ADAMTS-18基因沉默和过表达对肝癌细胞系MHCC-97H和HepG2增殖和侵袭能力的影响

发布时间：2018-05-29 07:57

本文选题：原发性肝细胞癌 + HBV　；参考：《广西医科大学》2017年硕士论文

【摘要】：目的:探讨ADAMTS-18基因对肝癌细胞系MHCC-97H、Hep G2体外增殖和侵袭能力的影响。方法:收集2016年经广西医科大学第一附属医院肝胆外科手术治疗和病理诊断为HBV阳性原发性肝细胞癌的癌组织与癌旁组织标本共62对。(1)用Trizol法分别提取癌与癌旁组织总RNA,分别利用q RT-PCR和免疫组化染色技术检验癌组织与癌旁组织中ADAMTS-18的m RNA和蛋白的表达水平差异,并结合患者临床病理特征做相关性分析。(2)提取MHCC-97H、Hep G2和HL-7702细胞系的总RNA,利用q RT-PCR检测细胞系中ADAMTS-18基因的m RNA表达量。(3)应用慢病毒介导的基因沉默和过表达技术分别构建MHCC-97H、Hep G2、HL-7702细胞系的ADAMTS-18基因沉默和表达稳转细胞株,并通过q RT-PCR和Western blot检测沉默和过表达效率。(4)应用MTT法、细胞划痕实验和Transwell小室体外侵袭实验分别检测ADAMTS-18基因沉默和过表达前后肝癌细胞系的体外增殖和迁移侵袭能力的变化。结果:(1)在HCC癌组织中的ADAMTS-18 m RNA水平和蛋白表达水平均显著高于癌旁组织(P0.05);(2)ADAMTS-18 m RNA在HL-7702、MHCC-97H、Hep G2、SMMC-7721、BEL-7404和QGY-7703细胞系中均有表达。(3)成功构建ADAMTS-18沉默和过表达的稳转细胞株MHCC-97H、Hep G2、HL-7702。(4)细胞划痕实验和Transwell小室侵袭实验结果显示,上调ADAMTS-18的表达后,MHCC-97H、Hep G2的侵袭能力显著提高(P0.05);在下调ADAMTS-18的表达后,MHCC-97H的侵袭能力明显降低(P0.05)。(5)MTT方法检测结果发现,上调或下调ADAMTS-18的表达后,MHCC-97H、Hep G2、HL-7702细胞系的增殖能力随之明显升高和下降(P0.05)。结论:1、ADAMTS-18基因在HBV相关性HCC癌组织中的表达水平显著高于癌旁组织;2、上调ADAMTS-18基因的表达可促进MHCC-97H的体外迁移侵袭能力;下调ADAMTS-18基因的表达能抑制MHCC-97H的体外迁移侵袭能力;3、上调ADAMTS-18基因可促进MHCC-97H、Hep G2和HL-7702细胞系的增殖能力。下调ADAMTS-18基因可抑制上述细胞系的增值能力。
[Abstract]:Aim: to investigate the effect of ADAMTS-18 gene on the proliferation and invasion of hepatocellular carcinoma cell line MHCC-97 Hep G2 in vitro. Methods: a total of 62 pairs of carcinomas and paracancerous tissues were collected from the primary hepatocellular carcinoma (HCC) specimens from the first affiliated Hospital of Guangxi Medical University in 2016 and pathologically diagnosed as HBV positive primary hepatocellular carcinoma (HCC). Cancer and paracancerous tissues were extracted by Trizol method. The expression levels of m RNA and protein of ADAMTS-18 in cancer tissues and adjacent tissues were detected by Q RT-PCR and immunohistochemical staining respectively. The total RNAs of MHCC-97 Hep G2 and HL-7702 cell lines were extracted, and the m RNA expression of ADAMTS-18 gene was detected by Q RT-PCR. The lentivirus mediated gene silencing and overexpression techniques were used, respectively. ADAMTS-18 gene silencing and expression stabilization of MHCC-97Hep G2HL-7702 cell line were constructed. MTT method, cell scratch assay and Transwell chamber invasion assay were used to detect the proliferation, migration and invasion ability of hepatoma cell lines before and after ADAMTS-18 gene silencing and overexpression, respectively. Results the level of ADAMTS-18 m RNA and protein expression in HCC tissues were significantly higher than those in adjacent tissues (P0.05ADAMTS-18m RNA) and in HL-7702MHCC-97Hep SMMC-7721 BEL-7404 and QGY-7703 cell lines.) the stable transformed cell line MHCC-97Hep Hep G2HL-7702.4 was successfully constructed with ADAMTS-18 silencing and overexpression. The results of scar test and Transwell chamber invasion test showed that, After upregulation of ADAMTS-18 expression, the invasion ability of MHCC-97Hep G2 increased significantly, and the invasion ability of MHCC-97H decreased significantly after down-regulation of ADAMTS-18 expression. The results of MTT assay showed that the proliferation ability of Hep G2HL-7702 cell line increased and decreased after up-regulating or down-regulating the expression of MHCC-97Hep G2HL-7702, and the proliferation ability of MHCC-97Hep G2HL-7702 cell line increased and decreased after down-regulating the expression of MHCC-97Hep Hep G2HL-7702. Conclusion the expression of ADAMTS-18 gene in HBV associated HCC tissues is significantly higher than that in adjacent HCC tissues. Upregulating the expression of ADAMTS-18 gene can promote the migration and invasion ability of MHCC-97H in vitro. Down-regulating the expression of ADAMTS-18 gene could inhibit the migration and invasion ability of MHCC-97H in vitro and up-regulate the proliferation of MHCC-97Hep G2 and HL-7702 cell lines. Down-regulation of ADAMTS-18 gene can inhibit the proliferation ability of the above cell lines.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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