低糖基化E-cadherins抑制舌癌细胞增殖和侵袭的机理研究

发布时间：2018-06-01 11:14

本文选题：糖基化 + E-cadherin　；参考：《山东大学》2014年硕士论文

【摘要】：目的：研究表明肿瘤细胞中E-cadherin胞外域发生高度异常N-糖基化,抑制肿瘤细胞之间形成成熟的黏附连接,使肿瘤细胞的增殖侵袭能力增强。本课题通过基因转染提高肿瘤细胞连接的稳定性来抑制肿瘤细胞的增殖和侵袭能力,其可能会成为肿瘤基因治疗的新思路。本课题的实验目的是通过低糖基化的E-cadherin质粒转染Ca127舌癌细胞研究转染质粒对肿瘤细胞的增殖和侵袭的影响及其发生机制。方法： 1.用编码有Flag标签的低糖基化E-cadherin(V13)和野生型E-cadherin (E-cad)的质粒分别转染Ca127细胞。通过G418筛选获得转染成功的细胞,使用Western blot检测外源性E-cadherin的表达。 2.通过CCK-8实验获得各组细胞生长曲线,检测不同组细胞的增殖能力。 3.通过单层细胞划痕愈合实验观察各组细胞迁移能力。 4.通过细胞体外侵袭实验检测各组细胞的侵袭能力。5.使用免疫沉淀法检测E-cadherins介导的AJs (adherens junctions)复合体中的α-catenin、β-catenin、γ-catenin和vinculin的含量。 6.通过细胞免疫荧光实验检测各组细胞中E-cadherin的表达情况。结果： 1.Western blot结果显示：外源性编码E-cadherin的基因都有显著表达,低糖基化E-cadherin的分子量比野生型E-cadherin的分子量要小。 2.CCK-8结果显示：72h后,相对于对照组细胞和野生型E-cadherin转染组细胞,低糖基化E-cadherin转染组细胞OD值明显降低,差异具有显著性。 3.单层细胞划痕愈合实验结果显示：与野生型转染组和未转染组细胞相比,低糖基化组细胞的划痕关闭速度明显减慢,差异显著。 4.体外细胞侵袭实验结果表明：低糖基化组细胞侵袭能力比野生型转染细胞小,野生型转染组细胞比未转染组侵袭能力降低,差异显著。 5.免疫沉淀实验结果显示：与野生型转染组细胞相比,低糖基化组细胞的胞内域有更多的α-catenin、β-catenin、γ-catenin和vinculin与其相连接,黏附连接更稳定,两者之间的差异具有显著性。 6.细胞免疫荧光结果显示：对照组中E-cadherin弥散性表达于细胞质中,野生型组细胞E-cadherin在细胞膜有部分集中性表达,但是表达不连续,低糖基化组细胞中E-cadherin在细胞膜上的表达是集中并连续的。结论：低糖基化E-cadherin(V13)基因转染比野生型E-cadherin(E-cad)基因转染Ca127舌癌细胞更能显著抑制舌癌细胞体外的增殖和侵袭能力。其发生机理是可能是因为V13能形成更加稳定的细胞黏附连接,从而抑制了肿瘤细胞的增殖和侵袭。
[Abstract]:Objective: The study showed that the E-cadherin extracellular domain in tumor cells was highly abnormal N-glycosylation, which inhibited the formation of mature adhesion between tumor cells, and enhanced the proliferation and invasion ability of tumor cells. In this study, gene transfection can improve the stability of tumor cell junctions to inhibit the proliferation and invasion of tumor cells, which may become a new way of tumor gene therapy. The purpose of this study was to investigate the effect of transfection of E-cadherin plasmid with low glycosylation on the proliferation and invasion of Ca127 tongue cancer cells and its pathogenesis. Methods: 1. The plasmids encoding low glycosylated E-cadherin (V13) and wild type E-cadherin (E-cadad) were transfected into Ca127 cells respectively. The transfected cells were screened by G418 and the expression of exogenous E-cadherin was detected by Western blot. 2. The cell growth curve was obtained by CCK-8 test, and the proliferation ability of different groups was measured. 3. The migration ability of each group was observed by scratch healing experiment of monolayer cells. 4. The invasiveness of each group was detected by cell invasion assay in vitro. The levels of 伪 -catenin, 尾 -catenin, 纬 -catenin and vinculin in E-cadherins mediated AJs junctions were detected by immunoprecipitation. 6. The expression of E-cadherin was detected by immunofluorescence assay. Results: 1.Western blot results showed that the genes encoding E-cadherin were significantly expressed, and the molecular weight of low-glycosylated E-cadherin was lower than that of wild-type E-cadherin. The results of 2.CCK-8 showed that compared with the control group and the wild-type E-cadherin transfection group, the OD value of the cells transfected with low-glycosylated E-cadherin was significantly lower than that of the control group and the wild-type E-cadherin transfection group. 3. The results of scratch healing of monolayer cells showed that compared with wild-type and non-transfected cells, the rate of scratch closure in low glycosylation group was significantly slower than that in wild-type transfection group and non-transfected group, and the difference was significant. 4. The results of in vitro cell invasion test showed that the invasion ability of the low glycosylation group was less than that of the wild type transfected cells, and the invasiveness of the wild type transfection group was lower than that of the untransfected group, and the difference was significant. 5. The results of immunoprecipitation showed that there were more 伪 -catenin, 尾 -catenin, 纬 -catenin and vinculin in the cells of low glycosylation group than those in the wild-type transfection group. 6. The results of cellular immunofluorescence showed that E-cadherin was diffusely expressed in the cytoplasm of the control group, while the E-cadherin in the wild-type group was partially concentrated in the cell membrane, but the expression was not continuous. The expression of E-cadherin on cell membrane was concentrated and continuous in low glycosylation group. Conclusion: Low glycosylated E-cadherin (V13) gene transfection could significantly inhibit the proliferation and invasion of Ca127 tongue cancer cells in vitro compared with wild-type E-cadherin E-cadhe gene transfection. The mechanism may be that V13 can form more stable cell adhesion, which inhibits the proliferation and invasion of tumor cells.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.86

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