【摘要】 目的探讨短发夹状干扰 RNA（shRNA）诱导 FMS 样酪氨酸激酶3（FLT3）的靶向抑制对急性单核细胞白血病（AMOL）细胞株 THP-1增殖、凋亡的影响。方法设计并体外转录合成 FLT3靶向 shRNA（FLT3-shRNA）,转染 THP-1细胞。以半定量逆转录 PCR（RT-PCR）、流式细胞法（FCM）检测细胞 FLT3在 mRNA、蛋白水平表达,细胞计数试剂8（CCK-8）检测细胞增殖活力,FCM 法检测细胞周期,DNA 梯度条带（DNA Ladder）和 Annexin V-FITC 染色法分析细胞凋亡。结果合成的 FLT3-shRNA 15 nmol/L 转染48 h 对 FLT3 mRNA 和蛋白的抑制率分别达（72.95±2.07）%、（65.39±5.57）%。shRNA 干扰后细胞增殖活力受到抑制,48 h 抑制率达（36.66±3.67）%,72 h 达（35.56±0.73）%。转染48 h 细胞周期出现 G₀/G₁期到 S 期的阻滞,FLT3-shRNA 组 G₀/G₁期的细胞百分比（65.71±4.47）%明显高于对照组,S 期（25.11±2.70）%低于对照组。FLT3-shRNA 作用48 h 有明显的凋亡条带出现,Annexin V-FITC 检测细胞的早期凋亡率（18.59±2.07）%明显高于对照组。结论由 shRNA 诱导的 FLT3靶向干扰可有效的抑制 THP-1细胞增殖、诱导凋亡,显示其在儿童 AMOL 治疗中具有潜在的价值。更多还原

【Abstract】 Objective FMS-like tyrosine kinase 3（FLT3）is a receptor tyrosine kinase that is constitutively activated in（70-90）% pediatric patients with acute myeloid leukemia（AML）and appears to confer an adverse prognosis.Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success,to address the specificity and resistance,new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice.The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA （shRNA）on myeloproliferation and apoptosis in an acute monocytic leukemia（AMOL）cell line THP-1. Methods FLT3-targeted small hairpin interfering RNA（FLT3-shRNA）was designed and synthesized by transcription system in vitro was transfected into THP-1 cells.Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry（FCM）to verify the efficacy on FLT3-shRNA interference at 48 h after transfection.Cell growth viability was measured at 24h, 48h and 72h after treatment with CCK-8.The distribution of cell cycle was assayed by FCM,and apeptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48h.Results FLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48h could obtain desirable downregulation of FLT3 expression,the inhibitory percentages of FLT3 mRNA and protein were（72.95±2.07）% and （65.39±5.57）%,respectively.The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were（36.66±3.67）% at 48 h,（35.56±0.73）% at 72h.FLT3-shRNA induced the inhibition of cell cycle from G₀/G₁ phase to S phase,the percentage of sub-G₀/G₁ phase（65.71±4.47）% was higher than those in the PBS-control group（52.23±2.98）%, NC-shRNA control group（51.81±1.44）%,P<0.01;the percentage of S phase（25.11±2.70）% was lower than those in the PBS-control group（34.41±4.07）% and NC-shRNA control group（32.50± 1.46）%,P<0.05.Furthermore treatment with FLT3-shRNA for 48h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was（18.59±2.07）% which was significantly higher than that in the PBS-control group（4.00 + 0.50）% and the NC-shRNA control group （6.06±0.70）%,P<0.001.Conclusion The suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation,and apoptosis induction on THP-1 cells,which indicates that this approach may bear the therapeutic potential on childhood AMOL.更多还原