Rap1GAP抑制黑色素瘤VEGF自分泌和旁分泌机制研究

发布时间：2018-04-08 17:21

本文选题：黑色素瘤　切入点：Rap1GAP　出处：《华中科技大学》2011年博士论文

【摘要】：目的：构建高表达Rap1GAP的黑色素瘤细胞系,研究Rap1GAP的高表达对黑色素瘤成瘤的影响及其机制。探讨高表达Rap1GAP的黑色素瘤细胞中,Rap1GAP对黑色素瘤细胞的合成、分泌VEGF,以及抑制VEGF作用的机制,并研究VEGF对黑色素瘤细胞行为学的影响。探讨高表达Rap1GAP的黑色素瘤细胞旁分泌VEGF的功能的作用,以及其对黑色素瘤的血管内皮细胞的影响。方法：以pcDNA-Rap1GAP-Flagged质粒和pcDNA3.1质粒转染黑色素瘤细胞,将上述细胞皮下注射于裸鼠建立肿瘤模型；比较不同Rap1GAP表达水平细胞成瘤生长的差异；通过免疫组化染色比较不同Rap1GAP表达水平下,新生血管的数量；应用Epac、PD98059以及LY294002等工具药物激活或抑制Rap1及其下游信号分子,用免疫印迹方法测定相关蛋白表达水平；并在不同条件下,用MTT方法比较VEGF对黑色素瘤细胞生长和增殖的作用及机制；用Transwell和酶谱分析检测其迁移能力的改变,TUNEL染色检测细胞凋亡等生物行为学改变。收集不同Rap1GAP水平的黑色素瘤细胞培养基上清,用ELISA方法检测培养基上清中的VEGF含量；用此培养基上清交叉培养内皮细胞,比较不同Rap1GAP水平的内皮细胞对其反应的差异：并研究黑色素瘤细胞培养基上清对内皮细胞中Rap1GTP水平的影响。并用Western Blot检测血管形成相关蛋白的表达水平差异。结果：高表达Rap1GAP的黑色素瘤细胞在成瘤瘤体体积,新生血管数量上较正常表达Rap1GAP的细胞显著下降；进一步研究表明,在高表达Rap1GAP的黑色素瘤中,HIF-α和VEGF表达显著下降。细胞免疫印迹结果与组织检测结果相同,体外实验同样表明,Rap1GAP可以抑制Rap1GTP的水平,同时降低HIF-α和VEGF的表达。Epac在体外实验中可以促进VEGF和p-ERK表达,但是这一效果可以被Rap1GAP和PD98059取消；VEGF在体外试验中可以增加Rap1活性,从而活化ERK和Akt通路,但这种作用同样为Rap1GAP抑制；进一步研究表明,VEGF可以抑制黑色素瘤中的凋亡蛋白,如Caspase3、Caspase 9的表达,并通过增加Cyclin D1等蛋白的表达而促进细胞增殖,同时提高MMP-9、MMP-2活性；细胞学实验结果与分子生物学实验结果一致,上述作用在高表达Rap1GAP的黑色素瘤细胞中均被抑制。ELISA结果显示,高表达Rap1GAP的黑色素瘤细胞培养基上清中,VEGF的含量较pcDNA3.1转染的细胞显著下降；而用黑色素瘤细胞培养基上清交叉培养后,Rap1GTP的水平较普通培养基培养下显著升高；同时,此升高作用可以被VEGF的抑制剂取消。结论：Rap1GAP对黑色素瘤的发生发展具有一定的抑制作用,其作用机制可能通过抑制VEGF和HIF-α表达。VEGF在黑色素瘤的生长过程中具有自分泌作用,其作用机制通过Akt和ERK途径实现；同时VEGF不断刺激黑色素瘤分泌而形成正反馈机制,从而使得细胞的增殖失控,最终导致肿瘤形成；ERK磷酸化后可以抑制VEGF的mRNA降解,从而在翻译水平上增加VEGF表达；Akt通路活化在黑色素瘤的发生发展过程中具有核心作用,并通过增加HIF-α表达而增加VEGF mRNA的转录水平；Rap1GAP可以抑制上述增加VEGF表达的翻译和转录作用；同时,Rap1GAP可以抑制VEGF刺激细胞引起的下游效应蛋白表达增加,而抑制VEGF的生物学效应。此外,黑色素瘤细胞能够通过旁分泌途径,诱导和趋化内皮细胞进入黑色素瘤,并诱导血管新生,获得满足肿瘤生长所需的营养。其机制可能与VEGF在内皮细胞中升高Rap1GTP水平相关。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5

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