内源性FOXP3抑制胶质瘤细胞增殖迁移和侵袭
发布时间：2019-01-24 22:32
【摘要】：背景:转录因子FOXP3(Transcription factor forkhead box P3)是调节性T细胞(Regulatory T cells,Tregs)的特异性因子,在Tregs的发育和功能发挥中起重要作用。最近的研究发现FOXP3也在某些上皮细胞和肿瘤细胞中表达。本课题组前期的研究发现神经胶质瘤细胞中存在FOXP3的表达,然而FOXP3在神经胶质瘤细胞中的确切功能和分子机制仍不清楚。目的:探讨FOXP3对人神经胶质瘤细胞株U87、LN229增殖、凋亡、迁移和侵袭等生物学行为的影响,阐明FOXP3在胶质瘤细胞中的功能作用,进一步加深对胶质瘤发生发展的相关分子及分子基础的理解。方法:用脂质体法将4个含有靶向FOXP3的sh RNA序列(命名为sh RNA1~4)和1个含有sh RNA无意义阴性对照序列(Scrambled)的质粒以及过表达FOXP3的质粒(p CMV6-FOXP3-GFP)和其空载对照质粒(p CMV6-Empty-GFP)转染入人胶质瘤U87细胞和LN229细胞,并在倒置荧光显微镜下观察转染效率;应用Western blot法检测转染后FOXP3蛋白的表达情况,并筛选出最有效的一个sh RNA干扰质粒;应用CCK-8法检测转染后U87细胞和LN229细胞增殖活性的变化;应用流式细胞术(FCM)定量测定转染后细胞的细胞周期和细胞凋亡的变化;应用Western blot法分别检测转染前后细胞凋亡相关蛋白caspases-3和caspases-7的表达情况;转染质粒后细胞的迁移和侵袭情况采用细胞迁移和侵袭实验方法进行检测。利用SPSS17.0软件对实验数据进行统计学分析,P0.05为差异有统计学意义。结果:1、转染后的细胞表达荧光,放在倒置荧光显微镜下观察转染效率为80%,符合转染的实验要求,细胞转染72h后荧光表达效率最高,转染效果最好。2、Western blot结果显示转染干扰质粒后空白对照组(Control)、sh RNA-1、sh RNA-2、sh RNA-3、sh RNA-4组和Scrambled组中FOXP3蛋白的表达受抑制最明显的是sh RNA-1组;转染过表达质粒后细胞FOXP3蛋白表达水平明显上调(P0.05)。3、CCK-8结果显示,下调FOXP3后U87细胞和LN229细胞增殖活性与对照组相比均显著升高;上调FOXP3后细胞增殖活性与对照组相比则显著降低(P0.05)。4、流式细胞术结果显示,下调FOXP3后U87细胞和LN229细胞凋亡率与对照组相比显著降低;上调FOXP3后细胞凋亡率与对照组比较均显著升高(P0.05)。5、Western blot法分别检测转染前后细胞凋亡相关蛋白caspases-3和caspases-7表达情况结果显示,上调FOXP3后细胞凋亡蛋白caspases-3和caspases-7表达明显升高,而下调FOXP3后caspases-3和caspases-7蛋白的表达则明显降低(P0.05)。6、PI染色流式细胞周期检测分析显示,上调FOXP3表达可以诱导U87、LN229细胞G0/G1期比例升高,S期比例降低,细胞周期阻滞在G0/G1期;下调FOXP3细胞周期没有显著变化(P0.05)。7、Transwell小室细胞迁移、侵袭实验结果显示,下调FOXP3后U87细胞和LN229细胞迁移和侵袭能力与对照组相比显著升高;上调FOXP3后细胞迁移和侵袭能力则显著降低(P0.05)。结论:1、转录因子FOXP3在胶质瘤细胞株U87、LN229中具有抑制肿瘤细胞增殖的作用。2、转录因子FOXP3在胶质瘤细胞株U87、LN229中可能通过影响凋亡蛋白caspases-3和caspases-7表达促进肿瘤细胞凋亡。3、上调胶质瘤细胞FOXP3的表达可以诱导U87、LN229细胞发生细胞周期阻滞,使U87、LN229细胞周期阻滞在G0/G1期,可能成为胶质瘤基因靶向治疗的新策略。4、FOXP3抑制U87细胞和LN229细胞迁移和侵袭。上调细胞FOXP3的表达抑制U87细胞和LN229细胞迁移和侵袭能力,下调细胞FOXP3的表达促进U87细胞和LN229细胞迁移和侵袭。
[Abstract]:Background: The transcription factor (FOXP3) is a specific factor for regulatory T cells (Tregs) and plays an important role in the development and function of Tregs. The recent study found that FOXP3 was also expressed in some epithelial and tumor cells. In the previous study, the expression of FOXP3 was found in glioma cells, but the exact function and molecular mechanism of FOXP3 in glioma cells was still unknown. Objective: To study the effects of FOXP3 on the proliferation, apoptosis, migration and invasion of human glioma cell line U87 and LN229, and to clarify the function of FOXP3 in glioma cells and to further enhance the understanding of relevant molecular and molecular basis of the development of glioma. Methods: Four (4) sh RNA sequences containing the target FOXP3 (named sh RNA1 ~ 4) and one plasmid (pCMV6-FOXP3-GFP) containing the non-significant negative control sequence (Scrambled) and its no-load control plasmid (pCMV6-Empty-GFP) were transfected into human glioma U87 and LN229 cells by a liposome method. The transfection efficiency was observed under an inverted fluorescence microscope. The expression of FOXP3 protein was detected by Western blot, and the most effective one of the sh RNA interference plasmids was selected. The changes of the proliferation activity of U87 and LN229 cells after transfection were detected by the CCK-8 method. The changes of cell cycle and apoptosis in transfected cells were determined by flow cytometry (FCM), and the expression of caspase-3 and caspastes-7 were detected by Western blot. The cell migration and invasion were tested by cell migration and invasion. The statistical analysis of the experimental data was carried out by using the SPSS17.0 software, and the difference was statistically significant. Results: 1. The transfected cells express the fluorescence, and the transfection efficiency is 80% under the inverted fluorescence microscope, and the fluorescence expression efficiency is the highest after the transfection of the cells for 72h, and the transfection effect is the best. The results of Western blot showed that the expression of FOXP3 protein in the control group, the sh RNA-1, the sh RNA-2, the sh RNA-3, the sh RNA-4 group and the Scrubb group was significantly higher than that of the sh RNA-1 group (P0.05). After the down-regulation of FOXP3, the proliferation activity of U87 cells and LN229 cells increased significantly compared with the control group, and the proliferation activity of the cells after up-regulation of FOXP3 was significantly lower than that in the control group (P0.05). 4. The flow cytometry showed that the apoptosis rate of U87 and LN229 after the down-regulation of FOXP3 was significantly lower than that of the control group. The expression of caspase-3 and caspastes-7 was significantly increased after up-regulation of the expression of caspase-3 and caspastes-7 after the up-regulation of FOXP3, and the expression of caspase-3 and caspastes-7 was significantly increased after the up-regulation of FOXP3. The expression of caspastes-3 and caspastes-7 after the down-regulation of FOXP3 was significantly lower (P0.05). The results of flow cytometry and flow cytometry showed that up-regulation of the expression of FOXP3 could induce the ratio of G0/ G1 phase of U87 and LN229 cells to increase, the proportion of S phase was decreased, and the cell cycle was blocked in G0/ G1 phase. The decrease of the cell cycle of FOXP3 was not significantly changed (P0.05). The cell migration and invasion of the cell in the Transwell chamber showed that the migration and invasion ability of U87 cells and LN229 cells after the down-regulation of FOXP3 was significantly higher than that in the control group, and the cell migration and invasion ability after up-regulation of FOXP3 was significantly lower (P0.05). Conclusion: 1. The transcription factor FOXP3 has the role of inhibiting the proliferation of tumor cells in the glioma cell lines U87 and LN229. Up-regulation of the expression of FOXP3 in glioma cells can induce cell cycle arrest of U87 and LN229 cells, and the cell cycle of U87 and LN229 is blocked in G0/ G1 phase, which may be a new strategy for the targeted therapy of glioma gene. Up-regulation of the expression of FOXP3 inhibited the migration and invasion of U87 cells and LN229 cells, and down-regulated the expression of FOXP3 to promote the migration and invasion of U87 cells and LN229 cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.41
本文编号：2414927
[Abstract]:Background: The transcription factor (FOXP3) is a specific factor for regulatory T cells (Tregs) and plays an important role in the development and function of Tregs. The recent study found that FOXP3 was also expressed in some epithelial and tumor cells. In the previous study, the expression of FOXP3 was found in glioma cells, but the exact function and molecular mechanism of FOXP3 in glioma cells was still unknown. Objective: To study the effects of FOXP3 on the proliferation, apoptosis, migration and invasion of human glioma cell line U87 and LN229, and to clarify the function of FOXP3 in glioma cells and to further enhance the understanding of relevant molecular and molecular basis of the development of glioma. Methods: Four (4) sh RNA sequences containing the target FOXP3 (named sh RNA1 ~ 4) and one plasmid (pCMV6-FOXP3-GFP) containing the non-significant negative control sequence (Scrambled) and its no-load control plasmid (pCMV6-Empty-GFP) were transfected into human glioma U87 and LN229 cells by a liposome method. The transfection efficiency was observed under an inverted fluorescence microscope. The expression of FOXP3 protein was detected by Western blot, and the most effective one of the sh RNA interference plasmids was selected. The changes of the proliferation activity of U87 and LN229 cells after transfection were detected by the CCK-8 method. The changes of cell cycle and apoptosis in transfected cells were determined by flow cytometry (FCM), and the expression of caspase-3 and caspastes-7 were detected by Western blot. The cell migration and invasion were tested by cell migration and invasion. The statistical analysis of the experimental data was carried out by using the SPSS17.0 software, and the difference was statistically significant. Results: 1. The transfected cells express the fluorescence, and the transfection efficiency is 80% under the inverted fluorescence microscope, and the fluorescence expression efficiency is the highest after the transfection of the cells for 72h, and the transfection effect is the best. The results of Western blot showed that the expression of FOXP3 protein in the control group, the sh RNA-1, the sh RNA-2, the sh RNA-3, the sh RNA-4 group and the Scrubb group was significantly higher than that of the sh RNA-1 group (P0.05). After the down-regulation of FOXP3, the proliferation activity of U87 cells and LN229 cells increased significantly compared with the control group, and the proliferation activity of the cells after up-regulation of FOXP3 was significantly lower than that in the control group (P0.05). 4. The flow cytometry showed that the apoptosis rate of U87 and LN229 after the down-regulation of FOXP3 was significantly lower than that of the control group. The expression of caspase-3 and caspastes-7 was significantly increased after up-regulation of the expression of caspase-3 and caspastes-7 after the up-regulation of FOXP3, and the expression of caspase-3 and caspastes-7 was significantly increased after the up-regulation of FOXP3. The expression of caspastes-3 and caspastes-7 after the down-regulation of FOXP3 was significantly lower (P0.05). The results of flow cytometry and flow cytometry showed that up-regulation of the expression of FOXP3 could induce the ratio of G0/ G1 phase of U87 and LN229 cells to increase, the proportion of S phase was decreased, and the cell cycle was blocked in G0/ G1 phase. The decrease of the cell cycle of FOXP3 was not significantly changed (P0.05). The cell migration and invasion of the cell in the Transwell chamber showed that the migration and invasion ability of U87 cells and LN229 cells after the down-regulation of FOXP3 was significantly higher than that in the control group, and the cell migration and invasion ability after up-regulation of FOXP3 was significantly lower (P0.05). Conclusion: 1. The transcription factor FOXP3 has the role of inhibiting the proliferation of tumor cells in the glioma cell lines U87 and LN229. Up-regulation of the expression of FOXP3 in glioma cells can induce cell cycle arrest of U87 and LN229 cells, and the cell cycle of U87 and LN229 is blocked in G0/ G1 phase, which may be a new strategy for the targeted therapy of glioma gene. Up-regulation of the expression of FOXP3 inhibited the migration and invasion of U87 cells and LN229 cells, and down-regulated the expression of FOXP3 to promote the migration and invasion of U87 cells and LN229 cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.41
【参考文献】
相关期刊论文 前1条
1 王之敏;陶承;;恶性脑胶质瘤分子靶向治疗研究进展[J];中国肿瘤;2006年03期
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