GRIM-19对头颈部皮肤鳞状细胞癌生长和侵袭的影响及机制研究

发布时间：2018-03-05 22:25

本文选题：GRIM-19　切入点：鳞状细胞癌　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：背景:头颈部鳞状细胞癌是一类最常见的皮肤癌症,在世界范围,每年约650000新发病例,每年死亡病例约35000人。头颈部鳞癌的发病率随着年龄的增加而增加。吸烟、饮酒、环境暴露和HPV感染已被确定为鳞癌发展的重要危险因素。虽然有手术、放疗和化疗等治疗办法的改进,但是晚期头颈部鳞癌患者的5年生存率仍然很低。有很多试验,试图找出在头颈部鳞癌的发生发展的分子机制,包括例如TP53突变失活、缺失和代谢产物的改变等。在头部和颈部肿瘤分子机制的发展,将进一步阐明,预计将有助于加速发展有效的抗癌制剂,和识别诊断或治疗的生物标志物。干扰素与维甲酸联合应用诱导细胞凋亡相关基因19(GRIM-19),是一种干扰素β/RA诱导基因产物,已被确定为一个的肿瘤抑制潜在基因,与抑制肿瘤生长相关。在皮肤鳞状细胞癌患者肿瘤组织中GRIM-19表达进行下调。然而,关于其在皮肤鳞状细胞癌中的作用却不太了解。在这里,重组真核表达质粒携带GRIM-19转染头颈部皮肤鳞状细胞癌细胞(HN5),探讨其对皮肤鳞状细胞癌细胞生长的作用,体外侵袭和迁移的几种方式。结果发现,上调GRIM-19可以显著抑制HN5细胞的细胞增殖、克隆形成、迁移和侵袭,以及诱导细胞凋亡。此外,上调GRIM-19也能抑制尿激酶型纤溶酶原激活物u-PA,金属蛋白酶分泌基质MMP2、MMP-9、血管内皮生长因子VEGF等。我们的研究还表明,GRIM-19表达下调将导致信号转导与转录激活因子3(STAT3)的激活。总之,过表达的GRIM-1 9基因可能是控制皮肤鳞状细胞癌细胞生长和侵袭的有效途径。目的:在本文的研究中,我们的目的是探讨在皮肤鳞状细胞癌细胞迁移和侵袭中,通过将过表达GRIM-19质粒转染至细胞,分析其对细胞增殖、凋亡的影响。本研究也开发了一种双表达质粒共同表达GRIM?19和P16,评价同时表达质粒转染的两种基因对头颈部皮肤鳞状细胞癌细胞在体外和体内的综合影响。我们的发现将使皮肤鳞状细胞癌的基因治疗有所发展,它是以GRIM-19为目标的研究与开发。材料与方法:应用脂质体法将p GRIM-19质粒及对照质粒转染至人头颈部皮肤鳞状细胞癌细胞HN5内。STAT3和GRIM-19的RNA表达水平用RT-PCR来测定。Western-Blot法检测相关蛋白的表达。细胞增殖采用MTT细胞增殖试剂盒评估。验证细胞成瘤能力用克隆能力实验。用流式细胞术和凋亡试剂盒检测细胞凋亡率的变化。用划痕试验检测GRIM-19细胞的迁移能力。Transwell侵袭实验用来检测细胞侵袭能力。血管内皮生长因子的生成量由酶联免疫吸附试验测量。应用脂质体法将p GRIM-19-P16共表达质粒及对照质粒转染至人头颈部鳞状细胞癌细胞HN5内,通过MTT法检测细胞增殖抑制情况,流式细胞术等观察细胞周期与凋亡,以RT-PCR、Western blot法检测目的基因与相关基因和蛋白的表达;复制裸鼠鳞状细胞癌皮下移植瘤模型,腹腔注射携带共表达质粒p GRIM-19-P16的细胞,进行抗肿瘤作用与机制的研究。结果:HN5细胞在经p GRIM 19转染后,GRIM-19表达增加。GRIM-19表达上调抑制STAT3的表达。GRIM-19表达上调抑制HN5细胞的增殖和克隆能力。GRIM-19表达上调促进HN5细胞的凋亡。GRIM-19表达上调抑制HN5细胞的迁移和侵袭能力。GRIM-19表达上调抑制HN5细胞中MMP-9,VEGF,u PA and MMP 2的表达水平。GRIM?19和P16的共表达质粒(p Grim19?P16)对HN5头颈部皮肤鳞状细胞癌细胞的增殖,克隆形成、迁移和侵袭效应,与单独转染p Grim?19或p P16效应比较有明显的抑制增强作用。此外,经p Grim19?P16基因转染的HN5细胞诱导的细胞凋亡相比于p Grim?19或p P16单独转染的在G0/G1期细胞周期阻滞效应水平提高。在体内实验中使用一个HN5移植肿瘤模型表明,腹腔注射p Grim19?P16与p Grim?19或p P16单独注射相比,有对肿瘤生长的抑制有增强作用。结论:本研究结论是:(1)上调GRIM-19抑制人类头颈部皮肤鳞状细胞癌细胞的增殖、克隆形成、迁移和侵袭,以及诱导细胞的凋亡。(2)在HN5细胞迁移和侵袭过程中GRIM-19过度表达,可以通过STAT3通路,使其介导的MMP-2、MMP-9,VEGF和u-PA表达降低。(3)在体外和体内试验中,同时表达GRIM?19和P16转染真核表达质粒能更有效地抑制的头颈部皮肤鳞状细胞癌的增长。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.91

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