本文選題：糖基化 + E-cadherin　； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：目的： 研究表明腫瘤細(xì)胞中E-cadherin胞外域發(fā)生高度異常N-糖基化,抑制腫瘤細(xì)胞之間形成成熟的黏附連接,使腫瘤細(xì)胞的增殖侵襲能力增強(qiáng)。本課題通過基因轉(zhuǎn)染提高腫瘤細(xì)胞連接的穩(wěn)定性來抑制腫瘤細(xì)胞的增殖和侵襲能力,其可能會(huì)成為腫瘤基因治療的新思路。本課題的實(shí)驗(yàn)?zāi)康氖峭ㄟ^低糖基化的E-cadherin質(zhì)粒轉(zhuǎn)染Ca127舌癌細(xì)胞研究轉(zhuǎn)染質(zhì)粒對(duì)腫瘤細(xì)胞的增殖和侵襲的影響及其發(fā)生機(jī)制。 方法： 1.用編碼有Flag標(biāo)簽的低糖基化E-cadherin(V13)和野生型E-cadherin (E-cad)的質(zhì)粒分別轉(zhuǎn)染Ca127細(xì)胞。通過G418篩選獲得轉(zhuǎn)染成功的細(xì)胞,使用Western blot檢測(cè)外源性E-cadherin的表達(dá)。 2.通過CCK-8實(shí)驗(yàn)獲得各組細(xì)胞生長(zhǎng)曲線,檢測(cè)不同組細(xì)胞的增殖能力。 3.通過單層細(xì)胞劃痕愈合實(shí)驗(yàn)觀察各組細(xì)胞遷移能力。 4.通過細(xì)胞體外侵襲實(shí)驗(yàn)檢測(cè)各組細(xì)胞的侵襲能力。5.使用免疫沉淀法檢測(cè)E-cadherins介導(dǎo)的AJs (adherens junctions)復(fù)合體中的α-catenin、β-catenin、γ-catenin和vinculin的含量。 6.通過細(xì)胞免疫熒光實(shí)驗(yàn)檢測(cè)各組細(xì)胞中E-cadherin的表達(dá)情況。 結(jié)果： 1.Western blot結(jié)果顯示：外源性編碼E-cadherin的基因都有顯著表達(dá),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結(jié)果顯示：72h后,相對(duì)于對(duì)照組細(xì)胞和野生型E-cadherin轉(zhuǎn)染組細(xì)胞,低糖基化E-cadherin轉(zhuǎn)染組細(xì)胞OD值明顯降低,差異具有顯著性。 3.單層細(xì)胞劃痕愈合實(shí)驗(yàn)結(jié)果顯示：與野生型轉(zhuǎn)染組和未轉(zhuǎn)染組細(xì)胞相比,低糖基化組細(xì)胞的劃痕關(guān)閉速度明顯減慢,差異顯著。 4.體外細(xì)胞侵襲實(shí)驗(yàn)結(jié)果表明：低糖基化組細(xì)胞侵襲能力比野生型轉(zhuǎn)染細(xì)胞小,野生型轉(zhuǎn)染組細(xì)胞比未轉(zhuǎn)染組侵襲能力降低,差異顯著。 5.免疫沉淀實(shí)驗(yàn)結(jié)果顯示：與野生型轉(zhuǎn)染組細(xì)胞相比,低糖基化組細(xì)胞的胞內(nèi)域有更多的α-catenin、β-catenin、γ-catenin和vinculin與其相連接,黏附連接更穩(wěn)定,兩者之間的差異具有顯著性。 6.細(xì)胞免疫熒光結(jié)果顯示：對(duì)照組中E-cadherin彌散性表達(dá)于細(xì)胞質(zhì)中,野生型組細(xì)胞E-cadherin在細(xì)胞膜有部分集中性表達(dá),但是表達(dá)不連續(xù),低糖基化組細(xì)胞中E-cadherin在細(xì)胞膜上的表達(dá)是集中并連續(xù)的。 結(jié)論： 低糖基化E-cadherin(V13)基因轉(zhuǎn)染比野生型E-cadherin(E-cad)基因轉(zhuǎn)染Ca127舌癌細(xì)胞更能顯著抑制舌癌細(xì)胞體外的增殖和侵襲能力。其發(fā)生機(jī)理是可能是因?yàn)閂13能形成更加穩(wěn)定的細(xì)胞黏附連接,從而抑制了腫瘤細(xì)胞的增殖和侵襲。
[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 偽 -catenin, 尾 -catenin, 緯 -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 偽 -catenin, 尾 -catenin, 緯 -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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.86

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 鄭家偉;李金忠;鐘來平;張志愿;;口腔鱗狀細(xì)胞癌臨床流行病學(xué)研究現(xiàn)狀[J];中國(guó)口腔頜面外科雜志;2007年02期

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本文編號(hào)：1963984