应用改良的RUUO模型探索Treg与梗阻性肾病的关系

发布时间：2018-06-19 22:49

  本文选题：置管 + 单侧输尿管梗阻再通　； 参考：《南京大学》2017年硕士论文

【摘要】：目的:用小鼠作为模型动物,探索改良现有的单侧输尿管梗阻再通(RUUO)模型的方法;在此基础上,研究氯沙坦对解除梗阻后的肾脏的纤维化的影响,以及调节性T细胞在这一过程中的变化。方法:将18只C57BL/6小鼠随机分为RUUO+氯沙坦组(n=6)、RUUO组(n=6)和假手术组(n=6)。在左侧输尿管梗阻(UUO)7天后采用套入+置管法实施再通手术(RUUO)。再通术后RUUO+氯沙坦组水饲法给予氯沙坦。UUO第5天、RUUO第4、7天行外周血流式细胞术检测调节性T细胞(Treg);RUUO术后7天,取术侧肾组织行HE、Masson染色,同时免疫组化检测肾脏α-SMA表达,用Western blot检测肾脏Foxp3蛋白的表达。结果:RUUO+氯沙坦组和RUUO组肾脏均无明显积水,体积较对侧缩小,色泽较浅,肾盂无明显积水,提示梗阻解除,尿路再通成功。HE染色结果示RUUO组肾小管上皮紊乱,刷状缘脱落,管腔扩张,管型形成;假手术组肾小管结构清晰,上皮完整;RUUO+氯沙坦组病理改变较手术组为轻。RUUO组肾小管损伤评分(3.9±0.74)较RUUO+氯沙坦组(2.4±0.48)为重(p0.05),假手术组肾小管损伤评分(0.5±0.45)较两者轻(p0.05)。Masson染色结果示假手术组未见明显蓝染区域,RUUO组可见明显蓝染区域,主要分布在肾小管间质中,而RUUO+氯沙坦组较RUUO组蓝染面积减少。RUUO+氯沙坦组、RUUO组和假手术组胶原面积所占比例分别是5.33±0.64%、9.60± 1.49%和1.17±0.41%,各组之间存在统计学区别(p0.05)。肾脏α-SMA免疫组化染色示假手术组α-SMA主要表达于小血管管壁,亦散见于肾小球周围和肾小管间质中,表达量较少。RUUO组α-SMA表达量明显增多,主要见于肾小管间质中。RUUO+氯沙坦组的表达量较RUUO组减少。RUUO+氯沙坦组α-SMA免疫组化的平均积分光密度为0.0138±0.0011,较RUUO组(0.0203±0.0012)小(P0.05),假手术组α-SMA的平均积分光密度为0.0038±0.0003,与其余两组相比有统计学差异(P0.05)。单侧输尿管结扎术(UUO)后第5天、再通手术(RUUO)后第4天以及第7天外周血Foxp3+Treg/CD4+T cell比例示RUUO+氯沙坦组、RUUO组和假手术组之间无统计学区别。Western blot结果示肾脏Foxp3蛋白在RUUO组表达量降低,其积分光密度与内参积分光密度之比为 0.0664±0.0080,分别低于 RUUO+氯沙坦组的 0.5987±0.2543(P0.05)和假手术组的0.9910±0.1484(P0.05);RUUO+氯沙坦组Foxp3蛋白的表达量低于假手术组(P0.05)。结论:套入+置管法可以建立起较为可靠的小鼠单侧输尿管梗阻再通模型;单侧输尿管梗阻再通后肾脏仍然处于损伤和纤维化状态;调节性T细胞的标志物Foxp3在RUUO肾脏中的表达量下调;氯沙坦可以提高RUUO肾脏Foxp3蛋白的表达量并且改善单侧输尿管梗阻再通后肾脏的纤维化指标。
[Abstract]:Objective: to study the effect of losartan on renal fibrosis after unilateral ureteral obstruction (UUO) was removed by using mice as model animals. And the changes of regulatory T cells during this process. Methods: eighteen C57BL / 6 mice were randomly divided into RUUO losartan group and sham operation group. After 7 days of UUO obstruction in left ureter recanalization was performed by intubation. Losartan 路UUO group was treated with losartan. UUO on the 5th day and RUUO on the 4th and 7th day after operation, the regulatory T cell Tregon was detected by peripheral blood flow cytometry 7 days after operation. The renal tissues were taken out for HE Masson staining, and the expression of 伪 -SMA in the kidney was detected by immunohistochemistry, and the expression of 伪 -SMA in the kidney was also detected by immunohistochemistry, and the expression of 伪 -SMA in the kidney was detected by immunohistochemistry. The expression of Foxp3 protein in kidney was detected by Western blot. Results there was no obvious hydronephrosis in the kidney of the two groups, the volume was smaller than the opposite side, the color of the renal pelvis was lighter, and there was no obvious hydronephrosis in the renal pelvis. The results indicated that the obstruction was relieved and the urethral recanalization was successful. The results of HE staining showed that the renal tubule epithelium in the RUUO group was disordered and the brush edge was shedding. The renal tubules in the sham-operation group were clear, and the tubules were dilated and formed. The pathological changes in the epithelial integrity group were lighter than those in the operation group (3.9 卤0.74) and the renal tubule injury score in the sham-operated group (0.5 卤0.45) was significantly higher than that in the RUUO losartan group (2.4 卤0.48). The results of Masson staining showed that there was no significant blue in the sham-operated group. The dyed area can be seen in the Ruud group, and the blue staining area can be seen. The percentage of collagen in RUUO losartan group and sham operation group was 5.33 卤0.64 卤1.49% and 1.17 卤0.41%, respectively. Renal 伪 -SMA immunohistochemical staining showed that 伪 -SMA was mainly expressed in the wall of small vessels, scattered around glomeruli and tubulointerstitium in sham-operated group, and the expression of 伪 -SMA was significantly increased in RUUO group. The average integral optical density of 伪 -SMA immunohistochemistry in the RUUO losartan group was 0.0138 卤0.0011, which was higher than that in the RUUO group (0.0203 卤0.0012). The mean integral optical density of 伪 -SMA in the sham-operated group was 0.0038 卤0.0003, and that in the other two groups was 0.0038 卤0.0003. There was a statistical difference between the two groups (P 0.05). The ratio of Foxp3 Treg-CD4T cell in peripheral blood on the 4th and 7th day after UUO (unilateral ureteral ligation) showed no statistical difference between RUUO Losartan group and sham-operated group. Western blot results showed that the expression of Foxp3 protein in kidney decreased in RUUO group. The ratio of integral optical density to integrated optical density of internal parameters was 0.0664 卤0.0080, which was lower than that of Rup losartan group (0.5987 卤0.2543 P0.05) and sham-operated group (0.9910 卤0.1484 P0.05P 0.05). The expression of Foxp3 protein in Ruluo losartan group was lower than that in sham operation group (P0.05. Conclusion: the recanalization model of unilateral ureteral obstruction in mice can be established by inserting catheter, and the kidney is still in the state of injury and fibrosis after the recanalization of unilateral ureteral obstruction. The expression of Foxp3, a marker of regulatory T cells, was down-regulated in RUUO kidney, and losartan could increase the expression of Foxp3 protein and improve the renal fibrosis index after the recanalization of unilateral ureteral obstruction.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
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本文编号：2041669