【摘要】：背景:轉(zhuǎn)錄因子FOXP3(Transcription factor forkhead box P3)是調(diào)節(jié)性T細(xì)胞(Regulatory T cells,Tregs)的特異性因子,在Tregs的發(fā)育和功能發(fā)揮中起重要作用。最近的研究發(fā)現(xiàn)FOXP3也在某些上皮細(xì)胞和腫瘤細(xì)胞中表達(dá)。本課題組前期的研究發(fā)現(xiàn)神經(jīng)膠質(zhì)瘤細(xì)胞中存在FOXP3的表達(dá),然而FOXP3在神經(jīng)膠質(zhì)瘤細(xì)胞中的確切功能和分子機(jī)制仍不清楚。目的:探討FOXP3對(duì)人神經(jīng)膠質(zhì)瘤細(xì)胞株U87、LN229增殖、凋亡、遷移和侵襲等生物學(xué)行為的影響,闡明FOXP3在膠質(zhì)瘤細(xì)胞中的功能作用,進(jìn)一步加深對(duì)膠質(zhì)瘤發(fā)生發(fā)展的相關(guān)分子及分子基礎(chǔ)的理解。方法:用脂質(zhì)體法將4個(gè)含有靶向FOXP3的sh RNA序列(命名為sh RNA1~4)和1個(gè)含有sh RNA無(wú)意義陰性對(duì)照序列(Scrambled)的質(zhì)粒以及過(guò)表達(dá)FOXP3的質(zhì)粒(p CMV6-FOXP3-GFP)和其空載對(duì)照質(zhì)粒(p CMV6-Empty-GFP)轉(zhuǎn)染入人膠質(zhì)瘤U87細(xì)胞和LN229細(xì)胞,并在倒置熒光顯微鏡下觀察轉(zhuǎn)染效率;應(yīng)用Western blot法檢測(cè)轉(zhuǎn)染后FOXP3蛋白的表達(dá)情況,并篩選出最有效的一個(gè)sh RNA干擾質(zhì)粒;應(yīng)用CCK-8法檢測(cè)轉(zhuǎn)染后U87細(xì)胞和LN229細(xì)胞增殖活性的變化;應(yīng)用流式細(xì)胞術(shù)(FCM)定量測(cè)定轉(zhuǎn)染后細(xì)胞的細(xì)胞周期和細(xì)胞凋亡的變化;應(yīng)用Western blot法分別檢測(cè)轉(zhuǎn)染前后細(xì)胞凋亡相關(guān)蛋白caspases-3和caspases-7的表達(dá)情況;轉(zhuǎn)染質(zhì)粒后細(xì)胞的遷移和侵襲情況采用細(xì)胞遷移和侵襲實(shí)驗(yàn)方法進(jìn)行檢測(cè)。利用SPSS17.0軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1、轉(zhuǎn)染后的細(xì)胞表達(dá)熒光,放在倒置熒光顯微鏡下觀察轉(zhuǎn)染效率為80%,符合轉(zhuǎn)染的實(shí)驗(yàn)要求,細(xì)胞轉(zhuǎn)染72h后熒光表達(dá)效率最高,轉(zhuǎn)染效果最好。2、Western blot結(jié)果顯示轉(zhuǎn)染干擾質(zhì)粒后空白對(duì)照組(Control)、sh RNA-1、sh RNA-2、sh RNA-3、sh RNA-4組和Scrambled組中FOXP3蛋白的表達(dá)受抑制最明顯的是sh RNA-1組;轉(zhuǎn)染過(guò)表達(dá)質(zhì)粒后細(xì)胞FOXP3蛋白表達(dá)水平明顯上調(diào)(P0.05)。3、CCK-8結(jié)果顯示,下調(diào)FOXP3后U87細(xì)胞和LN229細(xì)胞增殖活性與對(duì)照組相比均顯著升高;上調(diào)FOXP3后細(xì)胞增殖活性與對(duì)照組相比則顯著降低(P0.05)。4、流式細(xì)胞術(shù)結(jié)果顯示,下調(diào)FOXP3后U87細(xì)胞和LN229細(xì)胞凋亡率與對(duì)照組相比顯著降低;上調(diào)FOXP3后細(xì)胞凋亡率與對(duì)照組比較均顯著升高(P0.05)。5、Western blot法分別檢測(cè)轉(zhuǎn)染前后細(xì)胞凋亡相關(guān)蛋白caspases-3和caspases-7表達(dá)情況結(jié)果顯示,上調(diào)FOXP3后細(xì)胞凋亡蛋白caspases-3和caspases-7表達(dá)明顯升高,而下調(diào)FOXP3后caspases-3和caspases-7蛋白的表達(dá)則明顯降低(P0.05)。6、PI染色流式細(xì)胞周期檢測(cè)分析顯示,上調(diào)FOXP3表達(dá)可以誘導(dǎo)U87、LN229細(xì)胞G0/G1期比例升高,S期比例降低,細(xì)胞周期阻滯在G0/G1期;下調(diào)FOXP3細(xì)胞周期沒(méi)有顯著變化(P0.05)。7、Transwell小室細(xì)胞遷移、侵襲實(shí)驗(yàn)結(jié)果顯示,下調(diào)FOXP3后U87細(xì)胞和LN229細(xì)胞遷移和侵襲能力與對(duì)照組相比顯著升高;上調(diào)FOXP3后細(xì)胞遷移和侵襲能力則顯著降低(P0.05)。結(jié)論:1、轉(zhuǎn)錄因子FOXP3在膠質(zhì)瘤細(xì)胞株U87、LN229中具有抑制腫瘤細(xì)胞增殖的作用。2、轉(zhuǎn)錄因子FOXP3在膠質(zhì)瘤細(xì)胞株U87、LN229中可能通過(guò)影響凋亡蛋白caspases-3和caspases-7表達(dá)促進(jìn)腫瘤細(xì)胞凋亡。3、上調(diào)膠質(zhì)瘤細(xì)胞FOXP3的表達(dá)可以誘導(dǎo)U87、LN229細(xì)胞發(fā)生細(xì)胞周期阻滯,使U87、LN229細(xì)胞周期阻滯在G0/G1期,可能成為膠質(zhì)瘤基因靶向治療的新策略。4、FOXP3抑制U87細(xì)胞和LN229細(xì)胞遷移和侵襲。上調(diào)細(xì)胞FOXP3的表達(dá)抑制U87細(xì)胞和LN229細(xì)胞遷移和侵襲能力,下調(diào)細(xì)胞FOXP3的表達(dá)促進(jìn)U87細(xì)胞和LN229細(xì)胞遷移和侵襲。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王之敏;陶承;;惡性腦膠質(zhì)瘤分子靶向治療研究進(jìn)展[J];中國(guó)腫瘤;2006年03期

，

本文編號(hào)：2414927