底膜及足突病理变化，应用免疫组织化学方法检测HSPG、HPA的表达情况，结果应用图像分析系统IPP(Image-Pro Plus)进行半定量分析。实验数据用均数±标准差(x±S)表示，各组间比较采用单因素方差分析（满足正态性和方差齐性），两组间比较采用两独立样本t检验，不满足正态性和方差齐性的数据资料采用非参数检验，应用SPSS13.0统计分析软件处理实验数据，以P＜0.05为具有统计学意义。结果： 1.24小时尿蛋白定量隔日尾静脉注射C-BSA，连续4周后，模型组、高剂量舒洛地特组、低剂量舒洛地特组均出现大量蛋白尿，与正常对照组比较有显著性差异（P均0.05），，连续舒洛地特灌胃4周后，高剂量舒洛地特组与低剂量舒洛地特组24小时尿蛋白较前均降低，与模型组相比有显著性差异（P均0.05），与低剂量舒洛地特组相比，高剂量舒洛地特组24小时尿蛋白降低更明显（P0.05）。 2.肾组织病理形态学观察光镜及电镜下正常对照组肾小球结构无明显异常。模型组光镜下肾小球基底膜明显增厚，电镜下肾小球基底膜内大量电子致密物沉积，足细胞足突弥漫融合。与模型组相比，低剂量舒洛地特组与高剂量舒洛地特组光镜下肾小球基底膜轻度增厚，电镜下肾小球基底膜电子致密物沉积减少，足细胞足突融合减轻。 3.肾组织中HSPG、HPA免疫组化结果 HSPG在正常对照组肾小球基底膜上大量表达，模型组、低剂量舒洛地特组及高剂量舒洛地特组肾小球基底膜上有表达，较正常对照组表达降低，有统计学差异（P均0.05），模型组较低剂量舒洛地特组及高剂量舒洛地特组表达均降低，有统计学差异（P均0.05），低剂量舒洛地特组较高剂量舒洛地特组表达降低，有统计学差异（P0.05）。 HPA在模型组肾小球内皮及上皮细胞大量表达，正常对照组微量表达，低剂量舒洛地特及高剂量舒洛地特组有表达，较模型组表达均降低，有统计学差异（P均0.05），较正常对照组表达增加，有统计学差异（P均0.05），模型组与正常对照组相比表达明显增强，有显著性差异（P0.05）。 4.相关性分析对HSPG的表达水平与HPA的表达情况进行了Pearson相关分析，结果表明：HSPG与HPA的表达水平呈负相关（r=㧟0.932，P0.05）。结论： 1.舒洛地特可以降低膜性肾病大鼠尿蛋白程度。 2.舒洛地特可能通过抑制HPA在肾组织的表达，上调肾小球基底膜上的HSPG表达，从而具有改善肾小球基底膜的通透性，减轻肾脏损伤，降低蛋白尿的作用。 3.高剂量舒洛地特较低剂量舒洛地特治疗效果明显。
[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
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

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