【作者】 郭亚；

【导师】 郑志竑；

【作者基本信息】 福建医科大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目的:1、检测Notch1基因对人胶质瘤U251增殖和细胞周期的影响。2、探讨Notch1基因影响人胶质瘤U251细胞增殖和周期的可能机制。方法:1、利用Notch1-shRNA和pNL-NICD/EGFP慢病毒感染人胶质瘤U251细胞,RT-PCR和Western Blotting法筛选和鉴定Notch1表达下调或Notch1胞内段(Notch1 intracellular domain, NICD)表达上调的U251细胞。2、MTT法和PI单染流式细胞术分析Notch1对细胞增殖和细胞周期的影响。3、采用RT-PCR和Western blotting法检测NF-κB p65表达;EMSA法检测NF-κB的活性。4、采用RT-PCR法检测细胞周期相关基因,包括p53, p21, p27, p16, cyclinD1, cyclinE1, cyclinA2, cyclinB1, cyclinB2, CDK2, CDK4, CDK6, Cdc25C的表达情况,对mRNA表达水平变化的基因,进一步采用Western blotting法检测其蛋白表达水平。结果:1、Notch1-shRNA慢病毒表达载体能有效下调U251细胞Notch1的表达,而pNL-NICD/EGFP慢病毒表达载体能有效上调U251细胞NICD的表达。2、Notch1基因表达下调的细胞其细胞增殖能力受到明显抑制(P<0.01),并引起细胞G1期阻滞(P<0.01), S期细胞减少(P<0.01);在NICD表达上调的细胞其增殖能力明显增强(P<0.01),且引起G1期细胞减少(P<0.05),S期细胞增加(P<0.01)。3、Notch1基因表达下调的细胞其NF-κB p65 mRNA和蛋白表达水平显著降低(P<0.05),NF-κB DNA结合活性显著降低(P<0.01);在NICD表达上调的细胞其NF-κB p65mRNA和蛋白表达水平显著增加(P<0.01),NF-κB DNA结合活性显著增加(P<0.01)。4、Notch1基因表达下调的细胞其cyclinD1、cyclinA2、CDK2、Cdc25C的mRNA和蛋白表达水平显著降低(P<0.01);而在NICD表达上调的细胞,这些基因的mRNA和蛋白表达水平显著增加(P<0.05)。结论:1、Notch1基因与人胶质瘤U251细胞的增殖能力和周期调控密切相关。2、Notch1基因可能通过NF-κB通路影响U251细胞的增殖,且可能部分通过NF-κB p65起作用。3、Notch1基因可通过cyclinD1、cyclinA2、CDK2和Cdc25C影响U251细胞的周期进程。更多还原

【Abstract】 Objective:1、To investigate the effect of Notch1 gene on the change of proliferation and cell cycle in human glioma U251 cell.2、To research the possible mechanisms of Notch1 gene involved in proliferation and cell cycle in human glioma U251 cell.Methods:1、The lentiviral vectors, which expressed Notch1 shRNA or Notch1 intracellular domain (NICD), were transfected into U251 cell respectively. RT-PCR and Western Blotting were applied to monitor the validity of down-regulation of Notch1 expression and over-expression of NICD.2、MTT assay was performed to examine the cell proliferation. Flow cytometric analysis was used to detect the cell cycle.3、RT-PCR and Western Blotting were applied to monitor the expression of NF-κB p65. EMSA was performed to assay the NF-κB DNA-binding activity.4、RT-PCR was applied to monitor the expression of capital genes involved in cell cycle, which included p53, p21 , p27 , p16, cyclinD1, cyclinE1, cyclinA2, cyclinB1, cyclinB2, CDK2, CDK4, CDK6, Cdc25C. Then Western Blot was used to check the genes changed in mRNA expression.Results:1、The lentiviral vectors, which expressed Notch1 shRNA or NICD, were efficient in down-regulation of Notch1 expression and over-expression of NICD respectively.2、Down-regulation of Notch1 gene by RNAi inhibited cell proliferation remarkably(P<0.01), arrested cell cycle at G1 phase (P<0.01) and decreased the cell number of S phase(P<0.01). Over-expression of NICD enhanced the cell proliferation significantly (P<0.01), promoted cell cycle at G1 phase (P<0.05) and increased the cell number of S phase (P<0.01).3、Down-regulation of Notch1 gene by RNAi inhibited NF-κB p65 expression(P<0.05) and decreased NF-κB DNA-binding activity(P<0.01). Over-expression of NICD enhanced NF-κB p65 expression (P<0.01) and increased NF-κB DNA-binding activity (P<0.01) in U251 cell.4、Down-regulation of Notch1 gene by RNAi inhibited the expression of cyclinD1, cyclinA2 and Cdc25C (P<0.05). Over-expression of NICD enhanced the expression of cyclinD1, cyclinA2, CDK2 and Cdc25C (P<0.05) in U251 cells.Conclusion:1、Notch1 gene leads to the change of proliferation and cell cycle in human glioma U251 cell.2、Notch1 gene mybe enhance the cell proliferation via increasing NF-κB DNA-binding activity and which may be dependent on increasing the expression of NF-κB p65 partially in U251 cell.3、Notch1 gene maybe enhance the cell cycle via increasing the expression of cyclinD1, cyclinA2, CDK2 and Cdc25C in U251 cell.更多还原