本文選題：TRAIL信號(hào)通路 + sDR5-Fc�。� 參考：《河南大學(xué)》2016年碩士論文

【摘要】：研究背景:TNF相關(guān)的凋亡誘導(dǎo)配體(Tumor necrosis factor Related Apoptosis Inducing Ligand,TRAIL)是一種新型腫瘤壞死因子超家族成員之一。在細(xì)胞或組織中如果TRAIL與死亡受體(Death Receptor,DR)結(jié)合,就會(huì)引起炎癥及誘導(dǎo)細(xì)胞凋亡,它通常有五個(gè)重要的死亡受體,其中死亡受體4(DR4)和死亡受體(DR5)包含有被認(rèn)為是死亡結(jié)構(gòu)域的蛋白區(qū)域,這些死亡結(jié)構(gòu)域?qū)τ贒R4和DR5是至關(guān)重要的,它們是傳遞凋亡信號(hào)的重要物質(zhì)。DR4和DR5在配體TRAIL的刺激下與其結(jié)合,進(jìn)而招募具有死亡結(jié)構(gòu)區(qū)的FAS相關(guān)蛋白(Fas-associated Deathdomain,FADD),接著FADD招募Procaspase-8和Procaspase-10等,最終形成死亡誘導(dǎo)信號(hào)復(fù)合體(Death Induced Signal Complex,DISC),隨后Caspase8在Procaspase-8自動(dòng)催化加工下被激活并從DISC釋放,并進(jìn)一步激活Caspase3、Caspase6和Caspase7,且激活的Caspase3會(huì)進(jìn)一步誘導(dǎo)細(xì)胞凋亡。另外,TRAIL信號(hào)通路在肺癌、腎癌、肝癌細(xì)胞中的凋亡作用研究已得到了證實(shí),尤其是在急性腎臟損傷方面已成為研究的熱點(diǎn)。急性腎臟損傷(Acute Kidney Injury,AKI)由多種病因引起的快速腎功能紊亂和高度死亡率為特性的病理綜合征。而缺血再灌注(Ischemia Reperfusion,IR)損傷則是導(dǎo)致AKI的主要原因之一,IR損傷在腎臟的病理學(xué)非常復(fù)雜,很多疾病情況下都可能發(fā)生,具有較高的發(fā)病率,在這一損傷過程中常常伴隨著腎臟細(xì)胞的各種損傷,包括細(xì)胞變性、凋亡、壞死及炎癥細(xì)胞的浸潤等。雖然腎臟移植是近年來迅速發(fā)展的治療腎衰竭病人最有效的治療手段,然而IR損傷則是腎移植不可避免的,研究表明對(duì)IR損傷機(jī)制的研究及干預(yù)有助于減輕IR損傷。因此我們通過構(gòu)建腎臟IR損傷模型來研究sDR5-Fc阻斷TRAIL信號(hào)后對(duì)IR損傷的影響,通過檢測(cè)有關(guān)腎臟功能和組織形態(tài)來驗(yàn)證sDR5-Fc對(duì)腎臟是否具有保護(hù)作用,為臨床腎臟移植等手術(shù)提供良好的保護(hù)性藥物。目的:建立腎IR損傷的動(dòng)物模型,檢測(cè)模型中腎功能的血清指標(biāo),觀察腎組織切片形態(tài)學(xué)的變化和腎細(xì)胞的凋亡,研究TRAIL信號(hào)通路中各相關(guān)蛋白的分布以及基因的變化情況。方法:用健康成年6-8周雄性C57BL/6小鼠通過背部暴露左右兩側(cè)腎臟,分別夾閉左右腎蒂,建立腎臟IR損傷模型。小鼠模型分組情況為:A.Normal組,正常飼養(yǎng)不手術(shù);B.Sham組,只打開背部肌肉暴露腎臟而不缺血;C.PBS組,打開背部皮膚肌肉并且夾閉腎蒂22min使腎臟缺血;D.sDR5-Fc組,打開背部皮膚肌肉并且夾閉腎蒂22min使腎臟缺血,IR后2h和6h腹腔注射sDR5-Fc蛋白,24h時(shí)從心臟采取小鼠血液,分離小鼠血清,通過檢測(cè)各組血清中的BUN與Cr濃度來反應(yīng)小鼠腎臟功能以驗(yàn)證各組腎臟損傷情況,同時(shí)收集腎臟組織,分別于4%多聚甲醛和-80℃保存。分別用TUNEL法和HE染色來觀察腎細(xì)胞凋亡情況和腎組織形態(tài)變化情況,Western Blot確定組織中激活的Cleaved Caspase8的表達(dá)情況,免疫組織化學(xué)方法來檢測(cè)DR5、TRAIL、Cleaved Caspase8和Cleaved Caspase3蛋白的分布與表達(dá)情況,RT-PCR法檢測(cè)DR5和TRAIL蛋白的基因表達(dá)情況。結(jié)果:成功建立小鼠腎IR損傷模型一和模型二。通過檢測(cè)小鼠血清中BUN濃度,發(fā)現(xiàn)Sham組與Normal組沒有明顯差異;PBS組較Normal組和Sham組升高;腹腔注射sDR5-Fc后與PBS組比較BUN濃度下降,同時(shí)檢測(cè)小鼠血清中Cr濃度,發(fā)現(xiàn)各組濃度與BUN濃度結(jié)果一致,具有統(tǒng)計(jì)學(xué)差異,模型一和模型二結(jié)果一致。通過HE染色發(fā)現(xiàn),Normal組和Sham組形態(tài)正常;PBS組腎小管結(jié)構(gòu)被破壞,腎小管上皮細(xì)胞變性壞死,核脫落消失,細(xì)胞從基底膜脫落,但是基底膜處的基底膜輪廓尚存;在sDR5-Fc組中,上述情況不明顯,損傷區(qū)域減少,明顯少于PBS組,模型一和模型二結(jié)果一致。TUNEL檢測(cè)腎細(xì)胞凋亡發(fā)現(xiàn),Normal組與Sham組能觀察到少量綠色熒光點(diǎn),PBS組綠色熒光點(diǎn)數(shù)量較多,差異顯著,在sDR5-Fc組,能觀察到綠色熒光點(diǎn),與PBS組比明顯減少,差異顯著,模型一和模型二結(jié)果一致。免疫組化結(jié)果,分別檢測(cè)DR5、TRAIL、Cleaved Caspase8和Cleaved Caspase3蛋白的分布與表達(dá),其四種蛋白表達(dá)結(jié)果一致,Normal組和Sham組四種蛋白偶有表達(dá),PBS組陽性分布較多,sDR5-Fc組陽性分布明顯低于PBS組,模型一和模型二結(jié)果一致。WB結(jié)果顯示TRAIL信號(hào)通路下游分子激活的Cleaved Caspase8在各組內(nèi)均有表達(dá),PBS組比Normal組和Sham組表達(dá)量較多,sDR5-Fc組比PBS組表達(dá)量降低,模型一和模型二結(jié)果一致。RT-PCR從m RNA水平驗(yàn)證DR5和TRAIL基因的表達(dá)情況,結(jié)果證實(shí)在模型一中對(duì)于DR5基因PBS組比Normal組和Sham組基因變化升高,sDR5-Fc組比PBS組表達(dá)量降低,而TRAIL基因,PBS組比Normal和Sham組表達(dá)都升高,但是sDR5-Fc組比PBS組并沒有明顯降低。模型二中DR5基因表達(dá)與模型一較一致,TRAIL基因表現(xiàn)出PBS組比Normal和Sham組表達(dá)都升高,sDR5-Fc組比PBS組明顯降低。結(jié)論:在小鼠腎臟IR損傷模型中,sDR5-Fc蛋白對(duì)急性腎損傷具有良好的保護(hù)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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R692

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