Ma Xiao-xia, Wang Chun, Wei Ju, Qin You-wen, Yan Shi-ke, Gao Yan-rong, Cai Qi
Department of Hematology, Shanghai First People's Hospital, Shanghai 200080, China.
Zhonghua Xue Ye Xue Za Zhi. 2005 Jun;26(6):359-62.
To investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression.
The small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay.
The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively.
At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.
探讨RNA干扰对慢性髓性白血病（CML）bcr/abl癌基因表达的抑制作用。
体外合成小干扰RNA（siRNA）。通过电穿孔法将siRNA转染至稳定表达bcr/abl基因的K562细胞，未转染细胞和转染非特异性siRNA的细胞作为对照。以增强型绿色荧光蛋白（EGFP）质粒作为阳性对照，采用流式细胞术检测转染效率。通过实时定量RT-PCR和Western印迹法证明siRNA的抑制作用。采用MTT法检测细胞增殖，采用Annexin V-FITC法检测细胞凋亡。
转染效率约为70%。合成的siRNA在mRNA和蛋白水平均抑制CML bcr/abl癌基因表达。转染后24小时和48小时，siRNA可分别将K562细胞增殖抑制至47%和56%，并诱导细胞凋亡，从对照组的1.00%分别升至24小时的15.05%和48小时的19.4%。
在细胞水平上，化学合成的siRNA抑制CML bcr/abl癌基因表达为抗白血病研究提供了新方法。
