sDR5-Fc对小鼠肾脏缺血再灌注损伤的保护

发布时间：2018-06-18 10:29

本文选题：TRAIL信号通路 + sDR5-Fc　；参考：《河南大学》2016年硕士论文

【摘要】：研究背景:TNF相关的凋亡诱导配体(Tumor necrosis factor Related Apoptosis Inducing Ligand,TRAIL)是一种新型肿瘤坏死因子超家族成员之一。在细胞或组织中如果TRAIL与死亡受体(Death Receptor,DR)结合,就会引起炎症及诱导细胞凋亡,它通常有五个重要的死亡受体,其中死亡受体4(DR4)和死亡受体(DR5)包含有被认为是死亡结构域的蛋白区域,这些死亡结构域对于DR4和DR5是至关重要的,它们是传递凋亡信号的重要物质。DR4和DR5在配体TRAIL的刺激下与其结合,进而招募具有死亡结构区的FAS相关蛋白(Fas-associated Deathdomain,FADD),接着FADD招募Procaspase-8和Procaspase-10等,最终形成死亡诱导信号复合体(Death Induced Signal Complex,DISC),随后Caspase8在Procaspase-8自动催化加工下被激活并从DISC释放,并进一步激活Caspase3、Caspase6和Caspase7,且激活的Caspase3会进一步诱导细胞凋亡。另外,TRAIL信号通路在肺癌、肾癌、肝癌细胞中的凋亡作用研究已得到了证实,尤其是在急性肾脏损伤方面已成为研究的热点。急性肾脏损伤(Acute Kidney Injury,AKI)由多种病因引起的快速肾功能紊乱和高度死亡率为特性的病理综合征。而缺血再灌注(Ischemia Reperfusion,IR)损伤则是导致AKI的主要原因之一,IR损伤在肾脏的病理学非常复杂,很多疾病情况下都可能发生,具有较高的发病率,在这一损伤过程中常常伴随着肾脏细胞的各种损伤,包括细胞变性、凋亡、坏死及炎症细胞的浸润等。虽然肾脏移植是近年来迅速发展的治疗肾衰竭病人最有效的治疗手段,然而IR损伤则是肾移植不可避免的,研究表明对IR损伤机制的研究及干预有助于减轻IR损伤。因此我们通过构建肾脏IR损伤模型来研究sDR5-Fc阻断TRAIL信号后对IR损伤的影响,通过检测有关肾脏功能和组织形态来验证sDR5-Fc对肾脏是否具有保护作用,为临床肾脏移植等手术提供良好的保护性药物。目的:建立肾IR损伤的动物模型,检测模型中肾功能的血清指标,观察肾组织切片形态学的变化和肾细胞的凋亡,研究TRAIL信号通路中各相关蛋白的分布以及基因的变化情况。方法:用健康成年6-8周雄性C57BL/6小鼠通过背部暴露左右两侧肾脏,分别夹闭左右肾蒂,建立肾脏IR损伤模型。小鼠模型分组情况为:A.Normal组,正常饲养不手术;B.Sham组,只打开背部肌肉暴露肾脏而不缺血;C.PBS组,打开背部皮肤肌肉并且夹闭肾蒂22min使肾脏缺血;D.sDR5-Fc组,打开背部皮肤肌肉并且夹闭肾蒂22min使肾脏缺血,IR后2h和6h腹腔注射sDR5-Fc蛋白,24h时从心脏采取小鼠血液,分离小鼠血清,通过检测各组血清中的BUN与Cr浓度来反应小鼠肾脏功能以验证各组肾脏损伤情况,同时收集肾脏组织,分别于4%多聚甲醛和-80℃保存。分别用TUNEL法和HE染色来观察肾细胞凋亡情况和肾组织形态变化情况,Western Blot确定组织中激活的Cleaved Caspase8的表达情况,免疫组织化学方法来检测DR5、TRAIL、Cleaved Caspase8和Cleaved Caspase3蛋白的分布与表达情况,RT-PCR法检测DR5和TRAIL蛋白的基因表达情况。结果:成功建立小鼠肾IR损伤模型一和模型二。通过检测小鼠血清中BUN浓度,发现Sham组与Normal组没有明显差异;PBS组较Normal组和Sham组升高;腹腔注射sDR5-Fc后与PBS组比较BUN浓度下降,同时检测小鼠血清中Cr浓度,发现各组浓度与BUN浓度结果一致,具有统计学差异,模型一和模型二结果一致。通过HE染色发现,Normal组和Sham组形态正常;PBS组肾小管结构被破坏,肾小管上皮细胞变性坏死,核脱落消失,细胞从基底膜脱落,但是基底膜处的基底膜轮廓尚存;在sDR5-Fc组中,上述情况不明显,损伤区域减少,明显少于PBS组,模型一和模型二结果一致。TUNEL检测肾细胞凋亡发现,Normal组与Sham组能观察到少量绿色荧光点,PBS组绿色荧光点数量较多,差异显著,在sDR5-Fc组,能观察到绿色荧光点,与PBS组比明显减少,差异显著,模型一和模型二结果一致。免疫组化结果,分别检测DR5、TRAIL、Cleaved Caspase8和Cleaved Caspase3蛋白的分布与表达,其四种蛋白表达结果一致,Normal组和Sham组四种蛋白偶有表达,PBS组阳性分布较多,sDR5-Fc组阳性分布明显低于PBS组,模型一和模型二结果一致。WB结果显示TRAIL信号通路下游分子激活的Cleaved Caspase8在各组内均有表达,PBS组比Normal组和Sham组表达量较多,sDR5-Fc组比PBS组表达量降低,模型一和模型二结果一致。RT-PCR从m RNA水平验证DR5和TRAIL基因的表达情况,结果证实在模型一中对于DR5基因PBS组比Normal组和Sham组基因变化升高,sDR5-Fc组比PBS组表达量降低,而TRAIL基因,PBS组比Normal和Sham组表达都升高,但是sDR5-Fc组比PBS组并没有明显降低。模型二中DR5基因表达与模型一较一致,TRAIL基因表现出PBS组比Normal和Sham组表达都升高,sDR5-Fc组比PBS组明显降低。结论:在小鼠肾脏IR损伤模型中,sDR5-Fc蛋白对急性肾损伤具有良好的保护作用。
[Abstract]:Background: TNF related apoptosis inducing ligand (Tumor necrosis factor Related Apoptosis Inducing Ligand, TRAIL) is one of the new tumor necrosis factor superfamily members. In cells or tissues, if TRAIL is combined with the death receptor (Death Receptor), it can cause inflammation and induce apoptosis. It usually has five important factors. Death receptors, in which death receptors 4 (DR4) and death receptor (DR5) contain protein regions that are considered to be the domain of death, which are essential to DR4 and DR5, which are important substances that transmit apoptosis signals,.DR4 and DR5, to bind to the ligand TRAIL, and then recruit FAS with the dead structure area. The associated protein (Fas-associated Deathdomain, FADD), followed by FADD recruitment of Procaspase-8 and Procaspase-10, eventually forms a death induced signal complex (Death Induced Signal Complex, DISC). Subsequently, Caspase8 is activated and released under the automatic catalytic processing. Living Caspase3 can further induce apoptosis. In addition, the research on the apoptosis of TRAIL signaling pathway in lung cancer, kidney cancer and hepatoma cells has been confirmed, especially in acute renal injury. Acute renal injury (Acute Kidney Injury, AKI) is a rapid renal dysfunction and height caused by a variety of causes. Ischemia Reperfusion (IR) injury is one of the main causes of AKI. The IR damage is very complicated in the kidney pathology, and many diseases are likely to occur, with a high incidence, and often accompanied by various kinds of damage to the kidney cells during this damage process. It includes cell degeneration, apoptosis, necrosis and infiltration of inflammatory cells. Although renal transplantation is the most effective treatment for patients with renal failure in recent years, IR injury is inevitable in renal transplantation. Research shows that research and intervention on the mechanism of IR damage can help to reduce IR damage. Therefore, we build the renal IR loss by constructing kidney. The injury model is used to study the effect of sDR5-Fc blocking the TRAIL signal on the IR damage. By detecting the renal function and tissue morphology, it can be used to verify the protective effect of sDR5-Fc on the kidney and provide a good protective drug for the clinical renal transplantation. Objective: to establish an animal model of renal IR injury and to detect the serum finger of renal function in the model. To observe the morphological changes of the renal tissue sections and the apoptosis of renal cells, to study the distribution of the related proteins in the TRAIL signaling pathway and the change of the genes. Methods: the left and right kidney pedicles were clipped by the back exposure of the left and right kidneys on the back of the male C57BL/6 mice in the 6-8 weeks of healthy adults, and the renal IR damage model was established. The model group of the mice was divided into groups. A.Normal group, normal feeding, no operation, group B.Sham, only open the back muscles to expose the kidney without ischemia; group C.PBS, open the back skin muscles and clamp the renal pedicle 22min to make kidney ischemia; group D.sDR5-Fc, open the back skin muscles and clamp the kidney pedicle 22min to make renal ischemia, 2h and 6h intraperitoneally injected with sDR5-Fc protein after IR, 24h from the heart. The mice blood was taken and the mice serum was separated. The renal function of mice was tested by detecting the BUN and Cr concentrations in the serum of each group to verify renal damage in each group, and the renal tissue was collected at 4% paraformaldehyde and -80 centigrade respectively. The apoptosis of renal cells and the changes of renal tissue morphology were observed by TUNEL and HE staining, respectively. Western Blot determined the expression of Cleaved Caspase8 activated in the tissue. Immunohistochemical method was used to detect the distribution and expression of DR5, TRAIL, Cleaved Caspase8 and Cleaved Caspase3 proteins. The expression of the genes of DR5 and protein was detected by RT-PCR method. The concentration of BUN in the serum of mice was measured, and there was no significant difference between the Sham group and the Normal group; the PBS group was higher than the Normal group and the Sham group; the concentration of BUN decreased in the abdominal cavity and the PBS group, and the concentration of Cr in the serum of the mice was detected, and the consistency between the concentration and the BUN concentration was found to be the same, and the model one was in agreement with the model two. After HE staining, the morphology of group Normal and Sham group was normal, and the renal tubular structure of group PBS was destroyed, the epithelial cells of the renal tubule degeneration and necrosis, the nucleus exfoliated and disappeared, the cells were falling off the basement membrane, but the basilar membrane in the basement membrane still remained. In group sDR5-Fc, the above situation was not obvious, the damage area was less than that of the PBS group, model one and model two Results the same.TUNEL detection of renal cell apoptosis found that the Normal group and the Sham group could observe a small amount of green fluorescence point, and the number of green fluorescence points in the group PBS was more, the difference was significant. In group sDR5-Fc, the green fluorescence point could be observed and the ratio of the PBS group decreased significantly. The model one was the same as the model two. DR5, TRAI were detected by the immunohistochemical results respectively. The distribution and expression of L, Cleaved Caspase8 and Cleaved Caspase3 protein were the same. The four proteins in the Normal and Sham groups were even expressed, the positive distribution of the PBS group was more, the positive distribution of the sDR5-Fc group was obviously lower than the PBS group, and the model 1 was consistent with the model two and showed the activation of the downstream molecules of the TRAIL signal pathway. The expression of Caspase8 in group PBS was more than that of group Normal and Sham, and the expression of sDR5-Fc group was lower than that of PBS group. The expression of DR5 and TRAIL gene was verified by.RT-PCR from m RNA level. The results showed that the change of the DR5 gene group was higher than that of the DR5 and TRAIL genes in the model 1. The expression of -Fc group was lower than that of PBS group, while the expression of TRAIL gene and PBS group was higher than that of Normal and Sham group, but the sDR5-Fc group was not significantly lower than that of the PBS group. The DR5 gene expression in the model two was in accordance with the model one, and the TRAIL gene showed that PBS group was higher than that of Normal and other groups. In the R damage model, sDR5-Fc protein has a good protective effect on acute kidney injury.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R692

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