本文選題：黑色素瘤　切入點：Rap1GAP　出處：《華中科技大學(xué)》2011年博士論文

【摘要】：目的：構(gòu)建高表達Rap1GAP的黑色素瘤細胞系,研究Rap1GAP的高表達對黑色素瘤成瘤的影響及其機制。探討高表達Rap1GAP的黑色素瘤細胞中,Rap1GAP對黑色素瘤細胞的合成、分泌VEGF,以及抑制VEGF作用的機制,并研究VEGF對黑色素瘤細胞行為學(xué)的影響。探討高表達Rap1GAP的黑色素瘤細胞旁分泌VEGF的功能的作用,以及其對黑色素瘤的血管內(nèi)皮細胞的影響。 方法：以pcDNA-Rap1GAP-Flagged質(zhì)粒和pcDNA3.1質(zhì)粒轉(zhuǎn)染黑色素瘤細胞,將上述細胞皮下注射于裸鼠建立腫瘤模型；比較不同Rap1GAP表達水平細胞成瘤生長的差異；通過免疫組化染色比較不同Rap1GAP表達水平下,新生血管的數(shù)量；應(yīng)用Epac、PD98059以及LY294002等工具藥物激活或抑制Rap1及其下游信號分子,用免疫印跡方法測定相關(guān)蛋白表達水平；并在不同條件下,用MTT方法比較VEGF對黑色素瘤細胞生長和增殖的作用及機制；用Transwell和酶譜分析檢測其遷移能力的改變,TUNEL染色檢測細胞凋亡等生物行為學(xué)改變。收集不同Rap1GAP水平的黑色素瘤細胞培養(yǎng)基上清,用ELISA方法檢測培養(yǎng)基上清中的VEGF含量；用此培養(yǎng)基上清交叉培養(yǎng)內(nèi)皮細胞,比較不同Rap1GAP水平的內(nèi)皮細胞對其反應(yīng)的差異：并研究黑色素瘤細胞培養(yǎng)基上清對內(nèi)皮細胞中Rap1GTP水平的影響。并用Western Blot檢測血管形成相關(guān)蛋白的表達水平差異。 結(jié)果：高表達Rap1GAP的黑色素瘤細胞在成瘤瘤體體積,新生血管數(shù)量上較正常表達Rap1GAP的細胞顯著下降；進一步研究表明,在高表達Rap1GAP的黑色素瘤中,HIF-α和VEGF表達顯著下降。細胞免疫印跡結(jié)果與組織檢測結(jié)果相同,體外實驗同樣表明,Rap1GAP可以抑制Rap1GTP的水平,同時降低HIF-α和VEGF的表達。Epac在體外實驗中可以促進VEGF和p-ERK表達,但是這一效果可以被Rap1GAP和PD98059取消；VEGF在體外試驗中可以增加Rap1活性,從而活化ERK和Akt通路,但這種作用同樣為Rap1GAP抑制；進一步研究表明,VEGF可以抑制黑色素瘤中的凋亡蛋白,如Caspase3、Caspase 9的表達,并通過增加Cyclin D1等蛋白的表達而促進細胞增殖,同時提高MMP-9、MMP-2活性；細胞學(xué)實驗結(jié)果與分子生物學(xué)實驗結(jié)果一致,上述作用在高表達Rap1GAP的黑色素瘤細胞中均被抑制。ELISA結(jié)果顯示,高表達Rap1GAP的黑色素瘤細胞培養(yǎng)基上清中,VEGF的含量較pcDNA3.1轉(zhuǎn)染的細胞顯著下降；而用黑色素瘤細胞培養(yǎng)基上清交叉培養(yǎng)后,Rap1GTP的水平較普通培養(yǎng)基培養(yǎng)下顯著升高；同時,此升高作用可以被VEGF的抑制劑取消。 結(jié)論：Rap1GAP對黑色素瘤的發(fā)生發(fā)展具有一定的抑制作用,其作用機制可能通過抑制VEGF和HIF-α表達。VEGF在黑色素瘤的生長過程中具有自分泌作用,其作用機制通過Akt和ERK途徑實現(xiàn)；同時VEGF不斷刺激黑色素瘤分泌而形成正反饋機制,從而使得細胞的增殖失控,最終導(dǎo)致腫瘤形成；ERK磷酸化后可以抑制VEGF的mRNA降解,從而在翻譯水平上增加VEGF表達；Akt通路活化在黑色素瘤的發(fā)生發(fā)展過程中具有核心作用,并通過增加HIF-α表達而增加VEGF mRNA的轉(zhuǎn)錄水平；Rap1GAP可以抑制上述增加VEGF表達的翻譯和轉(zhuǎn)錄作用；同時,Rap1GAP可以抑制VEGF刺激細胞引起的下游效應(yīng)蛋白表達增加,而抑制VEGF的生物學(xué)效應(yīng)。此外,黑色素瘤細胞能夠通過旁分泌途徑,誘導(dǎo)和趨化內(nèi)皮細胞進入黑色素瘤,并誘導(dǎo)血管新生,獲得滿足腫瘤生長所需的營養(yǎng)。其機制可能與VEGF在內(nèi)皮細胞中升高Rap1GTP水平相關(guān)。
[Abstract]:Objective: to construct the expression of melanoma cell line Rap1GAP, Rap1GAP high expression on melanoma tumor. To investigate the effect and mechanism of high expression of Rap1GAP melanoma cells, synthesis of Rap1GAP on melanoma cells to secrete VEGF, and the mechanism of inhibition of VEGF function, and to study the effects of VEGF melanoma cell behavior. To investigate the expression of melanoma cells Rap1GAP paracrine VEGF function, and its effect on melanoma vascular endothelial cells.
Methods: pcDNA-Rap1GAP-Flagged plasmid and pcDNA3.1 plasmid was transfected into melanoma cells, the cells injected subcutaneously in nude mice to establish tumor model; compare the expression level of Rap1GAP cells into different tumor growth; by immunohistochemical staining of different Rap1GAP expression level, the number of new vessels; application of Epac, PD98059 and LY294002 tools such as drugs the activation or inhibition of Rap1 and its downstream signal molecules, to determine the expression levels of proteins by Western blot method; under different conditions, and the mechanism of VEGF on the growth and proliferation of melanoma cells by MTT method; analysis of the migration ability changes with Transwell and enzyme, TUNEL staining was used to detect cell apoptosis and other biological behavior learn to change. Melanoma cells collected from different Rap1GAP levels in supernatant of culture medium supernatant of culture medium, detection of VEGF by ELISA method Content; medium supernatant cultured endothelial cells by the cross culture differences between different Rap1GAP levels of endothelial cells on the reaction and study of melanoma cells effect of medium supernatant on Rap1GTP levels of endothelial cells. The difference of expression level and Western Blot detection of angiogenesis related proteins.
Results: the expression of Rap1GAP in melanoma cells in the tumor volume, the number of neovascularization than the normal expression of Rap1GAP cells decreased significantly; further study showed that high expression in melanoma Rap1GAP, expression of HIF- and VEGF decreased significantly. The results of imprinting and tissue cell immune detection results are the same, in vitro the experiments also show that Rap1GAP can inhibit the level of Rap1GTP, while reducing the expression of.Epac HIF- alpha and VEGF can promote the expression of VEGF and p-ERK in vitro, but this effect can be Rap1GAP and PD98059 cancel; VEGF can increase the activity of Rap1 in vitro, and the activation of ERK and Akt pathway, but this effect is the same inhibition of Rap1GAP; further study showed that VEGF can inhibit the apoptosis of melanoma proteins, such as Caspase3, the expression of Caspase 9, and by increasing the expression of Cyclin D1 protein and promote the fine At the same time, cell proliferation, increased MMP-9 and MMP-2 activity; cytological experimental results are consistent with the experimental results of the molecular biology, the role of the high expression of Rap1GAP melanoma cells were inhibited in.ELISA showed that the high expression of Rap1GAP melanoma cells in culture medium supernatant, VEGF content of pcDNA3.1 transfected cells decreased significantly; and with melanoma cell culture medium supernatant after cultured cross Rap1GTP levels, compared with ordinary medium significantly increased; at the same time, the increased effect may be VEGF inhibitors abolished.
Conclusion: Rap1GAP can inhibit the development of melanoma, and its mechanism may be through inhibition of VEGF and HIF- expression of.VEGF has an autocrine role in the growth process of melanoma, the mechanism is realized through Akt and ERK pathway; VEGF also continued to stimulate the secretion of melanoma and the formation of positive feedback mechanism thus, the uncontrolled proliferation, resulting in tumor formation; the degradation of mRNA phosphorylation of ERK could inhibit VEGF, thus increasing the expression of VEGF at the level of translation; the activation of Akt pathway plays a central role in the occurrence and development of melanoma, and by increasing the HIF- expression and increase the transcription level of VEGF mRNA; Rap1GAP can inhibit the increased expression of VEGF transcription and translation; at the same time, the expression of downstream effector protein Rap1GAP can inhibit VEGF induced stimulation of cells increased, the inhibition of VEGF In addition, the melanoma cells can induce and chemotaxis endothelial cells into melanoma through paracrine pathway, and induce angiogenesis, and obtain the nutrition that can meet the needs of tumor growth. The mechanism may be related to the increase of Rap1GTP level in endothelial cells by VEGF.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R739.5

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