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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 effects of silenced HOX A10 gene combined with small dose of Ara-C on proliferation and apoptosis of K562 cells by RNA interference (RNAi) technique, and to provide experimental evidence for gene therapy of leukemia. Methods Specific short hairpin RNA oligonucleotide chains targeting HOXA10 were designed and synthesized. The eukaryotic expression vector pGPHI / GFP / Neo-HOXA10 was constructed and sequenced. The cationic liposomes were transfected into K562 cells. After RNAi and low-dose Ara-C alone and in combination, the cell proliferation was detected by MTT assay and the apoptosis rate was detected by flow cytometry. Results The vector pGPHI / GFP / Neo-HOXA10 was successfully constructed and transfected into K562 cells. The results showed that the vector could effectively reduce the expression of HOXA10 mRNA. The ODR of HOXA10 / β-actin was (38.86 ± 4.49)%, Combined with low dose of Ara-C on K562 cells, the cell proliferation ability was significantly decreased and the cell proliferation inhibition rate (IR) was (75.58 ± 8.11)%, which was significantly different from the control group (P <0.05) Visible red fluorescence apoptotic cells increased significantly, the apoptotic rate was significantly increased (29.71 ± 1.24)%, compared with the control group, the difference was statistically significant (P <0.05). Conclusion RNAi targeting HOXA10 combined with low-dose Ara-C can effectively inhibit the proliferation and induce apoptosis of K562 cells, which is expected to become a new gene therapy program for leukemia.