本文選題：OTUB1　切入點(diǎn)：DCN　出處：《青島大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的:泛素-蛋白酶體途徑是調(diào)控細(xì)胞內(nèi)蛋白質(zhì)降解的重要途徑,涉及細(xì)胞周期調(diào)控、信號通路的轉(zhuǎn)導(dǎo)、細(xì)胞DNA修復(fù)、炎癥反應(yīng)及腫瘤發(fā)展等多個方面。去泛素化酶可逆向調(diào)控該途徑。飾膠蛋白聚糖(decorin,DCN)是細(xì)胞外基質(zhì)一種成分,能夠抑制腫瘤細(xì)胞生長、逆轉(zhuǎn)腫瘤細(xì)胞表型、抑制腫瘤血管生成。DCN主要通過泛素-蛋白酶體途徑進(jìn)行降解。卵巢腫瘤相關(guān)蛋白酶B1(OTU domain-containing ubiquitin aldehyde-binding protein B1,OTUB1)屬于去泛素化酶家族的成員,既可以抑制蛋白的泛素化,也可以促進(jìn)泛素化過程。本研究旨在探討OTUB1在結(jié)腸組織中的表達(dá),并進(jìn)一步探討OTUB1與DCN之間的表達(dá)趨向。方法:收集結(jié)腸癌、腺瘤、正常粘膜組織,采用免疫組織化學(xué)法檢測OTUB1在結(jié)腸組織的表達(dá)。應(yīng)用RT-PCR檢測結(jié)腸癌組織中DCN的m RNA表達(dá)。選定穩(wěn)定轉(zhuǎn)染OTUB1的結(jié)腸癌細(xì)胞系lovo和轉(zhuǎn)染空載質(zhì)粒結(jié)腸癌細(xì)胞系lovo(對照組)作為研究對象,采用實(shí)時熒光定量PCR(Real time quantitative PCR,real time qPCR)和Western blotting,在m RNA和蛋白水平上檢測OTUB1與DCN的表達(dá)。結(jié)果:(1)免疫組化:OTUB1在結(jié)腸癌組織中的表達(dá)明顯高于正常粘膜組織,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)RT-PCR結(jié)果顯示結(jié)腸癌組織中擴(kuò)增出的295bp目的DCN條帶明顯增亮,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)實(shí)時熒光定量PCR:轉(zhuǎn)染OTUB1的結(jié)腸癌細(xì)胞OTUB1與DCN的m RNA表達(dá)水平均上調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(4)Western-blot:與對照組lovo細(xì)胞相比較,穩(wěn)定轉(zhuǎn)染OTUB1的lovo細(xì)胞中OTUB1與DCN蛋白均有明顯表達(dá),差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:OTUB1蛋白在正常結(jié)腸粘膜、腺瘤、結(jié)腸癌組織中的表達(dá)呈遞增趨勢,OTUB1的過表達(dá)抑制了DCN的泛素化降解。
[Abstract]:Background & AIM: the ubiquitin proteasome pathway is an important pathway to regulate the degradation of proteins in cells, which involves cell cycle regulation, signal pathway transduction, and cell DNA repair. Inflammatory response and tumor development are many aspects. Desuginase can reverse regulate this pathway. Decorin DCNC is a component of extracellular matrix, which can inhibit the growth of tumor cells and reverse the phenotype of tumor cells. Inhibition of tumor angiogenesis. DCN is mainly degraded by ubiquitin proteasome pathway. Ovarian tumor-associated protease B1OTU domain-containing ubiquitin aldehyde-binding protein B1OTUB1) is a member of the family of diubiquitin enzymes, which can inhibit the ubiquitin of proteins. The aim of this study was to investigate the expression of OTUB1 in colon tissue and the expression trend between OTUB1 and DCN. Methods: to collect colon cancer, adenoma, normal mucous tissue. Immunohistochemical method was used to detect the expression of OTUB1 in colon tissue. RT-PCR was used to detect m RNA expression of DCN in colon cancer tissue. The colon cancer cell line lovo, which was stably transfected with OTUB1, and the colon cancer cell line, lovo-transfected with empty plasmid, were selected (control group). Group) as the subject of the study, The expression of OTUB1 and DCN at m RNA and protein levels was detected by real-time fluorescence quantitative PCR(Real time quantitative PCRRreal time qPCR) and Western blotting. The results showed that the expression of OTUB1 and DCN in colon cancer tissues was significantly higher than that in normal mucosa tissues. The results of RT-PCR showed that the 295bp DCN band amplified from colon cancer tissue was significantly enhanced, and the difference was statistically significant (P 0.05). The expression of m RNA in OTUB1 transfected colon cancer cell line OTUB1 and DCN were all upregulated. Compared with the control lovo cells, the expression of OTUB1 and DCN protein in lovo cells transfected with OTUB1 was significantly higher than that in the control group (P 0.05, P 0.05). Conclusion the expression of OTUB1 and DCN protein in normal colon mucosa and adenoma was significantly higher than that in control group. The overexpression of OTUB1 in colon cancer tissues inhibited the ubiquitin degradation of DCN.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.35

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本文編號：1608057