RNA干扰联合小剂量阿糖胞苷对K562细胞增殖、凋亡的影响

Effects of RNA Interference and Low Dose Cytarabine on Proliferation and Apoptosis of K562 Cells

目的探讨利用RNA干扰(RNAi)技术沉默同源盒(HOX)A10基因联合小剂量阿糖胞苷(Ara-C)对K562细胞增殖、凋亡的影响,为白血病的基因治疗提供实验依据。方法设计合成针对HOXA10的特异性短发卡RNA寡核苷酸链,构建pGPHI/GFP/Neo-HOXA10真核表达载体并测序,应用阳离子脂质体转染K562细胞。RNAi与小剂量Ara-C单独和联合应用后,应用四甲基偶氮唑蓝(MTT)法检测细胞增殖能力,流式细胞术检测细胞凋亡率。结果成功构建pGPHI/GFP/Neo-HOXA10载体,并转染K562细胞,结果显示该载体能有效降低HOXA10 mRNA表达水平,HOXA10/β-actin灰度比值(ODR)为(38.86±4.49)%;RNAi联合小剂量Ara-C作用于K562细胞后,细胞增殖能力明显下降,细胞增殖抑制率(IR)为(75.58±8.11)%,与对照组比较差异有统计学意义(P<0.05);荧光显微镜可见红色荧光凋亡细胞明显增加,凋亡率显著升高,可达(29.71±1.24)%,与对照组比较差异有统计学意义(P<0.05)。结论靶向HOXA10的RNAi技术联合小剂量Ara-C能有效抑制K562细胞增殖,并诱导其凋亡,有望成为白血病基因治疗的新方案。

Objective To investigate the effect of plasmid mediated RNA interference （RNAi） targeting HOXA10 and low dose cytarabine（ Ara - C） on proliferation and apoptosis of human chronic myeloid leukemia K562 cell line. Methods Effective and specific short RNA （shRNA） oligo targeting HOXA10 was designed and compounded. Then plasmid PGPHI/GFP/NeoHOXA10 was constructed and sequenced. It was transfected into K562 cells by positive ion liposome. The mRNA expression of HOXA10 was detected by semi - quantitative reverse transcriptase polymerase chain reaction,the cell proliferation was assayed by methy thiazolyl tetrazolium, the apoptosis was measured by flow cytometry. Results The plasmid pGPHI/GFP/Neo - HOXA10 was successfully constructed and transfected into K562 cells. The mRNA of HOXA10 was inhibited by this plasmid in K562 cells by optical density rate was （38.86 ±4.49） %. The inhibitory rate of cell proliferalion of group RNAi ＋ low dose Ara - C was （75, 58 ± 8.11 ） % and the apoptosis rate was （29.71 ± 1.24） %, they were different from the control group obviously（ P 〈 0. 05 ）. Conclusions The expression of HOXA10 can effectively silenced by the plasmid pGPHI/GFP/Neo - HOXA10,which combining with low dose Ara - C can inhibit the proliferation of K562 cell and promote its apoptosis, RNAi targeting and low dose Ara - C is possibly to be a new way of leukemia gene therapy.