siRNA对体外培养红系细胞α-珠蛋白基因表达的影响

Effect of small interfering RNA on the Alpha-globin gene expression in erythroid cells cultured in vitro

目的研究针对α-珠蛋白基因的小分子干扰RNA(si RNA)对体外培养红系细胞α-珠蛋白肽链mRNA表达的影响。方法将针对α-珠蛋白肽链设计、化学方法合成的si RNA通过脂质体转染体外培养正常人的红系细胞,在转染后24、48、72 h,用流式细胞术检测转染效率,RT-PCR检测α链mRNA表达水平。结果脂质体转染FCM标记的si RNA进入红系细胞后,24、48、72 h的转染效率分别为61.2%、44.3%、33.7%。si RNA作用于体外培养的红系细胞,经RT-PCR检测,转染后α-珠蛋白基因mRNA相对表达量下降,浓度越高,相对表达量越低。随着作用浓度增加,红系细胞台盼兰拒染率越低;且作用时间越长,台盼兰拒染率也越低。结论脂质体转染si RNA能特异性地抑制体外培养的红系细胞中α-珠蛋白基因表达,可能会成为β-珠蛋白生成障碍性贫血基因治疗的一个新靶点。

Objective To evaluate the effects of small interfering RNA（siRNA） targeting alpha-globin gene on the expression level of alpha-globin chain mRNA in erythroid cells cultured in vitro.Methods siRNA for alpha-globin gene was designed and chemical synthesized,and transferred into the normal adult donor originated erythroid cells cultured in vitro with liposome-induced gene transfection.After transfection,at the time of 24,48,72 h,the transfection efficiencies were inspected by flow cytometry,the level of mRNA in alpha-globin gene was detected by real-time quantitation PCR.Results Liposomal could effectively transfect the FCM-labeled siRNA into erythroid cells cultured in vitro.The transfection efficiencies were 61.2%,44.3%,33.7% at 24,48,72 h respectively.Results of RT-PCR indicated siRNA could down-regulated alpha-chain mRNA expression.The inhibition efficiency was higher as the siRNA concentration increased.The trypan blue exclusion rates of erythroid cells were decreased as the concentration increased and time lasts.Conclusion The siRNAs targeting alpha-globin gene transferred into erythroid cells cultured in vitro with liposome-induced gene transfection can down-regulate the alpha-chain mRNA expression level,which may be the new target for gene therapy in β-thalassemia.