本文選題：Sema3E　切入點(diǎn)：胃癌　出處：《上海交通大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:神經(jīng)導(dǎo)向蛋白Semaphorin 3E(Sema3E)在多種腫瘤中表達(dá)上調(diào)并能夠促進(jìn)腫瘤細(xì)胞轉(zhuǎn)移,抑制腫瘤細(xì)胞凋亡,但對(duì)其在胃癌發(fā)生過(guò)程中的研究尚屬空白,且Sema3E在腫瘤中異常表達(dá)的機(jī)制目前還未被闡明。本研究主要探討Sema3E在胃癌生長(zhǎng)、轉(zhuǎn)移和侵襲中的作用及其機(jī)制,并分析表觀遺傳學(xué)改變對(duì)Sema3E轉(zhuǎn)錄調(diào)控的影響。方法:1、采用聚合酶鏈?zhǔn)椒磻?yīng)和免疫組織化學(xué)等技術(shù)方法分析胃癌組織中Sema3E mRNA和蛋白表達(dá)水平與正常組織相比的變化情況。2、使用DAC,TSA,MGCD0103,MC1568,C646等特異性化學(xué)抑制劑,過(guò)表達(dá)和基因敲減組蛋白乙�；竝300,熒光素酶報(bào)告系統(tǒng)等方法研究表觀遺傳學(xué)變化在SEMA3E轉(zhuǎn)錄調(diào)控中的作用機(jī)制。3、構(gòu)建SEMA3E真核表達(dá)載體,研究過(guò)表達(dá)Sema3E對(duì)胃癌細(xì)胞增殖能力,克隆形成能力,裸鼠皮下成瘤能力,遷移能力以及體外重組基底膜侵襲能力的影響,并對(duì)其分子機(jī)制進(jìn)行分析。結(jié)果:1、聚合酶鏈?zhǔn)椒磻?yīng)和免疫組織化學(xué)等實(shí)驗(yàn)結(jié)果顯示Sema3E的mRNA水平和蛋白水平在胃癌樣本中表達(dá)明顯下調(diào)。2、I類(lèi)組蛋白去乙�；�(HDAC)和組蛋白乙酰化酶p300參與SEMA3E在胃癌細(xì)胞中的轉(zhuǎn)錄調(diào)控。3、過(guò)表達(dá)Sema3E能夠有效抑制BGC-823、MGC-803、MKN-45等胃癌細(xì)胞株在體外的細(xì)胞增殖能力和克隆形成能力;抑制MGC-803體外遷移能力和重組基底膜侵襲能力;并在體內(nèi)抑制裸鼠皮下移植瘤的生長(zhǎng)。Sema3E通過(guò)細(xì)胞周期G0/G1期阻滯,促進(jìn)腫瘤細(xì)胞凋亡和降低細(xì)胞信號(hào)通路中ERK、AKT等酶的磷酸化水平,發(fā)揮了對(duì)胃癌細(xì)胞增殖的抑制作用;通過(guò)抑制twist表達(dá)進(jìn)而促進(jìn)E-Cadherin表達(dá)上調(diào),抑制ERK和AKT磷酸化進(jìn)而抑制胃癌細(xì)胞的遷移和重組基底膜侵襲。結(jié)論:本研究表明,胃癌中Sema3E表達(dá)下調(diào)與表觀遺傳學(xué)調(diào)控的失衡密切相關(guān),胃癌中組蛋白去乙酰化酶和組蛋白乙�；副磉_(dá)異常導(dǎo)致Sema3E的表達(dá)下調(diào);Sema3E對(duì)胃癌發(fā)生發(fā)展具有重要作用。綜上所述,在胃癌中低表達(dá)的Sema3E可以作為診斷胃癌的候選標(biāo)志物;同時(shí),其抑癌基因的特點(diǎn)和功能,將為闡明表觀遺傳學(xué)異常與胃癌發(fā)生發(fā)展機(jī)制的關(guān)系奠定重要的理論基礎(chǔ),并為臨床治療提供新的思路。
[Abstract]:Objective: the expression of neurotropin Semaphorin _ 3E _ 2 Sema3E is up-regulated in many kinds of tumors and can promote the metastasis of tumor cells and inhibit the apoptosis of tumor cells, but the study on the expression of neurotropin Semaphorin _ (3E) Sema3E in gastric carcinogenesis is still blank. The mechanism of abnormal expression of Sema3E in gastric cancer has not been elucidated at present. This study mainly studies the role of Sema3E in the growth, metastasis and invasion of gastric cancer and its mechanism. The effects of epigenetic changes on Sema3E transcriptional regulation were analyzed. Methods the expression of Sema3E mRNA and protein in gastric cancer tissues was analyzed by polymerase chain reaction (PCR) and immunohistochemistry. Case .2Specific chemical inhibitors, such as Dacron TSAN MGCD0103, MC1568C646, were used. Overexpression and gene knockdown histone acetylase p300 and luciferase report system were used to study the mechanism of epigenetic changes in SEMA3E transcriptional regulation. The eukaryotic expression vector of SEMA3E was constructed and the ability of overexpression of Sema3E to gastric cancer cell proliferation was studied. The effects of clone forming ability, subcutaneous tumorigenesis ability, migration ability and invasion ability of recombinant basement membrane in nude mice in vitro. The molecular mechanism was analyzed. Results the mRNA and protein levels of Sema3E were significantly down-regulated in gastric cancer samples, including the expression of histone I deacetylase (HDAC) and histone type I deacetylase (HDAC-), and the results of immunohistochemistry and polymerase chain reaction showed that the expression of mRNA and protein in gastric cancer samples was significantly down-regulated. Histone acetylase p300 was involved in the transcriptional regulation of SEMA3E in gastric cancer cells. Overexpression of Sema3E could effectively inhibit the proliferation and clone formation of gastric cancer cell lines such as BGC-823, MGC-803 and MKN-45 in vitro. To inhibit the migration ability of MGC-803 in vitro and the invasion ability of recombinant basement membrane, and to inhibit the growth of subcutaneous xenograft tumor in nude mice in vivo. Sema3E could promote the apoptosis of tumor cells and decrease the phosphorylation level of ERKN AKT and other enzymes in cell signal pathway through G _ 0 / G _ 1 arrest of cell cycle. It can inhibit the proliferation of gastric cancer cells, promote the up-regulation of E-Cadherin expression by inhibiting the expression of twist, inhibit the phosphorylation of ERK and AKT, and thus inhibit the migration of gastric cancer cells and the invasion of recombinant basement membrane. The down-regulation of Sema3E expression in gastric cancer is closely related to the imbalance of epigenetic regulation. Abnormal expression of histone deacetylase and histone acetylase leads to down-regulation of Sema3E expression. The low expression of Sema3E in gastric cancer can be used as a candidate marker for the diagnosis of gastric cancer, and the characteristics and functions of its tumor suppressor gene will lay an important theoretical foundation for elucidating the relationship between epigenetic abnormalities and the mechanism of carcinogenesis and development of gastric cancer. And to provide new ideas for clinical treatment.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.2

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1 陳惠;神經(jīng)導(dǎo)向蛋白Semaphorin 3E在胃癌發(fā)生發(fā)展中的作用和機(jī)制的研究[D];上海交通大學(xué);2015年

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本文編號(hào)：1567378