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目的:采用基因芯片技术,观察RNA干涉法抑制人脑胶质瘤U251细胞MSP58基因表达后细胞周期相关基因的变化,从而了解MSP58在细胞周期相关基因通路中的可能定位。方法:将特异性的MSP58干涉表达载体pSilencer3.1-MSP58转染人脑胶质瘤细胞系U251,同时构建相应的阴性对照载体pSilenc-er3.1-NC以及空载体pSilencer3.1-H1neo。运用半定量RT-PCR及蛋白质印迹法,检测稳定转染和未转染干涉载体的胶质瘤细胞MSP58的mRNA和蛋白表达水平。运用基因芯片技术分析pSilencer3.1-MSP58转染后细胞周期相关基因的变化。结果:成功建立了具有稳定下调MSP58基因表达的U251-S细胞、阴性对照U251-NC细胞以及含有空载体的U251-H1neo细胞。干涉组细胞MSP58的表达无论在mRNA水平还是在蛋白质水平均较阴性对照组及空载体组细胞显著降低,P<0.01。基因芯片技术共筛选细胞周期相关基因128个,其中上调及下调>2倍的共有33个基因。结论:干涉MSP58基因表达后,胶质瘤细胞增殖的抑制与细胞周期相关基因表达的变化有关。
OBJECTIVE: To observe the possible changes of MSP58 gene expression in human glioma U251 cells by using gene chip technique and find out the possible location of MSP58 in cell cycle related gene pathway. METHODS: The specific MSP58 interference expression vector pSilencer3.1-MSP58 was transfected into human glioma cell line U251, and the corresponding negative control vector pSilenc-er3.1-NC and empty vector pSilencer3.1-H1neo were constructed. Semi-quantitative RT-PCR and Western blotting were used to detect mRNA and protein expression levels of MSP58 in glioma cells stably transfected and non-transfected. The changes of cell cycle related genes after pSilencer3.1-MSP58 transfection were analyzed by gene chip technology. Results: U251-S cells with stable down-regulation of MSP58 gene expression, U251-NC cells with negative control and U251-H1neo cells with empty vector were successfully established. The expression of MSP58 in the intervention group was significantly lower than that in the negative control group and empty vector group, both at the mRNA level and the protein level (P <0.01). A total of 128 cell cycle related genes were screened by gene chip technology, of which 33 were up-regulated and down-regulated> 2-fold. Conclusion: The inhibition of proliferation of glioma cells after interference with MSP58 gene expression is related to the changes of cell cycle related gene expression.