E3泛素化连接酶CHIP在胃癌发生发展中的作用及机制研究

发布时间：2018-03-19 02:26

本文选题：胃癌　切入点：CHIP　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的本研究试图阐明E3泛素化连接酶CHIP在胃癌细胞株AGS细胞中的作用以及探讨其潜在的生物学机制。方法应用q RT-PCR、Western blot以及IHC等方法检测胃癌组织中CHIP的表达情况。采用慢病毒感染宿主细胞的方法转染MSCV-GFP-h CHIP质粒并建立稳定高表达CHIP的胃癌细胞株AGS-h CHIP。通过x CELLigence系统实时动态观察CHIP高表达后细胞生长、细胞迁移以及细胞侵袭能力的变化。应用流式细胞术检测CHIP高表达后细胞周期的变化。采用Ki-67染色法以及TUNEL法检测CHIP高表达后细胞增殖以及细胞凋亡的变化。通过细胞划痕实验观察CHIP高表达后细胞迁移能力的变化。采用明胶酶谱MMP活性检测方法检测CHIP高表达后细胞内金属蛋白酶活性的变化。应用q RT-PCR及Western blot技术检测相关基因在m RNA水平以及蛋白水平的表达。通过Western blot技术检测CHIP高表达后对细胞内AKT信号通路相关蛋白以及TRAF-NF-κB信号通路相关家族成员蛋白水平的表达。结果胃癌组织中CHIP在m RNA水平以及蛋白质水平的表达均较正常组织呈相对低表达,且CHIP的表达量与胃癌的分化程度及TNM分期有显著统计学差异(P0.05)。在胃癌细胞株AGS中分别稳定转染MSCV-GFP-h CHIP质粒和MSCV-GFP质粒,成功获得实验组稳定高表达CHIP的AGS-h CHIP细胞和对照组AGS-control细胞。AGS-h CHIP细胞的生长速度明显慢于AGS-control细胞,并且迁移、侵袭能力均较AGS-control细胞显著减弱。AGS-h CHIP细胞与AGS-control细胞在细胞周期上无明显差异。AGS-h CHIP细胞的增殖能力较AGS-control细胞显著减弱。AGS-h CHIP细胞的凋亡细胞百分比较AGS-control细胞明显增多。AGS-h CHIP细胞中ITGB1基因在m RNA和蛋白质水平的表达均较AGS-control细胞显著下调。AGS-h CHIP细胞中的MMP-2、MMP-9蛋白质水平的表达均较AGS-control细胞显著下调,同时明胶酶谱MMP活性检测方法检测显示AGS-h CHIP细胞中MMP-2和MMP-9的表达显著低于AGS-control细胞。AGS-h CHIP细胞中Bcl-2基因在m RNA和蛋白质水平的表达均较AGS-control细胞明显下调,其余相关基因Bcl-xl、c IAP1、Bax、A20、Bim、Mcl-1在m RNA水平的表达无明显变化。AGS-h CHIP细胞中p AKT(Ser473)及p AKT(Thr308)的蛋白表达水平较AGS-control细胞呈明显下调。AGS-h CHIP细胞中Rel A、p50、Rel B及TRAF-2的蛋白表达水平较AGS-control细胞显著下调。结论CHIP在胃癌的发生发展中扮演着抑癌基因的角色,可以作为判断胃癌侵袭、转移以及患者预后的参考指标。在胃癌细胞株AGS细胞中高表达CHIP能显著抑制细胞的生长、迁移以及侵袭等恶性生物学行为。高表达CHIP显著抑制了胃癌细胞株AGS细胞的生长,与细胞增殖能力减弱及细胞凋亡增多相关,Bcl-2基因的下调及AKT信号通路活性的抑制可能是其潜在的原因。高表达CHIP显著抑制了AGS胃癌细胞株中TRAF-2-NF-κB信号通路的活性,导致了NF-κB信号通路下游一系列靶基因如ITGB1、MMP-2及MMP-9表达水平的下调,这可能是CHIP抑制胃癌细胞恶性生物学行为的重要机制之一。CHIP有望作为胃癌分子靶向治疗的新靶点,应用CHIP高表达技术可以成为一种潜在的胃癌基因治疗的手段。
[Abstract]:Objective to elucidate the E3 ubiquitin ligase CHIP in gastric cancer cell line AGS cells and investigate its biological mechanisms underlying this study. Methods Q RT-PCR CHIP was used to detect the expression of Western in gastric cancer tissue blot and IHC method. The lentivirus infected host cells transfected with MSCV-GFP-h plasmid CHIP method and the establishment of gastric cancer cells line AGS-h CHIP. CHIP high stable expression of growth by real-time x CELLigence system dynamic observation of the high expression of CHIP cells, changes of cell migration and cell invasion. CHIP detected by flow cytometry after high expression of cell cycle. Ki-67 staining was used to detect CHIP method and TUNEL method of high expression of apoptosis and cell proliferation by cell scratch experimental observation of high CHIP expression changes after cell migration. Gelatin zymography detection method to detect the activity of MMP CHIP High expression of metalloproteinase activity in cells was detected by Q. RT-PCR and Western blot related genes in M RNA mRNA and protein expression detected by CHIP Western. Blot technology of high expression on intracellular AKT signaling pathway related protein and member of the TRAF-NF- B signaling pathway related proteins. Results the expression level of CHIP in gastric cancer at M RNA level and protein expression level were lower than that of the normal tissue showed relatively low expression, differentiation and expression of TNM and gastric cancer and CHIP staging with significant difference (P0.05) in gastric cancer cell line AGS were transfected with MSCV-GFP-h CHIP plasmid and MSCV-GFP plasmid, the experimental group successfully obtained high stability the expression of CHIP AGS-h CHIP cells and AGS-control cells of control group.AGS-h CHIP cell growth was slower than AGS-control cells, and the migration, invasion to Force was significantly lower in the AGS-control cells decreased the percentage of apoptotic AGS-control cells.AGS-h cells CHIP cells and AGS-control cells in the cell cycle did not differ significantly between the.AGS-h CHIP cell proliferation ability of AGS-control cells was significantly decreased compared with.AGS-h CHIP cells significantly increased the expression of ITGB1.AGS-h gene in CHIP cells in M RNA and protein levels were significantly lower than that of AGS-control cells.AGS-h CHIP MMP-2 in the cells and MMP-9 protein expression levels were significantly lower than that of AGS-control cells, and zymography method for detection of MMP activity detection showed that expression of MMP-2 and MMP-9 AGS-h in CHIP cells was significantly lower than that of Bcl-2 AGS-control cell.AGS-h gene in CHIP cells in m RNA and protein levels were significantly lower than that of AGS-control cells, the C IAP1, gene Bcl-xl, Bax, A20, Bim, Mcl-1 expression in m level of RNA had no obvious change .AGS-h CHIP P AKT cells (Ser473) and P AKT (Thr308) protein expression level was higher than that of AGS-control cells showed a significant down-regulation of.AGS-h CHIP cells in Rel A, P50, Rel B and TRAF-2 protein expression was significantly down regulated in AGS-control cells. Conclusion CHIP in gastric cancer suppressor gene plays a role in the development of that can be used to judge the invasion, metastasis and prognosis of patients with the reference index. The high expression of CHIP can inhibit cell growth in gastric cancer cell line AGS cells, the malignant biological behavior of migration and invasion. The high expression of CHIP significantly inhibited the growth of gastric cancer cell line AGS cells, associated with cell proliferation and apoptosis increased weakened the down-regulation of AKT signaling pathway, and the activity of Bcl-2 gene may be the underlying cause. The high expression of CHIP significantly inhibited the TRAF-2-NF- activity of the B signaling pathway in gastric cancer cell lines AGS in the guide The NF- B signaling pathway downstream of a series of target genes such as ITGB1, MMP-2 and MMP-9 expression, which may be an important mechanism of CHIP inhibits the malignant biological behavior of gastric cancer cells.CHIP gastric cancer is expected as a new target for molecular therapy, high expression of CHIP can be used as a potential gene therapy of gastric cancer means.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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