益肾活血方对FSGS大鼠模型足细胞损伤的保护作用及其机制研究

发布时间：2018-05-22 15:46

本文选题：阿霉素模型 + 局灶节段性肾小球硬化　；参考：《北京中医药大学》2017年硕士论文

【摘要】：目的:通过观察益肾活血方对FSGS大鼠模型足细胞损伤的保护作用,探讨其机制研究。方法:采用随机对照的实验方法,实验动物选用SD大鼠两次尾静脉注射阿霉素(4mg/kg/次)的方法制备的FSGS大鼠模型。二次尾静脉注射间隔2周,于第二次尾静脉注射后7、14天测24小时尿蛋白定量,大于100mg/24h提示造模成功,将提示造模成功的大鼠常规饲养至第二次尾静脉注射后8周末,检测24小时尿蛋白定量。剔除造模失败、尾部溃烂严重或死亡大鼠,造模组剩余35只,计入实验。根据大鼠第二次尾静脉注射后第8周末24小时尿蛋白定量、体重,随机分为:模型组(9只)、贝那普利组(8只)、益肾活血方低剂量组(9只)、益肾活血方高剂量组(9只);另设10只同周龄正常SD大鼠为正常组;各组连续干预8周。实验过程中观察记录大鼠一般情况、体重、24小时尿蛋白定量水平;治疗前眼眶取血检测血浆白蛋白(ALB)、血肌酐(Scr)、尿素氮(BUN)水平;治疗8周后大鼠麻醉、腹主动脉取血,分别冻存和固定肾脏,检测血清中ALB、Scr、BUN水平;行HE、PAS染色观察肾脏病理形态变化,检测肾小球硬化程度;行Western blotting检测WT1(Wilms' tumor 1)蛋白表达;行逆转录-多聚酶链反应(RT-PCR)检测大鼠肾脏podocin、nephrinmRNA表达。所有数据采用SPSS 20.0统计学软件进行分析处理。结果:①益肾活血方能够明显改善大鼠的一般生存状态。②体重上,成模后,正常组与造模组之间即有统计学差异(P0.01);药物干预后,高剂量组与贝那普利组体重仍有增加,模型组自治疗后4周(实验第12周)开始有下降趋势,治疗8周高剂量组、贝那普利组与模型组之间有统计学差异(P0.05)。③尿蛋白上,益肾活血方治疗组、贝那普利组与模型组之间有统计学差异(P0.05);各治疗组之间没有差异。④血浆白蛋白上,治疗后高剂量组与贝那普利组均较前有改善,与正常组之间没有统计学差异(P0.05)。⑤肾功能上,血肌酐:高剂量组改善最为明显,与模型组有明显统计学差异(P0.01),并且与低剂量组与统计学差异(P0.05);尿素氮:贝那普利组改善最为明显,与模型组相比,高剂量、贝那普利组有统计学差异(P0.05)。⑥组织病理学上,肾小球硬化指数、基质比值治疗后均有改善,虽然与正常组相比仍有明显统计学差异(P0.01),但较模型组有统计学改善(P0.05)。⑦蛋白及基因表达上,治疗后,益肾活血方治疗组、贝那普利组WT1表达较模型组增加,有统计学差异(P0.05);益肾活血方高剂量组、贝那普利组nephrin和podocinmRNA水平与模型组相比有明显统计学差异(P0.01)。结论:益肾活血方具有改善FSGS大鼠低蛋白血症、蛋白尿和肾功能,减轻肾小球硬化程度的作用,其机制可能是增加nephrin、podocin基因表达和WT1蛋白表达。
[Abstract]:Aim: to investigate the protective effect of Yishen Huoxue recipe on podocyte injury in FSGS rats. Methods: the FSGS rat model was established by injecting adriamycin 4 mg / kg twice into the tail vein of SD rats. Two weeks after the second caudal vein injection, 24 hours urine protein was measured at 714 days after the second caudal vein injection, which was larger than that indicated by 100mg/24h. The rats who indicated the success of the second caudal vein injection were fed regularly until 8 weeks after the second caudal vein injection. Urine protein was measured in 24 hours. The remaining 35 rats in the model group were included in the experiment. According to the 24 hour urine protein quantity at the end of the eighth week after the second caudal vein injection, The rats were randomly divided into three groups: model group (n = 9), benazepril group (n = 8), low dose group (n = 9), high dose group (n = 9) and control group (n = 10). During the course of the experiment, we observed and recorded the general situation of rats, the weight of rats was measured by 24 hours urine protein; before treatment, the blood samples were taken from orbit to detect the levels of plasma albumin, creatinine, bun and bun; after 8 weeks of treatment, the rats were anesthetized, and the abdominal aorta was taken to get blood. Frozen and fixed kidneys were used to detect the level of serum ALB Scrn bun, the histopathologic changes of kidney and glomerulosclerosis were observed by HEPAS staining, and WT1 Wilms' tumor 1 protein expression was detected by Western blotting. The expression of podocinine mRNA in rat kidney was detected by reverse transcription-polymerase chain reaction (RT PCR). All the data were analyzed by SPSS 20.0 statistical software. Results the general living state of rats was significantly improved by using the "1: 1 Yishen Huoxue recipe". After the model was established, there was a significant difference between the normal group and the model group (P 0.01), and after the intervention of the drug, the weight of the high dose group and benazepril group still increased. In the model group, there was a downward trend since 4 weeks after treatment (the 12th week of the experiment). After 8 weeks of treatment, there was a significant difference between the benazepril group and the model group (P 0.05.3 urine protein), and the treatment group with Yishen Huoxue recipe. There was statistical difference between benazepril group and model group (P 0.05), there was no difference of 4. 4 plasma albumin between each treatment group, after treatment, there was improvement in high dose group and benazepril group, and there was no significant difference in renal function between benazepril group and normal group. Serum creatinine: the improvement was most obvious in the high dose group, which was significantly different from the model group (P 0.01), and was significantly different from the low dose group and the statistical difference (P 0.05), the urea nitrogen: benazepril group was the most obvious improvement, compared with the model group, the high dose was higher than that in the model group. In benazepril group, the histopathology of benazepril was significantly different. Glomerulosclerosis index and matrix ratio were all improved after treatment. Compared with the normal group, the expression of WT1 in the Yishen Huoxue prescription group and benazepril group was increased after treatment, but the protein and gene expression of P0.05.7 protein were significantly improved compared with the model group, although there was still significant difference between the treatment group and the normal control group, the expression of WT1 in the benazepril group was significantly higher than that in the model group. The levels of nephrin and podocinmRNA in the high dose group and benazepril group were significantly different from those in the model group. Conclusion: Yishen Huoxue recipe can improve hypoproteinemia, proteinuria and renal function and alleviate glomerular sclerosis in FSGS rats. Its mechanism may be to increase the expression of nephrininpodocin gene and WT1 protein.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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