【摘要】：腫瘤抗原OVA12是本實驗室應(yīng)用SEREX(Serological analysis of recombinant cDNA expression library)方法從人卵巢癌cDNA表達(dá)文庫篩選得到的一個新的CT(cancer/testis)抗原。前期研究表明,OVA12抗原在多種腫瘤組織和腫瘤細(xì)胞株中高表達(dá),在正常組織(睪丸除外)及正常細(xì)胞株中不表達(dá)或低表達(dá);將OVA12基因轉(zhuǎn)染SMMC-7721細(xì)胞后,細(xì)胞增殖速度加快,裸鼠體內(nèi)成瘤能力增強。結(jié)果顯示OVA12與腫瘤的發(fā)生發(fā)展存在密切的關(guān)聯(lián),因此對OVA12生物學(xué)功能和作用機制的深入研究有著重要的理論意義和臨床應(yīng)用價值。本課題主要從以下三個方面進(jìn)行研究:1)從mRNA及蛋白水平檢測多種正常和腫瘤細(xì)胞及組織中OVA12的表達(dá)水平,明確其表達(dá)譜特征;應(yīng)用病毒感染的方式構(gòu)建OVA12穩(wěn)定高表達(dá)及干擾細(xì)胞株,通過體內(nèi)外實驗進(jìn)一步探討OVA12在腫瘤發(fā)生發(fā)展中的生物學(xué)功能。結(jié)果顯示OVA12在多種腫瘤組織和細(xì)胞株中高表達(dá),而在正常組織和細(xì)胞中不表達(dá)或低表達(dá)。體內(nèi)外結(jié)果證實OVA12在腫瘤細(xì)胞株高表達(dá)或者干擾后能夠顯著促進(jìn)或抑制腫瘤細(xì)胞增殖能力及抗凋亡能力,并能通過上調(diào)Mcl-1和survivin蛋白的表達(dá)增加腫瘤細(xì)胞對5-FU的耐藥性;2)以O(shè)VA12穩(wěn)定高表達(dá)和干擾細(xì)胞株為研究對象,檢測與細(xì)胞增殖、凋亡密切相關(guān)的信號分子的表達(dá)變化。結(jié)果顯示OVA12表達(dá)降低能夠顯著提高p53蛋白的表達(dá),而OVA12高表達(dá)能夠抑制p53蛋白的表達(dá),但不影響p53 mRNA水平;與對照組相比,p53熒光素酶基因報告系統(tǒng)檢測顯示OVA12干擾細(xì)胞株p53轉(zhuǎn)錄活性明顯增加,而OVA12高表達(dá)細(xì)胞株其轉(zhuǎn)錄活性顯著降低。OVA12能夠調(diào)控p53蛋白的K48位多聚泛素鏈形式,促進(jìn)p53在蛋白酶體的泛素化降解,并能夠影響腫瘤細(xì)胞對抗腫瘤藥物順鉑(依賴p53信號通路誘導(dǎo)腫瘤細(xì)胞凋亡)的敏感性。結(jié)果提示OVA12蛋白能夠負(fù)向調(diào)控p53蛋白;3)利用特異性的p53 shRNA干擾阻斷p53信號通路,應(yīng)用CCK-8等相關(guān)體內(nèi)外實驗方法檢測p53蛋白表達(dá)降低后對OVA12生物學(xué)功能的影響,同時檢測調(diào)控p53信號通路上游的多個重要蛋白表達(dá)變化,探討OVA12調(diào)控p53信號通路的作用機制。結(jié)果顯示與對照組相比,在OVA12干擾細(xì)胞株內(nèi),特異性阻斷p53信號通路能有效的逆轉(zhuǎn)OVA12表達(dá)降低所導(dǎo)致的細(xì)胞增殖能力降低、G1/S期阻滯等生物學(xué)行為,體內(nèi)裸鼠成瘤實驗與體外實驗結(jié)果一致,證實OVA12發(fā)揮其生物學(xué)功能依賴于p53信號通路;OVA12能夠影響腫瘤抑癌基因p14ARF的表達(dá),促進(jìn)MDM2從細(xì)胞核到細(xì)胞漿的輸出,增強MDM2和p53的相互作用,加速p53蛋白的降解。上述一系列結(jié)果表明,OVA12蛋白是一種在腫瘤組織中呈特征性高表達(dá)的CT抗原,具有促進(jìn)腫瘤細(xì)胞增殖和抑制細(xì)胞凋亡的作用;提示OVA12通過負(fù)向調(diào)控p53信號通路作用機制發(fā)揮其生物學(xué)功能。
[Abstract]:Tumor antigen OVA12 is a new CT (cancer/testis) antigen selected from cDNA expression library of human ovarian cancer by SEREX (Serological analysis of recombinant cDNA expression library) method in our laboratory. Previous studies have shown that OVA12 antigen is highly expressed in various tumor tissues and tumor cell lines, but not expressed or low in normal tissues (except testis) and normal cell lines. After transfection of OVA12 gene into SMMC-7721 cells, cell proliferation rate is accelerated. The tumorigenic ability of nude mice was enhanced. The results show that OVA12 is closely related to the occurrence and development of tumor, so it has important theoretical significance and clinical application value to further study the biological function and mechanism of OVA12. In this study, we studied the following three aspects: (1) detect the expression of OVA12 in various normal and tumor cells and tissues from the mRNA and protein levels, and clarify the characteristics of OVA12 expression profile; The stable high expression and interfering cell line of OVA12 were constructed by virus infection, and the biological function of OVA12 in tumorigenesis and development was further studied by in vivo and in vitro experiments. The results showed that OVA12 was highly expressed in various tumor tissues and cell lines, but not expressed or low in normal tissues and cells. The results in vitro and in vivo confirmed that OVA12 could significantly promote or inhibit the proliferation and anti-apoptotic ability of tumor cells after high expression or interference. It can increase the drug resistance of tumor cells to 5-FU by upregulating the expression of Mcl-1 and survivin protein. The stable high expression of OVA12 and interfering cell lines were studied. The expression of signal molecules closely related to cell proliferation and apoptosis were detected. The results showed that the decreased expression of OVA12 could significantly increase the expression of p53 protein, while the high expression of OVA12 could inhibit the expression of p53 protein, but did not affect the level of p53 mRNA. Compared with the control group, p53 luciferase gene reporting system showed that the OVA12 interference cell line p53 transcription activity was significantly increased, while the transcription activity of OVA12 high expression cell line decreased significantly. OVA12 could regulate the K48 position polyubiquitin chain form of p53 protein. It can promote the degradation of p53 in proteasome and affect the sensitivity of tumor cells to cisplatin (which is dependent on p53 signaling pathway to induce apoptosis). The results suggest that OVA12 protein can negatively regulate p53 protein 3) by using specific p53 shRNA interference to block p53 signaling pathway, and CCK-8 and other related methods in vitro and in vivo were used to detect the effect of p53 protein expression on the biological function of OVA12. At the same time, the expression changes of several important proteins in the upstream of p53 signaling pathway were detected, and the mechanism of OVA12 regulating p53 signaling pathway was discussed. The results showed that the specific blocking of p53 signaling pathway in OVA12 interfering cell lines could effectively reverse the biological behaviors such as the decrease of cell proliferation induced by the decrease of OVA12 expression and G 1 / S phase arrest, compared with the control group. The results of tumorigenesis in nude mice in vivo and in vitro showed that the biological function of OVA12 depended on the p53 signaling pathway: OVA12 could affect the expression of tumor suppressor gene p14ARF and promote the output of MDM2 from nucleus to cytoplasm. Enhance the interaction between MDM2 and p53, accelerate the degradation of p53 protein. These results suggest that OVA12 protein is a characteristic high expression CT antigen in tumor tissues, which can promote the proliferation of tumor cells and inhibit cell apoptosis. The results suggest that OVA12 exerts its biological function through the mechanism of negative regulation of p53 signaling pathway.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R96

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