生长抑制因子5抑制肺癌细胞增殖和侵袭作用及其机制的研究

发布时间：2018-04-15 10:36

本文选题：生长抑制因子5 + 肺癌细胞　；参考：《第四军医大学》2015年硕士论文

【摘要】：目的:肺癌已经成为导致人类死亡的主要癌症之一,其发病率和死亡率逐年增高。大多数肺癌患者确诊时已处于癌症晚期,其中50%以上的患者失去手术机会。而现有的治疗方法对大多数肺癌患者疗效甚微,因此亟需新的治疗策略。研究表明,肺癌的发生、发展以及侵袭和转移是个多基因参与、多步骤发生的过程,其中抑癌基因的失活在肿瘤的生长和侵袭过程中发挥重要作用。生长抑制因子(Inhibitor of Growth)属于候选抑癌基因家族,目前发现了五个成员,包括ING1、ING2、ING3、ING4、ING5。研究表明,ING家族成员参与调控细胞生长、细胞凋亡和DNA损伤修复等过程。ING5作为该家族最新成员于2003年被首次报道,结果显示ING5可以与P53相互作用并促进P53转录活化,从而引起结肠癌细胞周期阻滞和凋亡;此外,ING5参与构成两种组蛋白乙酰基转移酶(Histone acetyltransferase,HAT)复合体,并且参与组蛋白修饰和染色质重构,通过与组蛋白结合而作为连接组蛋白和HAT的桥梁分子,提示ING5可能通过调控基因的表达而发挥抑癌作用;课题组前期研究发现ING5可以和细胞周期调控蛋白INCA1相互作用,抑制小鼠胚胎成纤维细胞(MEF细胞)增殖,并促进CD95抗体引起的细胞凋亡,而在INCA1基因敲除的MEF细胞中上述作用则消失,表明ING5抑制细胞生长的作用依赖于和其他蛋白质的相互作用。但是ing5如何发挥抑癌作用及其机制仍不明确,ing5在肺癌发生发展中的作用尚未见报道。肿瘤细胞的恶性生物学行为包括其增殖活性、迁移和侵袭能力,与临床肿瘤的生长和转移密切相关。本实验通过建立ing5过表达和基因敲减的肺癌细胞株,从细胞和在体水平研究ing5对肺癌细胞增殖和侵袭能力的影响及可能的机制。方法:实验一以a549和h1299两株肺癌细胞为研究对象,采用慢病毒介导的方法分别建立a549细胞ing5过表达细胞系(a549-ing5)和h1299细胞ing5基因敲减细胞系(h1299-shing5)。在细胞水平采用细胞增殖和克隆形成实验研究ing5对肺癌细胞增殖能力的影响;采用细胞划痕、迁移和侵袭实验研究ing5对肺癌细胞侵袭能力的影响。实验二以裸鼠为研究对象,采用皮下接种和尾静脉注射a549-control和a549-ing5细胞的方法分别建立裸鼠皮下移植瘤模型和尾静脉注射肺癌细胞裸鼠体内转移模型,研究ing5在体内对肺癌细胞增殖和转移能力的影响。实验三为了探讨ing5是否通过抑制上皮间质转化(epithelial-mesenchymaltransition,emt)发挥抑制肺癌侵袭作用,采用倒置显微镜和扫描电镜观察a549-control和a549-ing5形态学变化;qrt-pcr和westerblot检测ing5对emt标志蛋白分子在mrna和蛋白水平的影响。结果实验一1、成功构建了a549细胞ing5过表达细胞系和h1299细胞ing5基因敲减细胞系:与a549-control比较,细胞系a549-ing5中ing5表达显著增高;与h1299-shcontorl相比,细胞系h1299-shing5中ing5表达显著降低。2、ing5过表达显著抑制了a549细胞的增殖能力和克隆形成能力,而ing5基因敲减则显著提高了h1299细胞的增殖能力和克隆形成能力。3、划痕实验和迁移实验结果表明ING5过表达的A549细胞迁移愈合能力明显低于对照肺癌细胞,侵袭实验结果表明ING5过表达可显著抑制A549细胞侵袭能力;而ING5基因敲减的H1299细胞迁移和侵袭能力显著高于对照肺癌细胞。实验二1、裸鼠皮下荷瘤模型实验结果表明:ING5过表达显著抑制裸鼠皮下成瘤及移植瘤的生长,ING5过表达肺癌细胞肿瘤体积较对照组显著缩小。2、尾静脉注射肺癌细胞裸鼠体内转移模型结果表明:A549-control组100%形成了肺部转移瘤,注射A549-ING5细胞40%形成转移瘤,且肿瘤转移灶数量明显低于对照组。实验三1、形态学研究结果表明:ING5过表达后细胞形态发生明显变化,在倒置显微镜下观察发现上皮样细胞明显增多,并聚集成簇;电镜下观察发现ING5过表达后伪足明显减少甚至消失。2、qRT-PCR和Western Blot结果显示:ING5在mRNA和蛋白水平促进E-cadherin的表达,抑制N-cadherin、Snail和Slug的表达。结论1、ING5可以抑制肺癌细胞的增殖、迁移和侵袭作用。2、ING5可以抑制肺癌细胞在裸鼠体内成瘤及生长和转移能力。3、ING5通过抑制肺癌细胞EMT机制而抑制其侵袭能力。
[Abstract]:Objective: lung cancer has become a major cause of cancer death. The morbidity and mortality increased year by year. Most patients are diagnosed with lung cancer in advanced cancer, of which more than 50% of the patients lost the chance of operation. The current treatment methods for most patients with lung cancer treatment ineffective, so the new strategy needed treatment. The study shows that the lung cancer the occurrence, development, invasion and metastasis is a multistep process involving multiple genes, which occur, inactivation of tumor suppressor genes play an important role in the growth and invasion of tumor. The growth inhibitory factor (Inhibitor of Growth) is a candidate tumor suppressor gene family, five members have been found so far include ING1 ING2, ING3, ING4, ING5. study showed that ING family members are involved in the regulation of cell growth, cell apoptosis and DNA damage repair process of.ING5 as a new member of the family for the first time in 2003 Together, the results show that ING5 can interact with P53 and promote P53 transcription activation, causing the node cycle arrest and apoptosis of colon cancer cells; in addition, ING5 is involved in the composition of the two kinds of histone acetyltransferase (Histone acetyltransferase HAT) complex, and involved in histone modification and chromatin remodeling, and as a bridge connecting the molecular group protein and HAT combined with proteins, suggesting that ING5 may regulate gene expression and play the role of tumor suppressor; previous study found that ING5 and cell cycle regulatory protein INCA1 interaction, inhibition of mouse embryonic fibroblasts (MEF cells) proliferation and promote apoptosis induced by CD95 antibody, and in INCA1 in addition to the role of gene knock in MEF cells disappeared, showed that ING5 inhibits the growth of cells and other dependent protein interactions. But ING5 how to play the role of tumor suppressor and The mechanism is still unclear, the ING5 role in the development of lung cancer has not been reported. The biological behavior of malignant tumor cells including proliferation, migration and invasion, and is closely related to tumor growth and metastasis. The expression of ING5 and gene knockdown cells from lung cancer cell lines, and influence study on the level of ING5 on the proliferation and invasion of lung cancer cells and its possible mechanism. Methods: experiment with A549 and H1299 two cell lines as the research object, A549 cell ING5 expression cell lines were established by lentivirus mediated (a549-ing5) and H1299 cell ING5 gene knockdown cell lines (h1299-shing5). The cell proliferation and clone formation at the cellular level experimental study of effect of ING5 on lung cancer cell proliferation; cell migration and invasion by scratches, experimental study of ING5 on the invasion of lung cancer cells In experiment two. The influence of nude mice as the research object, were established in nude mice and tail vein injection in nude mice lung cancer cell metastasis in vivo model by subcutaneous inoculation and intravenous injection of a549-control and a549-ing5 cells, the in vivo effects of ING5 on proliferation and metastasis of lung cancer cells. In experiment three, in order to investigate whether ING5 through inhibition of epithelial mesenchymal transition (epithelial-mesenchymaltransition, EMT) play a role in inhibiting invasion of lung cancer, observe the a549-control and a549-ing5 morphological changes under inverted microscope and scanning electron microscope; qRT-PCR and westerblot detection of ING5 EMT marks the effect of protein molecules at mRNA and protein levels. The results of Experiment 1, the successful construction of A549 cells overexpressing ING5 and H1299 cell lines cell ING5 gene knockdown cell lines: compared with a549-control, a549-ing5 and ING5 expression in the cell lines increased significantly; and Compared to h1299-shcontorl, h1299-shing5 and ING5 expression in the cell lines significantly decreased.2, overexpression of ING5 significantly inhibited the proliferation and colony formation ability of A549 cells, while ING5 knockdown significantly increased the proliferation and colony formation ability of H1299 cells.3, scratch assay and migration experiment results show that ING5 expression of A549 cell migration and wound healing the ability of lung cancer cells invasion was significantly lower than the control, experimental results show that overexpression of ING5 can inhibit the invasion ability of A549 cells; ING5 gene on H1299 cell migration and invasion by lung cancer cells was significantly higher than that of control. In experiment two, 1, the experimental results showed that the tumor bearing nude mice model of overexpression of ING5 significantly inhibited the tumor formation and transplantation the growth of tumor, overexpression of ING5 lung cancer cell volume reduced significantly.2, intravenous injection of nude mice lung cancer cell metastasis in vivo model results Show: A549-control group of 100% forms of lung metastases, injection of A549-ING5 cells 40% formation of metastasis, and tumor metastasis number was significantly lower than the control group. The experimental results show that three 1 Morphological Studies: overexpression of ING5 in cell morphology changed significantly, observed epithelioid cells were increased under the microscope, and clustered; the over expression of ING5 after pseudopodia was decreased or even disappeared..2 were observed under electron microscope, qRT-PCR and Western Blot showed that ING5 expression in mRNA and protein levels, promote E-cadherin inhibition of N-cadherin, expression of Snail and Slug. Conclusion 1, ING5 can inhibit the proliferation of lung cancer cells, migration and invasion of.2, ING5 can inhibit lung cancer cell in vivo tumor growth and metastasis and inhibit the invasion ability of.3 and ING5 through the inhibition mechanism of EMT lung cancer cells.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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