低氧环境对胶质瘤细胞增殖和侵袭的影响

发布时间：2018-04-30 18:37

本文选题：低氧 + 胶质瘤　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:(1)探讨21%氧气浓度和5%氧气浓度的培养环境对胶质瘤细胞的增殖、自我更新能力和侵袭迁移能力的影响;(2)初步探究常氧及低氧环境下Wnt通路与肿瘤增殖及侵袭的关系。方法:(1)于21%氧气浓度和5%氧气浓度培养箱分别培养胶质瘤细胞,经过1d、2d、3d、4d、5 d、6d、7d后,显微镜下观察并拍照,CCK-8实验测定细胞增殖,细胞成球实验和单克隆实验测定成球能力,Real Time-q PCR实验检测Wnt通路相关m RNA水平及Western Blot实验检测相关蛋白表达水平;(2)于21%氧气浓度和5%氧气浓度培养箱培养培养胶质瘤细胞,培养24小时后,分别以划痕实验测定细胞迁移能力,细胞侵袭实验测定细胞侵袭能力,Real Time-q PCR实验检测EMT相关m RNA水平及Western Blot实验检测EMT相关蛋白表达水平。结果:(1)以CCK-8实验证明低氧环境下胶质瘤细胞增殖活力增强,细胞成球实验及单克隆实验证明胶质瘤细胞在5%的低氧气浓度条件下自我更新能力和21%氧气浓度组增强,PCR实验及Western实验证实Wnt/β-catenin经典通路标志蛋白β-catenin表达上调,而介导该蛋白数量平衡的GSK-3β的表达减少。作为是经典Wnt通路下游靶蛋白c-Myc明显上调。低氧环境能够促进肿瘤细胞增殖;(2)通过细胞划痕实验的观察和对细胞迁移距离的计算,可以确定经低氧环境处理的肿瘤细胞的迁移速率明显高于常氧组。而在细胞侵袭试验中5%低氧气浓度组细胞的穿膜细胞数多于21%浓度组。结合WB实验和PCR实验,低氧环境处理的胶质瘤细胞中EMT(Epithelial-mesenchymal transition,EMT)正性上游调节因子的表达增加,E-cad表达下调而N-cad表达明显增加。因此可以进一步认为低氧微环境通过对肿瘤细胞的EMT过程的启动和维持参与到肿瘤的侵袭和迁移过程。结论:(1)低氧气浓度能够启动Wnt通路并上调有关的下游靶蛋白使胶质瘤的增殖能力,自我更新能力增强。(2)低氧气浓度上调胶质瘤细胞中的Vim、N-cad蛋白上调,下调E-cad等,启动和维持EMT过程,提高胶质瘤细胞侵袭和迁移能力。本课题为国家自然科学基金项目(编号:81271316):平滑肌前体细胞移植介导内皮祖细胞基因甲基化作用早期干预动脉硬化性脑梗塞的分子机制研究。
[Abstract]:Objective to investigate the effects of 21% oxygen concentration and 5% oxygen concentration on the proliferation, self-renewal and invasion of glioma cells. (2) to explore the relationship between Wnt pathway and tumor proliferation and invasion in normoxic and hypoxic environment. Methods glioma cells were cultured in 21% oxygen concentration incubator and 5% oxygen concentration incubator respectively. After 1 d ~ 2 d ~ 3 d ~ 4 ~ 5 d ~ 5 ~ 5 d ~ (-1) ~ 6 d ~ (-1) after 7 days, CCK-8 cell proliferation was observed and photographed under microscope, and CCK-8 assay was used to determine the proliferation of glioma cells. Glioma cells were cultured in 21% oxygen and 5% oxygen concentration incubators by real Time-q PCR assay and Western Blot assay. After cultured for 24 hours, the cell migration ability was measured by scratch assay, the cell invasion ability was measured by real Time-q PCR assay, and the expression of EMT related protein was detected by Western Blot assay and real Time-q PCR assay. Results CCK-8 assay showed that the proliferation activity of glioma cells was increased under hypoxia. Cell sphericity and monoclonal assay showed that glioma cells had the ability of self-renewal at low oxygen concentration of 5%, and the enhancement of the expression of 尾 -catenin in Wnt/ 尾 -catenin classic pathway was confirmed by Western assay and enhanced polymerase chain reaction with 21% oxygen concentration, and the expression of 尾 -catenin in glioma cells was up-regulated. However, the expression of GSK-3 尾 mediated the quantitative balance of the protein decreased. C-Myc, the downstream target protein of classical Wnt pathway, was upregulated significantly. Through the observation of cell scratch test and the calculation of cell migration distance, it can be concluded that the migration rate of tumor cells treated with hypoxic environment is significantly higher than that of normoxic group. In the cell invasion test, the number of transmembrane cells in the 5% low oxygen concentration group was more than that in the 21% concentration group. In combination with WB assay and PCR assay, the expression of positive upstream regulatory factor of EMT(Epithelial-mesenchymal transitionship was increased in cultured glioma cells treated with hypoxia. The expression of E-cad was down-regulated and the expression of N-cad was increased significantly. It is suggested that hypoxia microenvironment is involved in tumor invasion and migration through the initiation and maintenance of the EMT process of tumor cells. Conclusion low oxygen concentration can activate the Wnt pathway and up-regulate the downstream target proteins and enhance the ability of glioma to proliferate and self-renew. 2) low oxygen concentration can up-regulate the Vim-N-cad protein and down-regulate E-cad in glioma cells. To initiate and maintain the EMT process and improve the invasion and migration of glioma cells. This paper is a project of National Natural Science Foundation of China (No.: 81271316): the molecular mechanism of endothelial progenitor cell gene methylation mediated by smooth muscle precursor cell transplantation in early intervention of arteriosclerotic cerebral infarction.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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