论文部分内容阅读
目的探讨RNA干扰技术沉默热休克蛋白90β(heat shock protein90beta,Hsp90β)基因表达对Jurkat细胞生长的抑制作用。方法根据Hsp90β基因全长cDNA序列Hsp90B1(NM_003299.1),设计并构建针对Hsp90β基因的siRNA真核表达载体,应用LipofectamineTMLTX转染入Jurkat细胞后,Real-time PCR检测Hsp90βmRNA水平的变化,筛选出沉默效果最好的Hsp90βsiRNA干扰片段;Western印迹法检测Hsp90β蛋白表达水平;MTT法和流式细胞仪检测干扰前后对Jurkat细胞生长抑制作用。结果成功构建Hsp90β基因特异性的真核表达载体pSOS-Hsp90βi,转染Jurkat细胞后发现pSOS-Hsp90βi2的沉默效果最明显,Jurkat细胞Hsp90βmRNA及蛋白表达均明显降低(P<0.05)。Hsp90β基因表达沉默后,Jurkat细胞增殖明显受抑(P<0.01),实验组与对照组的细胞凋亡率分别为:(2.69±0.34)%、(3.63±0.38)%、(19.56±0.62)%,细胞凋亡率比空白对照组及转染pSOS-Hsp90βi control组明显增加,差异具有显著意义(P<0.05)。结论靶向Hsp90β的siRNA下调Hsp90β表达对人白血病Jurkat细胞有明显的生长抑制作用。
Objective To investigate the inhibitory effect of heat shock protein 90beta (Hsp90β) gene silencing on Jurkat cell growth by RNA interference. Methods According to the full-length Hsp90B1 cDNA sequence of Hsp90B1 (NM_003299.1), a siRNA eukaryotic expression vector targeting Hsp90β gene was designed and constructed. After transfected into Jurkat cells with LipofectamineTM LXTX, the expression of Hsp90βmRNA was detected by Real-time PCR and silenced The best Hsp90βsiRNA interference fragment was detected by Western blotting. The expression of Hsp90βprotein was detected by Western blotting. The growth inhibition of Jurkat cells was detected by MTT assay and flow cytometry. Results The eukaryotic expression vector pSOS-Hsp90βi of Hsp90β gene was constructed successfully. The transfection of Jurkat cells showed that the silencing effect of pSOS-Hsp90βi2 was the most obvious. The mRNA and protein expression of Hsp90β in Jurkat cells were significantly decreased (P <0.05). The apoptosis rate of Jurkat cells was significantly inhibited after Hsp90β gene silencing (P <0.01). The apoptotic rates of experimental group and control group were (2.69 ± 0.34)%, (3.63 ± 0.38)% and (19.56 ± 0.62) %, The apoptosis rate was significantly higher than the blank control group and transfected pSOS-Hsp90βi control group, the difference was significant (P <0.05). Conclusion siRNA targeting Hsp90β can down-regulate the expression of Hsp90β in human leukemia Jurkat cells.