發(fā)布時(shí)間：2018-09-09 18:09

【摘要】：宮頸癌是全球婦女第二大常見(jiàn)的惡性腫瘤，雖然在過(guò)去的30年發(fā)生率和死亡率都有所下降，但是近年有年輕化的趨勢(shì)，嚴(yán)重威脅婦女的健康。目前已經(jīng)明確持續(xù)感染高危型人乳頭瘤病毒（HPV）是宮頸癌發(fā)生的重要原因，然而并不是所有感染HPV的患者都發(fā)展為宮頸癌。因此人們認(rèn)為除了HPV，還存在其他影響宮頸癌發(fā)生的因素。 研究發(fā)現(xiàn)，巨噬細(xì)胞移動(dòng)抑制因子（MIF）在宮頸癌細(xì)胞中高表達(dá)，且通過(guò)基因敲除、異種移植等實(shí)驗(yàn)技術(shù)證實(shí)了MIF在宮頸癌發(fā)生發(fā)展中的作用。同時(shí)越來(lái)越多的研究表明上皮間質(zhì)轉(zhuǎn)化（EMT）與上皮細(xì)胞惡性腫瘤的發(fā)生、發(fā)展有關(guān)，EMT不僅參與了宮頸癌的侵襲，而且與腫瘤的惡性進(jìn)展有關(guān)。此外，MIF過(guò)度表達(dá)可以使E-鈣粘蛋白表達(dá)減少、波形蛋白表達(dá)增加，此外還可以增加Snail的表達(dá)，這些都與EMT的發(fā)生一致。 本研究以宮頸癌SiHa細(xì)胞株為研究對(duì)象，通過(guò)分析過(guò)表達(dá)MIF的SiHa細(xì)胞中Snail、E-鈣粘蛋白、波形蛋白的表達(dá)情況，以及細(xì)胞的增殖和遷移能力，來(lái)探討過(guò)表達(dá)MIF對(duì)宮頸癌上皮間質(zhì)轉(zhuǎn)化的影響目的：探討過(guò)表達(dá)MIF是否可以促進(jìn)宮頸癌SiHa細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化，進(jìn)而促進(jìn)其增殖和遷移。方法： 1.采用脂質(zhì)體法將重組質(zhì)粒轉(zhuǎn)染至SiHa細(xì)胞，轉(zhuǎn)染48小時(shí)后用熒光顯微鏡觀察轉(zhuǎn)染效率，并用RT-PCR方法進(jìn)行驗(yàn)證。 2.用RT-PCR方法檢測(cè)各組細(xì)胞（轉(zhuǎn)染重組質(zhì)粒pEGFP-N1-MIF實(shí)驗(yàn)組；轉(zhuǎn)染空質(zhì)粒pEGFP-N1的陰性對(duì)照組；未轉(zhuǎn)染的空白對(duì)照組）中Snail、E-鈣粘蛋白、波形蛋白mRNA的表達(dá)水平。 3.用免疫細(xì)胞化學(xué)方法檢測(cè)各組細(xì)胞中Snail、E-鈣粘蛋白、波形蛋白的蛋白表達(dá)水平。 4.用CCK-8法檢測(cè)各組細(xì)胞的增殖能力。 5.用Boyden小室檢測(cè)各組細(xì)胞的遷移能力。 6.采用SPSS17.0統(tǒng)計(jì)軟件處理，數(shù)據(jù)以均數(shù)標(biāo)準(zhǔn)差表示，不同組別細(xì)胞中MIF、E-鈣粘蛋白、Snail、波形蛋白的mRNA及蛋白的表達(dá)以及細(xì)胞遷移能力的變化采用單因素方差分析，，不同時(shí)間、不同組別細(xì)胞增殖能力的變化采用析因設(shè)計(jì)方差分析。P＜0.05表示差別有統(tǒng)計(jì)學(xué)意義。結(jié)果： 1.轉(zhuǎn)染6小時(shí)后，即可在熒光顯微鏡下觀察到綠色熒光，且隨時(shí)間增加熒光強(qiáng)度逐漸增強(qiáng)，48小時(shí)時(shí)最強(qiáng)。48小時(shí)轉(zhuǎn)染效率約為（621.9）%。 2.RT-PCR檢測(cè)結(jié)果顯示，實(shí)驗(yàn)組細(xì)胞中MIF mRNA的表達(dá)水平高于各對(duì)照組（p0.01）。 3.RT-PCR方法測(cè)得實(shí)驗(yàn)組細(xì)胞中Snail、波形蛋白mRNA的表達(dá)水平高于各對(duì)照組（p0.01），E-鈣粘蛋白的mRNA表達(dá)水平低于各對(duì)照組（P＜0.01）。 4.免疫細(xì)胞化學(xué)方法測(cè)得實(shí)驗(yàn)組細(xì)胞中Snail、波形蛋白的蛋白表達(dá)水平高于各對(duì)照組（P0.05），而E-鈣粘蛋白的蛋白表達(dá)水平低于各對(duì)照組（P0.05）。 5.CCK-8法檢測(cè)結(jié)果顯示：實(shí)驗(yàn)組細(xì)胞與各對(duì)照組細(xì)胞相比，細(xì)胞增殖能力明顯增強(qiáng)（P0.05），且隨轉(zhuǎn)染時(shí)間延長(zhǎng)，促增殖作用更加明顯；但是陰性對(duì)照組與空白對(duì)照組相比，差別無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 6.Boyden小室趨化實(shí)驗(yàn)結(jié)果顯示：實(shí)驗(yàn)組與各對(duì)照組相比，細(xì)胞遷移能力明顯增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。結(jié)論： 1.重組真核表達(dá)載體成功轉(zhuǎn)染SiHa細(xì)胞。 2.過(guò)表達(dá)MIF促進(jìn)SiHa細(xì)胞中Snail、波形蛋白的表達(dá)，抑制E-鈣粘蛋白的表達(dá)。 3.過(guò)表達(dá)MIF促進(jìn)SiHa細(xì)胞增殖和遷移。 綜上所述，過(guò)表達(dá)MIF促進(jìn)了上皮細(xì)胞向間質(zhì)細(xì)胞的轉(zhuǎn)化，并增強(qiáng)了SiHa細(xì)胞增殖和遷移能力。本實(shí)驗(yàn)可能為研究宮頸癌發(fā)生、發(fā)展的分子機(jī)制提供線索，為早期基因診斷及治療提供有效標(biāo)志物。
[Abstract]:Cervical cancer is the second most common malignancy among women in the world. Although the incidence and mortality of cervical cancer have declined in the past 30 years, there has been a trend towards younger age in recent years, which seriously threatens women's health. Patients infected with HPV develop cervical cancer, so it is believed that there are other factors affecting cervical cancer besides HPV.
Studies have shown that macrophage migration inhibitory factor (MIF) is highly expressed in cervical cancer cells, and the role of MIF in the development of cervical cancer has been confirmed by gene knockout and xenotransplantation. In addition, over-expression of MIF can decrease the expression of E-cadherin and increase the expression of vimentin. In addition, it can also increase the expression of Snail, which is consistent with the occurrence of EMT.
In this study, we studied the expression of Snail, E-cadherin, vimentin, and the proliferation and migration of cervical cancer SiHa cells overexpressing MIF. Objective: To investigate the effect of overexpression of MIF on epithelial-mesenchymal transformation of cervical cancer. Cytosolic epithelial mesenchymal transition can further promote its proliferation and migration.
1. The recombinant plasmid was transfected into SiHa cells by liposome method. The transfection efficiency was observed by fluorescence microscope 48 hours after transfection and verified by RT-PCR.
2. The expression levels of Snail, E-cadherin and vimentin mRNA were detected by RT-PCR in the cells of each group.
3. The expression of Snail, E-cadherin and vimentin were detected by immunocytochemistry.
4. the proliferation ability of cells in each group was detected by CCK-8.
5. the cell migration ability of each group was detected by Boyden chamber.
6. SPSS17.0 statistical software was used to process the data. The expression of MIF, E-cadherin, Snail, vimentin mRNA and protein and the change of cell migration ability in different groups were analyzed by one-way ANOVA. The change of cell proliferation ability in different groups at different time was analyzed by factorial design ANOVA. 05, the difference was statistically significant.
1. Green fluorescence was observed under fluorescence microscope 6 hours after transfection, and the fluorescence intensity increased gradually with time. The transfection efficiency was about (621.9)% at 48 hours.
2.RT-PCR test showed that the expression level of MIF mRNA in the experimental group was higher than that in the control group (P0.01).
3. The expression of Snail and vimentin mRNA in the cells of the experimental group was higher than that of the control group (p0.01), and the expression of E-cadherin mRNA was lower than that of the control group (P < 0.01).
4. The expression of Snail and vimentin in the cells of the experimental group was higher than that of the control group (P 0.05), while the expression of E-cadherin was lower than that of the control group (P 0.05).
5. The results of CCK-8 assay showed that compared with the control group, the proliferation ability of the cells in the experimental group increased significantly (P 0.05), and the effect of promoting proliferation was more obvious with the extension of transfection time; but there was no significant difference between the negative control group and the blank control group (P > 0.05).
6. Boyden cell chemotaxis test showed that the migration ability of the experimental group was significantly enhanced compared with the control group, the difference was statistically significant (P 0.05).
1. recombinant eukaryotic expression vector was successfully transfected into SiHa cells.
2. over expression of MIF promotes the expression of Snail and vimentin in SiHa cells, and inhibits the expression of E- cadherin.
3. over expression of MIF promotes proliferation and migration of SiHa cells.
In conclusion, overexpression of MIF promotes the transformation of epithelial cells into mesenchymal cells and enhances the proliferation and migration of SiHa cells.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33

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