發(fā)布時(shí)間：2018-07-15 14:29

【摘要】：細(xì)胞間連接是多細(xì)胞有機(jī)體中相鄰細(xì)胞之間通過細(xì)胞質(zhì)膜相互聯(lián)系以及發(fā)揮協(xié)同作用的重要基礎(chǔ),在人類和動物體內(nèi)主要存在四種形式,緊密連接是其中較為重要的一種。緊密連接是細(xì)胞/細(xì)胞間連接復(fù)合物的最頂端的組成成分,是液體和溶質(zhì)通過細(xì)胞間隙的主要屏障,并在調(diào)節(jié)細(xì)胞增殖、分化和凋亡之間的平衡方面發(fā)揮關(guān)鍵作用,它主要由claudins(CLDNs)、閉合蛋白(occludin)、連接粘附分子(JAMs)等膜蛋白及緊密黏連蛋白(ZO-1、ZO-2和Z0-3)等胞漿蛋白組成,其中CLDNs是緊密連接最主要的成分,主要決定緊密連接的結(jié)構(gòu)和功能。在腫瘤中CLDNs的表達(dá)經(jīng)常發(fā)生改變,例如,CLDN1在食管癌中表達(dá)下調(diào),CLDN 7在胰腺導(dǎo)管癌中表達(dá)下調(diào),CLDN4在膀胱癌中表達(dá)和定位均發(fā)生改變。因此,推測CLDN s表達(dá)或定位的改變導(dǎo)致緊密連接結(jié)構(gòu)和功能受到破壞,從而促進(jìn)腫瘤的發(fā)生和轉(zhuǎn)移。CLDN 4是CLDNs家族27個(gè)成員之一,同時(shí)也是產(chǎn)氣莢膜梭菌腸毒素(CPE)C末端片段的受體。CLDN 4通過細(xì)胞外環(huán)能夠與相鄰細(xì)胞表面的CLDN 4相互作用,并為細(xì)胞旁擴(kuò)散提供良好的屏障。有研究表明,CLDN 4在食管癌和胰腺癌中表達(dá)上調(diào),在結(jié)腸癌和胃癌中表達(dá)下調(diào)。我們的前期工作中發(fā)現(xiàn),與癌旁組織相比,喉癌組織中CLDN 4表達(dá)明顯下調(diào),甲基化特異性蛋白Me CP2表達(dá)明顯上調(diào),且兩者的表達(dá)存在一定相關(guān)性;給予喉癌hep-2細(xì)胞去甲基化制劑5-aza-d C處理之后CLDN4表達(dá)上調(diào),并且CLDN4表達(dá)上調(diào)可以抑制hep-2細(xì)胞的遷移及侵襲能力,那么5-aza-d C上調(diào)CLDN4表達(dá)是否是通過直接改變其DNA甲基化狀態(tài)實(shí)現(xiàn)的,以及CLDN4是否具有調(diào)節(jié)喉癌hep-2細(xì)胞的遷移和侵襲等惡性表型的作用是本研究丞待解決的關(guān)鍵問題。目的:探討喉癌hep-2細(xì)胞中CLDN4表達(dá)下調(diào)與DNA甲基化的關(guān)系以及CLDN4表達(dá)下調(diào)對喉癌hep-2細(xì)胞惡性表型的影響。方法:采用甲基化特異性PCR(methylation specific PCR,MSP)法檢測5-aza-d C作用hep-2細(xì)胞對CLDN4基因DNA甲基化狀態(tài)的影響;采用CCK8實(shí)驗(yàn)、劃痕實(shí)驗(yàn)及Transwell實(shí)驗(yàn)分別檢測RNAi技術(shù)沉默5-aza-d C作用組CLDN4基因?qū)ep-2細(xì)胞增殖、遷移和侵襲能力的影響。結(jié)果:1、低表達(dá)CLDN4的hep-2細(xì)胞中檢測到CLDN4基因的DNA甲基化,而5-aza-d C作用組hep-2細(xì)胞未檢測到CLDN4基因的DNA甲基化;2、沉默5-aza-d C作用組的CLDN4基因后,hep-2細(xì)胞的增殖、遷移和侵襲能力均明顯增強(qiáng)。結(jié)論:喉癌hep-2細(xì)胞中CLDN4表達(dá)下調(diào)與DNA甲基化有關(guān);CLDN4表達(dá)下調(diào)可以促進(jìn)喉癌hep-2細(xì)胞的惡性表型。
[Abstract]:Intercellular junction is an important basis for the interrelation and synergism of adjacent cells in multicellular organisms through the cytoplasmic membrane. There are four main forms in human and animal bodies, among which the close connection is one of the most important. Tight junctions are the top component of cellular / intercellular junction complexes, are the main barrier of liquid and solute passing through the intercellular space, and play a key role in regulating the balance between cell proliferation, differentiation and apoptosis. It is mainly composed of membrane proteins such as claudins (CLDNs), (occludin), binding adhesion molecules (jams) and cytosolic proteins such as tight adhesion proteins (ZO-1, ZO-2 and Z0-3), among which CLDNs are the most important components, which mainly determine the structure and function of tight junctions. The expression of CLDNs was frequently changed in tumors, such as the down-regulated expression of CLDN7 in esophageal carcinoma and the change of the expression and localization of CLDN4 in pancreatic ductal carcinoma. Therefore, it is assumed that changes in the expression or localization of CLDN s lead to the destruction of the tight junction structure and function, thus promoting the occurrence and metastasis of tumors. CLDN4 is one of the 27 members of the CLDNs family. At the same time, CLDN4, the receptor of the C-terminal fragment of Clostridium perfringens enterotoxin (CPE), can interact with CLDN4 on the surface of adjacent cells through the outer loop of the cell, and provide a good barrier for cell diffusion. Studies have shown that CLDN 4 is up-regulated in esophageal and pancreatic cancer, and down-regulated in colon and gastric cancer. In our previous work, we found that the expression of CLDN4 was down-regulated and the expression of methylation-specific protein me CP2 was significantly up-regulated in laryngeal carcinoma tissues compared with paracancerous tissues, and there was a certain correlation between the expression of CLDN4 and Me CP2. The expression of CLDN4 was upregulated after hep-2 cells were treated with 5-aza-d C, and the up-regulation of CLDN4 could inhibit the migration and invasion of hep-2 cells. So, whether the up-regulation of CLDN4 expression by 5-aza-d C was achieved by directly changing the methylation state of hep-2 cells? Whether CLDN4 can regulate the migration and invasion of hep-2 cells in laryngeal carcinoma is the key problem to be solved in this study. Aim: to investigate the relationship between the down-regulation of CLDN4 expression and methylation in laryngeal carcinoma hep-2 cells and the effect of the down-regulation of CLDN4 expression on the malignant phenotype of hep-2 cells. Methods: the effect of 5-aza-d C on the methylation status of hep-2 cells was detected by methylation specific (methylation specific PCR, and the proliferation of hep-2 cells was detected by CCK8 test, scratch test and Transwell test respectively. The effect of migration and invasiveness. Results DNA methylation of CLDN4 gene was detected in hep-2 cells with low expression of CLDN4, but no methylation of CLDN4 gene was detected in hep-2 cells treated with 5-aza-d C. Migration and invasion were significantly enhanced. Conclusion: the down-regulation of CLDN4 expression in laryngeal carcinoma hep-2 cells may promote the malignant phenotype of hep-2 cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.65

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