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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 G_0/G_1期到 S 期的阻滞,FLT3-shRNA 组 G_0/G_1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。
Objective To investigate the effect of short hairpin RNA (shRNA) -induced target inhibition of FTS-like tyrosine kinase 3 (FLT3) on proliferation and apoptosis of acute monocytic leukemia (AMOL) cell line THP-1. Methods FLT3-targeting shRNA (FLT3-shRNA) was designed and synthesized in vitro and transfected into THP-1 cells. The expression of FLT3 was detected by semi-quantitative RT-PCR and flow cytometry (FCM). Cell proliferation was detected by cell counting reagent 8 (CCK-8). Cell cycle was detected by FCM. Apoptosis was analyzed by DNA Ladder and Annexin V-FITC staining. Results The inhibitory rates of FLT3 mRNA and protein were up to (72.95 ± 2.07)% and (65.39 ± 5.57)% at 48 h after transfection with 15 nmol / L FLT3-shRNA. After shRNA interference, the cell proliferation activity was inhibited, the inhibitory rate reached (36.66 ± 3.67)% at 48 h and reached (35.56 ± 0.73)% at 72 h. The cell cycle of G0 / G1 phase to S phase was 48 h after transfection, the percentage of cells in G0 / G1 phase was significantly higher in FLT3-shRNA group (65.71 ± 4.47%) than that in control group (25.11 ± 2.70)% In the control group. The obvious apoptotic bands appeared after 48 h of FLT3-shRNA treatment. The early apoptotic rate of Annexin V-FITC cells (18.59 ± 2.07)% was significantly higher than that of the control group. CONCLUSIONS: FLT3-targeted interference induced by shRNA can effectively inhibit THP-1 cell proliferation and induce apoptosis, indicating that it has potential value in the treatment of AMOL in children.