舒洛地特对膜性肾病大鼠肾脏中podocin、CD2AP、nephrin表达的影响

发布时间：2018-07-05 00:19

本文选题：膜性肾病（MN） + 阳离子化牛血清白蛋白（C-BSA）　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：膜性肾病(membranons nephropathy，MN)是成人性肾病综合征的常见病因,其病理特点为局限于肾小球上皮下免疫复合物沉积。临床表现为大量蛋白尿。本研究通过注射阳离子化牛血清白蛋白的方法，建立SD大鼠膜性肾病模型，并应用舒洛地特进行干预，观察大鼠24小时尿蛋白定量以及膜性肾病肾小球podocin、CD2AP、nephrin表达的变化，从而探讨舒洛地特对膜性肾病干预的新靶点，为人类防治膜性肾病提供新的理论依据。方法：选择8周龄清洁级健康雄性SD大鼠40只，体重160±20g，普通饲料适应性饲养1周后，测24小时尿蛋白均为正常，采用随机法将其分为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。除正常对照组外，其余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复制膜性肾病大鼠模型。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。连续给药4周后，再次留取各组大鼠的24小时尿液，检测24小时尿蛋白定量，随后将大鼠处死，留取肾组织，应用光镜、电镜观察大鼠肾小球病变情况，采用免疫组织化学方法检测podocin、CD2AP、nephrin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。结果： 1.实验大鼠的一般情况正常对照组的大鼠饮食、尿量均正常，反应灵敏，眼睛有神，肌肉丰满。其余三组大鼠经尾静脉注射C-BSA后，出现饮食减少、精神萎靡、毛发脱落、少动抱团，大部分大鼠出现阴囊水肿。实验最后一周时，与正常对照组相比，造模各组大鼠体形较小，毛发脱落（腹部为重）。 2.24小时尿蛋白定量 24小时尿蛋白定量，在连续尾静脉注射C-BSA4周后，模型组、高剂量舒洛地特组、低剂量舒洛地特组与正常对照组比较均显著升高（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小时尿蛋白较前均有所降低，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）。 3.肾组织病理形态学观察光镜：正常对照组大鼠肾小球形态基本正常，毛细血管袢开放良好，无固有细胞增多，无基底膜增厚；模型组大鼠肾小球固有细胞增多，基底膜明显增厚；高剂量舒洛地特组和低剂量舒洛地特组大鼠的肾小球固有细胞轻度增多，基底膜增厚较模型组减轻。电镜：正常对照组大鼠的足细胞结构清晰，足突排列整齐；模型组大鼠的肾小球基底膜弥漫增厚，上皮下电子致密物沉积，足细胞足突广泛融合或消失；低剂量舒洛地特组大鼠肾小球基底膜不规则增厚，上皮下电子致密物沉积，足突部分融合；高剂量舒洛地特组大鼠基底膜轻度增厚，上皮下少量电子致密物沉积，足突部分融合。 4.免疫组化结果从免疫组化染色中可以观察到podocin、CD2AP、nephrin三种蛋白表达于肾小球上皮下。该三种蛋白在正常对照组肾小球上均有大量表达，模型组、低剂量舒洛地特组及高剂量舒洛地特组肾小球上也均有表达，但较正常对照组表达降低，有统计学意义（P均0.05），模型组中podocin、CD2AP、nephrin的表达较低剂量舒洛地特组及高剂量舒洛地特组表达均降低，，有统计学意义（P均0.05），低剂量舒洛地特组与高剂量舒洛地特组相比，podocin、CD2AP、nephrin表达较低，具有统计学意义（P均0.05）。结论： 1.膜性肾病大鼠足细胞nephrin、podocin及CD2AP表达下降，并与足细胞足突融合程度及蛋白尿程度变化一致。提示nephrin、podocin及CD2AP可能参与膜性肾病肾小球足细胞损伤和蛋白尿产生。 2.舒洛地特可以降低膜性肾病大鼠24小时尿蛋白含量。 3.舒洛地特可以上调膜性肾病大鼠肾组织中podocin、CD2AP、nephrin的表达，提示舒洛地特对膜性肾病的肾脏保护作用可能与维持足细胞裂隙隔膜结构和肾小球滤过功能的完整性有关。 4.舒洛地特治疗大鼠膜性肾病尿蛋白及肾脏保护作用具有剂量依懒性。
[Abstract]:Objective: membranons nephropathy (MN) is a common cause of adult nephrotic syndrome. Its pathological features are limited to the deposition of subcutaneous immuno complex on the glomeruli. The clinical manifestation is a large number of proteinuria. This study established a model of membranous nephropathy in SD rats by injection of cationic bovine serum albumin and applied sulo. To observe the changes of the 24 hour urine protein and the expression of podocin, CD2AP and nephrin in the glomeruli of membranous nephropathy, and to explore the new target for the intervention of membranous nephropathy and provide a new theoretical basis for the prevention and treatment of membranous nephropathy in human.
Methods: 40 healthy male SD rats of 8 weeks old were selected, the body weight was 160 + 20g. After 1 weeks of common feed adaptation, the urine protein was normal in 24 hours. The normal control group was divided into 4 groups, the normal control group, the model group, the high dose sulodiide group and the low dose sulodiide group, with 10 rats in each group. The other groups were used in addition to the normal control group. After 1 weeks of pre immunization with cationic bovine serum albumin (C-BSA), the rat model of membranous nephropathy was replicated by intravenous injection of C-BSA in the tail vein of the next day. The normal control group was injected with the same volume of normal saline in the caudal vein for 4 weeks. The urine of 24 hours of urine was left in each group of offspring, and the urine protein was measured for 24 hours. The high dose of sulalate group was given after the success of the model. The sullock 20mg/kg was given to the stomach. The low dose sullock group was given the sullock 10mg/kg. The model group and the normal control group were given the same volume of normal saline. The rats in each group were free to eat and drinking water. After 4 weeks of continuous administration, the urine of the rats in each group was retained for 24 hours, and the urine protein was measured at 24 hours. Then the rats were killed and the kidney group was left to take the kidney group. The expression of podocin, CD2AP and nephrin was detected by immunohistochemical method, and the results of immunohistochemical staining were used to detect the expression of podocin, CD2AP and nephrin. The results were semi quantitative analysis by using the image analysis system IPP (Image-Pro Plus).
Result:
1. the general situation of experimental rats
The rats in the normal control group have normal diet, the urine volume is normal, the reaction is sensitive, the eyes have the God, the muscle is plump. After the tail vein injection of C-BSA in the rest of the rats, the diet decreases, the spirit is dispirited, the hair falls off, the scrotal edema is seldom moved. The last week of the experiment, compared with the normal control group, the shape of the model rats is compared with the normal control group, the shape of the model rats is compared with the normal control group, the shape of the model rats is compared with the normal control group, the model rats body shape is compared with the normal control group. Small, hair shedding (the abdomen is heavy).
2.24 hour urine protein quantitative
24 hours urine protein quantitative, after C-BSA4 weeks of continuous tail vein injection, model group, high dose suludate group, low dose suludate group and normal control group were significantly higher (P 0.05). After 4 weeks of continuous sulo gavage, the high dose of sullock group and low dosage of sullock group 24 hours urine protein lower than before, and the model. There was a significant difference between the two groups (P = 0.05). Compared with the low dose sullow group, the 24 hour urine protein of the high dose sullow group decreased more significantly (P0.05).
Pathological observation of 3. renal tissues
In the normal control group, the glomerular morphology of the normal control group was basically normal, the capillary loops were open well, no more innate cells and thickening of the basement membrane. The glomerular innate cells in the model group were increased and the basement membrane was thickened, and the glomerular innate cells in the high dose sullad group and low dose sullad group were slightly increased and the basement membrane was slightly increased. The thickening is less than that of the model group.
In the normal control group, the podocyte structure was clear and the foot process was arranged neatly. The glomerular basement membrane in the model group was thickened with thickening of the glomerular basement membrane, the epithelia electron dense deposits were deposited, the podocyte poddate was widely fused or disappeared; the glomerular basement membrane unconventional in the low dose suludate group was thickened, the epithelia electron dense deposit, the foot process In the high dose sulsulide group, the basement membrane was slightly thickened, a small amount of electron dense deposits under the epithelium, and some fusion of foot processes.
4. immunohistochemical results
Three proteins of podocin, CD2AP, and nephrin were expressed in the subcutaneous of glomeruli from immunohistochemical staining. The three proteins were expressed in a large number of glomeruli in the normal control group. In the model group, the glomeruli were also expressed in the low dose sulosate group and the high dose sulosate group, but the expression was lower than that in the normal control group, and the statistics were statistically lower. Statistics showed that there was a statistical difference between the normal control group and the normal control group. The significance (P 0.05), the expression of podocin, CD2AP, nephrin in the model group was lower than the low dose sullock group and high dose sullock group. The expression was statistically significant (P 0.05). The low dose sullock group was lower in podocin, CD2AP, nephrin than in the high dose sullock group (P 0.05).
Conclusion:
The expression of nephrin, podocin and CD2AP in the podocytes of 1. membranous nephrotic rats decreased, and was consistent with the degree of podocyte fusion and proteinuria. It suggested that nephrin, podocin and CD2AP may be involved in the damage of glomerulonephritis and the production of proteinuria in membranous nephropathy.
2. sulpiride could reduce the 24 hour urine protein level in membranous nephropathy rats.
3. sulonate can increase the expression of podocin, CD2AP and nephrin in renal tissue of rats with membranous nephropathy, suggesting that the protective effect of sulonate on membranous nephropathy may be related to the maintenance of the integrity of the structure of the septum septum and the integrity of glomerular filtration function.
4. sulpiride treatment of urinary albumin and renal protection in rats with membranous nephropathy is dose dependent.
【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

【参考文献】