本文選題：GRIM-19　切入點：鱗狀細(xì)胞癌　出處：《吉林大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景:頭頸部鱗狀細(xì)胞癌是一類最常見的皮膚癌癥,在世界范圍,每年約650000新發(fā)病例,每年死亡病例約35000人。頭頸部鱗癌的發(fā)病率隨著年齡的增加而增加。吸煙、飲酒、環(huán)境暴露和HPV感染已被確定為鱗癌發(fā)展的重要危險因素。雖然有手術(shù)、放療和化療等治療辦法的改進(jìn),但是晚期頭頸部鱗癌患者的5年生存率仍然很低。有很多試驗,試圖找出在頭頸部鱗癌的發(fā)生發(fā)展的分子機(jī)制,包括例如TP53突變失活、缺失和代謝產(chǎn)物的改變等。在頭部和頸部腫瘤分子機(jī)制的發(fā)展,將進(jìn)一步闡明,預(yù)計將有助于加速發(fā)展有效的抗癌制劑,和識別診斷或治療的生物標(biāo)志物。干擾素與維甲酸聯(lián)合應(yīng)用誘導(dǎo)細(xì)胞凋亡相關(guān)基因19(GRIM-19),是一種干擾素β/RA誘導(dǎo)基因產(chǎn)物,已被確定為一個的腫瘤抑制潛在基因,與抑制腫瘤生長相關(guān)。在皮膚鱗狀細(xì)胞癌患者腫瘤組織中GRIM-19表達(dá)進(jìn)行下調(diào)。然而,關(guān)于其在皮膚鱗狀細(xì)胞癌中的作用卻不太了解。在這里,重組真核表達(dá)質(zhì)粒攜帶GRIM-19轉(zhuǎn)染頭頸部皮膚鱗狀細(xì)胞癌細(xì)胞(HN5),探討其對皮膚鱗狀細(xì)胞癌細(xì)胞生長的作用,體外侵襲和遷移的幾種方式。結(jié)果發(fā)現(xiàn),上調(diào)GRIM-19可以顯著抑制HN5細(xì)胞的細(xì)胞增殖、克隆形成、遷移和侵襲,以及誘導(dǎo)細(xì)胞凋亡。此外,上調(diào)GRIM-19也能抑制尿激酶型纖溶酶原激活物u-PA,金屬蛋白酶分泌基質(zhì)MMP2、MMP-9、血管內(nèi)皮生長因子VEGF等。我們的研究還表明,GRIM-19表達(dá)下調(diào)將導(dǎo)致信號轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子3(STAT3)的激活�？傊�,過表達(dá)的GRIM-1 9基因可能是控制皮膚鱗狀細(xì)胞癌細(xì)胞生長和侵襲的有效途徑。目的:在本文的研究中,我們的目的是探討在皮膚鱗狀細(xì)胞癌細(xì)胞遷移和侵襲中,通過將過表達(dá)GRIM-19質(zhì)粒轉(zhuǎn)染至細(xì)胞,分析其對細(xì)胞增殖、凋亡的影響。本研究也開發(fā)了一種雙表達(dá)質(zhì)粒共同表達(dá)GRIM?19和P16,評價同時表達(dá)質(zhì)粒轉(zhuǎn)染的兩種基因?qū)︻^頸部皮膚鱗狀細(xì)胞癌細(xì)胞在體外和體內(nèi)的綜合影響。我們的發(fā)現(xiàn)將使皮膚鱗狀細(xì)胞癌的基因治療有所發(fā)展,它是以GRIM-19為目標(biāo)的研究與開發(fā)。材料與方法:應(yīng)用脂質(zhì)體法將p GRIM-19質(zhì)粒及對照質(zhì)粒轉(zhuǎn)染至人頭頸部皮膚鱗狀細(xì)胞癌細(xì)胞HN5內(nèi)。STAT3和GRIM-19的RNA表達(dá)水平用RT-PCR來測定。Western-Blot法檢測相關(guān)蛋白的表達(dá)。細(xì)胞增殖采用MTT細(xì)胞增殖試劑盒評估。驗證細(xì)胞成瘤能力用克隆能力實驗。用流式細(xì)胞術(shù)和凋亡試劑盒檢測細(xì)胞凋亡率的變化。用劃痕試驗檢測GRIM-19細(xì)胞的遷移能力。Transwell侵襲實驗用來檢測細(xì)胞侵襲能力。血管內(nèi)皮生長因子的生成量由酶聯(lián)免疫吸附試驗測量。應(yīng)用脂質(zhì)體法將p GRIM-19-P16共表達(dá)質(zhì)粒及對照質(zhì)粒轉(zhuǎn)染至人頭頸部鱗狀細(xì)胞癌細(xì)胞HN5內(nèi),通過MTT法檢測細(xì)胞增殖抑制情況,流式細(xì)胞術(shù)等觀察細(xì)胞周期與凋亡,以RT-PCR、Western blot法檢測目的基因與相關(guān)基因和蛋白的表達(dá);復(fù)制裸鼠鱗狀細(xì)胞癌皮下移植瘤模型,腹腔注射攜帶共表達(dá)質(zhì)粒p GRIM-19-P16的細(xì)胞,進(jìn)行抗腫瘤作用與機(jī)制的研究。結(jié)果:HN5細(xì)胞在經(jīng)p GRIM 19轉(zhuǎn)染后,GRIM-19表達(dá)增加。GRIM-19表達(dá)上調(diào)抑制STAT3的表達(dá)。GRIM-19表達(dá)上調(diào)抑制HN5細(xì)胞的增殖和克隆能力。GRIM-19表達(dá)上調(diào)促進(jìn)HN5細(xì)胞的凋亡。GRIM-19表達(dá)上調(diào)抑制HN5細(xì)胞的遷移和侵襲能力。GRIM-19表達(dá)上調(diào)抑制HN5細(xì)胞中MMP-9,VEGF,u PA and MMP 2的表達(dá)水平。GRIM?19和P16的共表達(dá)質(zhì)粒(p Grim19?P16)對HN5頭頸部皮膚鱗狀細(xì)胞癌細(xì)胞的增殖,克隆形成、遷移和侵襲效應(yīng),與單獨(dú)轉(zhuǎn)染p Grim?19或p P16效應(yīng)比較有明顯的抑制增強(qiáng)作用。此外,經(jīng)p Grim19?P16基因轉(zhuǎn)染的HN5細(xì)胞誘導(dǎo)的細(xì)胞凋亡相比于p Grim?19或p P16單獨(dú)轉(zhuǎn)染的在G0/G1期細(xì)胞周期阻滯效應(yīng)水平提高。在體內(nèi)實驗中使用一個HN5移植腫瘤模型表明,腹腔注射p Grim19?P16與p Grim?19或p P16單獨(dú)注射相比,有對腫瘤生長的抑制有增強(qiáng)作用。結(jié)論:本研究結(jié)論是:(1)上調(diào)GRIM-19抑制人類頭頸部皮膚鱗狀細(xì)胞癌細(xì)胞的增殖、克隆形成、遷移和侵襲,以及誘導(dǎo)細(xì)胞的凋亡。(2)在HN5細(xì)胞遷移和侵襲過程中GRIM-19過度表達(dá),可以通過STAT3通路,使其介導(dǎo)的MMP-2、MMP-9,VEGF和u-PA表達(dá)降低。(3)在體外和體內(nèi)試驗中,同時表達(dá)GRIM?19和P16轉(zhuǎn)染真核表達(dá)質(zhì)粒能更有效地抑制的頭頸部皮膚鱗狀細(xì)胞癌的增長。
[Abstract]:Background: head and neck squamous cell carcinoma is the most common type of skin cancer in the world, each year about 650000 new cases each year, deaths of about 35000 people. The incidence of head and neck squamous cell carcinoma rate increased with age. Smoking, drinking, environmental exposure and HPV infection has been identified as an important risk of squamous cell carcinoma development the factors. Although there are surgery, radiotherapy and chemotherapy and other treatment improvement measures, but the 5 year survival rate of patients with advanced head and neck squamous cell carcinoma is still very low. There are a lot of experiments, trying to find out the molecular mechanism of the occurrence and development of head and neck squamous cell carcinoma, including cases such as TP53 inactivating mutations, deletion and metabolic changes. In the development of head and neck cancer molecular mechanism, will be further clarified, is expected to help accelerate the development of effective anticancer agents, biological markers and identification of diagnosis or treatment. The combined application of formic acid and interferon induced cell dimension Apoptosis related gene 19 (GRIM-19), is a kind of interferon beta /RA induced gene products have been identified as a potential tumor suppressor gene, and inhibit the growth of tumor. The expression of GRIM-19 in cutaneous squamous cell carcinoma in tumor tissue is reduced. However, with respect to the skin squamous cell carcinoma but the role of don't know. Here, the recombinant eukaryotic expression plasmid carrying the skin of head and neck squamous cell carcinoma cells transfected with GRIM-19 (HN5), to investigate its effect on cell growth of cutaneous squamous cell carcinoma, several ways of migration and invasion in vitro. The results showed that upregulation of GRIM-19 can significantly inhibit HN5 cell proliferation, colony formation, migration and invasion and induce apoptosis. Furthermore, upregulation of GRIM-19 also inhibited the expression of urokinase type plasminogen activator u-PA, matrix metalloproteinase secretion of MMP2, MMP-9, vascular endothelial growth factor VEGF. We The study also showed that the GRIM-19 expression will lead to signal transducer and activator of transcription factor 3 (STAT3) activation. In short, over expression of GRIM-1 9 gene may be an effective way to control skin squamous cell carcinoma cell growth and invasion. Objective: in this study, our aim is to explore the migration and invasion of the skin squamous cell carcinoma cells, the expression of GRIM-19 plasmid transfected into cells by, analysis of cell proliferation and apoptosis. This study also developed a double expression plasmid co expression of GRIM? 19 and P16 at the same time, the evaluation expression plasmid transfected two genes in head and neck squamous cell carcinoma cells in the comprehensive effect in vitro and in vivo. We found that will enable the development of gene therapy for squamous cell carcinoma of the skin, it is the research and development with the aim of GRIM-19. Materials and methods: application of P transfected GRIM-19 plasmid and to According to the poll transfection of squamous cell carcinoma of the skin of the neck in HN5 cells.STAT3 and GRIM-19 expression of RNA RT-PCR was determined by.Western-Blot assay. Cell proliferation related proteins were detected by MTT cell proliferation assay kit. The tumor formation ability evaluation verified by cloning ability experiment. Flow cytometry was used to detect changes in cell apoptosis and kit the rate of apoptosis. Cell invasion assay is used to detect the invasion ability of GRIM-19 cells to detect the scratch test the migration ability of.Transwell. Generation of vascular endothelial growth factor by ELISA measurement. The application of P transfected GRIM-19-P16 expression plasmid and control plasmid was transfected into human head and neck squamous cell carcinoma cell HN5, inhibition by MTT cell proliferation assay, flow cytometry and observe the cell cycle and apoptosis, RT-PCR, Western and blot method to detect gene related Gene and protein expression; replication model of squamous cell carcinoma transplanted subcutaneously in nude mice, co expression plasmid GRIM-19-P16 carrying P cells by intraperitoneal injection of anti tumor effect and mechanism. Results: HN5 cells in the P GRIM 19 after transfection, the expression of GRIM-19 increased the expression of.GRIM-19 up-regulated.GRIM-19 expression inhibited STAT3 expression inhibition of HN5 cells the proliferation and cloning.GRIM-19 expression to induce the apoptosis of.GRIM-19 HN5 cells expression inhibits HN5 cell migration and invasion inhibition of MMP-9.GRIM-19 expression in HN5 cells, VEGF expression level of.GRIM u PA and MMP 2? P16 19 and co expression plasmid (P Grim19? P16 HN5) in head and neck squamous cell carcinoma cell proliferation, colony formation, migration and invasion effect, and transfected with P Grim? 19 or P P16 effect more obvious enhancement inhibition. In addition, the P Grim19 P16 gene transfer? Apoptotic cells stained HN5 cells induced by P compared to Grim? 19 or P transfected with P16 alone increased in cell cycle arrest in G0/G1 phase. The effect of the level of the use of a HN5 transplantation tumor model in vivo showed that intraperitoneal injection of P Grim19? P16 and P Grim or P P16? 19 compared to single injection, there is growth the inhibition of tumor enhancement. Conclusion: the conclusion of this study is: (1) the upregulation of GRIM-19 inhibition in human head and neck squamous cell carcinoma cell proliferation, colony formation, migration and invasion and induce apoptosis. (2) in HN5 cell migration and invasion process of GRIM-19 overexpression can through STAT3 pathway, which is mediated by MMP-2, MMP-9, VEGF and u-PA expression decreased. (3) in vitro and in vivo, and the expression of GRIM? 19 and P16 transfected with eukaryotic expression plasmid of skin of head and neck squamous cell carcinoma can effectively restrain the growth.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.91

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本文編號：1572178