摘要
目的:采用基因芯片技术,比较RNA干扰法封闭U937细胞中PNAS-2基因前后,与凋亡有关基因的变化情况,从而了解PNAS-2在凋亡通路中的可能定位。方法:将急性单核细胞白血病细胞株U937分别转染已构建的靶向封闭PNAS-2的干扰质粒组和对照质粒组,合并使用流式细胞仪(FCM)筛选出GFP阳性细胞,并运用半定量PCR及实时PCR方法验证RNAi效果。采用affymetrix u133A 2.0基因芯片技术比较2组细胞间基因表达的差异。进一步通过半定量RT-PCR对其中的2条上调基因(IER3、CCL2)和6条下调基因(CASP8、CD74、VEGF、DNASE2、PRTN3、TERT)的表达情况进行验证。结果:半定量及实时PCR检测结果表明,合并使用G418及FCM筛选后得到的转染细胞在90%以上,干扰质粒组对PNAS-2的抑制率达到70%以上;基因芯片筛选结果提示共有1651条出现上调,1404条出现下调。其中比值≥2倍的基因共有336条,上调114条,下调222条。根据功能分类初步筛选出与凋亡相关基因共22条,上调基因11条,下调基因11条。RT-PCR检测证实基因芯片结果可靠。结论:抑制PNAS-2表达后促进U937细胞凋亡的机制可能与c-Jun过表达、JNK通路的激活及VEGF基因下调有关。
Objective To study the apoptosis-related gene changes using genechip after the suppression of PNAS-2 gene expression by RNA interference in U937 leukemia cell line in order to understand the role of PNAS-2 in apoptosis pathway. Methods After transfecting with interference and control recombinant plasmids into U937 cells, G418 with GFP^＋ cell subpopulation sorted by FCM was selected to purify the tansfected cells. Both semiquantitative PCR and reahime PCR （RT-PCR） were used to evaluate the RNAi efficiency. The affymetrix u133A 2.0 genechip was used to study the gene changes between the interference group and the control transfected group. And then 2 up-regulated and 6 down-regulated genes were selected and the results of genechip were confirmed through RT-PCT. Results Confocal microscopy comfirmed that almost pure tranfected U937 cells, were gotten and both semiquantitative PCR and RT-PCR confirmed that the inhibition rate was over 70%. The result of microarray indicated 1651 genes were up-regulated and 1404 downregulated in the PNAS-2-shRNA transfected group in comparison with the control group, among which 22 apoptosis relate genes＇ change rates were above 2 times including llup-regulated and 11 downregulated. The results were confirmed reliable by RT-PCR. Conclusions The mechanism of U937 cell apoptosis induced by the inhibition of PNAS-2 may be concerned with over-expression of c-jun and activation of JNK pathway, and also may be concerned with the down-regulation of VEGF gene.
出处
《诊断学理论与实践》
2007年第2期137-142,共6页
Journal of Diagnostics Concepts & Practice
基金
国家自然科学基金资助项目(30670881)
上海市教委科研项目(05BZ37)
