过表达MIF促进宫颈癌SiHa细胞发生上皮间质转化的研究

发布时间：2018-09-09 18:09

【摘要】：宫颈癌是全球妇女第二大常见的恶性肿瘤，虽然在过去的30年发生率和死亡率都有所下降，但是近年有年轻化的趋势，严重威胁妇女的健康。目前已经明确持续感染高危型人乳头瘤病毒（HPV）是宫颈癌发生的重要原因，然而并不是所有感染HPV的患者都发展为宫颈癌。因此人们认为除了HPV，还存在其他影响宫颈癌发生的因素。研究发现，巨噬细胞移动抑制因子（MIF）在宫颈癌细胞中高表达，且通过基因敲除、异种移植等实验技术证实了MIF在宫颈癌发生发展中的作用。同时越来越多的研究表明上皮间质转化（EMT）与上皮细胞恶性肿瘤的发生、发展有关，EMT不仅参与了宫颈癌的侵袭，而且与肿瘤的恶性进展有关。此外，MIF过度表达可以使E-钙粘蛋白表达减少、波形蛋白表达增加，此外还可以增加Snail的表达，这些都与EMT的发生一致。本研究以宫颈癌SiHa细胞株为研究对象，通过分析过表达MIF的SiHa细胞中Snail、E-钙粘蛋白、波形蛋白的表达情况，以及细胞的增殖和迁移能力，来探讨过表达MIF对宫颈癌上皮间质转化的影响目的：探讨过表达MIF是否可以促进宫颈癌SiHa细胞发生上皮间质转化，进而促进其增殖和迁移。方法： 1.采用脂质体法将重组质粒转染至SiHa细胞，转染48小时后用荧光显微镜观察转染效率，并用RT-PCR方法进行验证。 2.用RT-PCR方法检测各组细胞（转染重组质粒pEGFP-N1-MIF实验组；转染空质粒pEGFP-N1的阴性对照组；未转染的空白对照组）中Snail、E-钙粘蛋白、波形蛋白mRNA的表达水平。 3.用免疫细胞化学方法检测各组细胞中Snail、E-钙粘蛋白、波形蛋白的蛋白表达水平。 4.用CCK-8法检测各组细胞的增殖能力。 5.用Boyden小室检测各组细胞的迁移能力。 6.采用SPSS17.0统计软件处理，数据以均数标准差表示，不同组别细胞中MIF、E-钙粘蛋白、Snail、波形蛋白的mRNA及蛋白的表达以及细胞迁移能力的变化采用单因素方差分析，，不同时间、不同组别细胞增殖能力的变化采用析因设计方差分析。P＜0.05表示差别有统计学意义。结果： 1.转染6小时后，即可在荧光显微镜下观察到绿色荧光，且随时间增加荧光强度逐渐增强，48小时时最强。48小时转染效率约为（621.9）%。 2.RT-PCR检测结果显示，实验组细胞中MIF mRNA的表达水平高于各对照组（p0.01）。 3.RT-PCR方法测得实验组细胞中Snail、波形蛋白mRNA的表达水平高于各对照组（p0.01），E-钙粘蛋白的mRNA表达水平低于各对照组（P＜0.01）。 4.免疫细胞化学方法测得实验组细胞中Snail、波形蛋白的蛋白表达水平高于各对照组（P0.05），而E-钙粘蛋白的蛋白表达水平低于各对照组（P0.05）。 5.CCK-8法检测结果显示：实验组细胞与各对照组细胞相比，细胞增殖能力明显增强（P0.05），且随转染时间延长，促增殖作用更加明显；但是阴性对照组与空白对照组相比，差别无统计学意义（P＞0.05）。 6.Boyden小室趋化实验结果显示：实验组与各对照组相比，细胞迁移能力明显增强，差异有统计学意义（P0.05）。结论： 1.重组真核表达载体成功转染SiHa细胞。 2.过表达MIF促进SiHa细胞中Snail、波形蛋白的表达，抑制E-钙粘蛋白的表达。 3.过表达MIF促进SiHa细胞增殖和迁移。综上所述，过表达MIF促进了上皮细胞向间质细胞的转化，并增强了SiHa细胞增殖和迁移能力。本实验可能为研究宫颈癌发生、发展的分子机制提供线索，为早期基因诊断及治疗提供有效标志物。
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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