【摘要】：目的:1.研究micro RNA-146a對人神經(jīng)膠質(zhì)瘤U251細(xì)胞耐藥株的作用。2.探討Notch1基因與micro RNA-146a在人膠質(zhì)瘤耐藥性中的相關(guān)性。方法:1.通過慢病毒包裝、轉(zhuǎn)染、篩選,分別獲得高表達NICD的U251細(xì)胞株及其空載plvx的U251細(xì)胞株,并通過Western Blotting及免疫熒光染色進行鑒定。2.micro RNA芯片檢測和篩選NICD高表達和空載plvx的U251細(xì)胞及對照U251細(xì)胞中差異性表達的micro RNAs。3.RT-q PCR檢測鑒定NICD高表達和空載plvx的U251細(xì)胞及對照U251細(xì)胞中mi RNA-146a的表達情況。4.懫用替莫唑胺(TMZ)濃度遞增的作用方式誘導(dǎo)U251細(xì)胞,初始誘導(dǎo)劑量0.25μg/ml(0.2875μM),最高誘導(dǎo)劑量為20μg/ml(103.0μM),獲得穩(wěn)定的對TMZ耐藥的U251細(xì)胞,命名為U251TR,用CCK-8法檢測U251,U251TR的細(xì)胞增殖抑制率,計算半數(shù)抑制濃度IC50及耐藥指數(shù)(RF)。5.RT-q PCR檢測U251TR及其對照U251細(xì)胞中mi RNA-146a的表達情況。6.用脂質(zhì)體介導(dǎo)的轉(zhuǎn)染方法將mi R-146a-mimics轉(zhuǎn)入U251TR,稱為轉(zhuǎn)染組細(xì)胞(U251TR-mimics),熒光顯微法檢測轉(zhuǎn)染組轉(zhuǎn)染效率,RT-q PCR檢測U251TR及U251TR-mimics中mi R-146a的表達情況,CCK-8法檢測U251TR-mimics細(xì)胞增殖抑制率,計算IC50及RF。7.Western Blotting檢測U251,U251TR及U251TR-mimics細(xì)胞中Notch1基因的表達情況。 8.通過生物信息學(xué)預(yù)測靶向調(diào)控Notch1的mi RNA-146a,然后通過雙熒光素酶報告基因?qū)嶒灱癢estern blotting實驗對mi R-146a進行靶標(biāo)的驗證。結(jié)果:1.成功建立了高表達NICD和空載plvx的U251細(xì)胞株。2.Micro RNA芯片結(jié)果顯示NICD高表達的U251細(xì)胞中mi RNA-146a與對照組相比明顯降低(P0.05)。3.成功建立了對TMZ耐藥的U251TR,U251TR細(xì)胞IC50(320.68μM)是未經(jīng)誘導(dǎo)U251細(xì)胞IC50(30.54μM)的10.5倍(P0.05),其耐藥指數(shù)(RF)約為11。4.RT-q PCR結(jié)果顯示,mi R-146a在NICD高表達組細(xì)胞及其對照組細(xì)胞中的表達情況與micro RNA芯片結(jié)果一致;RT-q PCR顯示耐藥株U251TR中mi R-146a相對表達量低于U251(P0.05)。5.轉(zhuǎn)染組mi R-146a-mimics的轉(zhuǎn)染效率在90%以上;轉(zhuǎn)染組細(xì)胞U251TR-mimics IC50(160.79μM)是未經(jīng)誘導(dǎo)的U251細(xì)胞IC50(30.54μM)的5.26倍(P0.05),其耐藥指數(shù)(RF)約為5。6.雙熒光素酶報告基因?qū)嶒灪蚖estern blotting實驗均顯示mi R-146a-5p作用后,熒光素酶活性及Notch1下調(diào)明顯(P0.05)。結(jié)論:成功構(gòu)建了對TMZ耐藥的U251TR細(xì)胞,其耐藥指數(shù)(RF)約為10,U251TR細(xì)胞中mi R-146a相對表達量低于U251細(xì)胞;將mi R-146a-mimics轉(zhuǎn)入U251TR細(xì)胞后,其耐藥指數(shù)下降。雙熒光素酶報告基因和Western blotting結(jié)果均顯示mi R-146a靶向調(diào)控Notch1。mi RNA-146a可能通過Notch1信號通路在人膠質(zhì)瘤細(xì)胞U251耐藥形成機制中發(fā)揮一定的作用。
[Abstract]:Objective: 1. To study the effect of micro RNA-146a on human glioma cell line U251. 2. To investigate the relationship between Notch1 gene and micro RNA-146a in human glioma resistance. Methods: 1. U251 cell lines with high expression of NICD and U251 cell lines with no plvx were obtained by packaging, transfection and screening of lentivirus. Western Blotting and immunofluorescence staining were used to identify the high expression of NICD in U251 cells with high NICD expression and non-loaded U251 cells and U251 cells without plvx and control U251 cells. Micro RNAs.3.RT-q PCR detection was used to identify the high expression of NICD in U251 cells. The expression of mi RNA-146a in U251 cells and control U251 cells without plvx. 4. U251 cells were induced by the increasing concentration of temozolidomide (TMZ). The initial induction dose was 0.25 渭 g/ml (0.2875 渭 M),) and the maximum inducing dose was 20 渭 g/ml (103.0 渭 M),) to obtain stable TMZ resistant U251 cells named U251 TR. CCK-8 assay was used to detect the proliferation inhibition rate of U251TR cells. The expression of mi RNA-146a in U251TR and its control U251 cells was detected by calculating the half inhibitory concentration (IC50) and drug resistance index (RF). 5.RT-q PCR). The transfection efficiency of mi R-146a-mimics was detected by fluorescence microscopy, and the expression of mi R-146a in U251TR and U251TR-mimics was detected by RT-q PCR. CCK-8 assay was used to detect the proliferation inhibition rate of U251TR-mimics cells, and the expression of Notch1 gene in U251TR and U251TR-mimics cells was calculated by IC50 and RF.7.Western Blotting. 8. Mi RNA-146a, targeting Notch1 was predicted by bioinformatics. Then the target of mi R-146a was verified by double luciferase reporter gene experiment and Western blotting experiment. The result is 1: 1. U251 cell lines with high expression of NICD and no plvx were successfully established. The results of 2.Micro RNA microarray showed that mi RNA-146a in U251 cells with high NICD expression was significantly lower than that in control group (P0.05). The IC50 (320.68 渭 M) of U251 TR-U251TR cells resistant to TMZ was 10. 5 times as much as that of uninduced U251 cells IC50 (30.54 渭 M) (P0.05). The drug resistance index (RF) of U251 TR- U251TR cells was about the same as that of 11.4.RT-q PCR. The expression of mi R-146a in the cells with high expression of NICD and its control group was consistent with the results of micro RNA microarray. RT-q PCR showed that the relative expression of mi R-146a in U251TR was lower than that in U251 (P0.05). The transfection efficiency of mi R-146a-mimics in transfection group was more than 90%, and the transfection group U251TR-mimics IC50 (160.79 渭 M) was 5.26 times as high as the uninduced U251 cell IC50 (30.54 渭 M) (P0.05), and its drug resistance index (RF) was about 5.6. Double luciferase reporter gene experiment and Western blotting assay showed that luciferase activity and Notch1 decreased significantly after mi R-146a-5p treatment (P0.05). Conclusion: U251TR cells resistant to TMZ were successfully constructed, and the relative expression of mi R-146a in U251TR cells was lower than that in U251 cells, and the drug resistance index decreased after mi R-146a-mimics was transferred to U251TR cells. The results of double luciferase reporter gene and Western blotting showed that mi R-146a could play a role in the formation of drug resistance in human glioma cell line U251 through Notch1 signaling pathway.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R739.41

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