论文部分内容阅读
FMS样酪氨酸激酶3(FLT3)是一种酪氨酸激酶受体,在大多数急性髓系白血病(AML)患者中持续激活,与患者预后差密切相关。为探讨3种FLT3靶向短发夹状干扰RNA(shRNA)对急性髓系白血病细胞株THP-1的沉默效应,设计和体外转录合成3个FLT3靶向shRNA(shRNA1、shRNA2、shRNA3),体外转染THP-1细胞;以RT-PCR法检测FLT3mRNA水平表达,用流式细胞术、免疫荧光测定法检测FLT3蛋白的表达。结果显示:shRNA1、shRNA3可显著下调FLT3mRNA的表达,其中shRNA1的抑制作用较强。25nmol/LshRNA1转染48小时对FLT3mRNA的抑制率是(72.95±2.07)%,作用可达72小时。5nmol/L及以上浓度的shRNA1对FLT3mRNA表达有下调作用,作用存在量-效关系。15nmol/LshRNA1的抑制率是(67.53±0.66)%。FLT3蛋白位于细胞膜上,shRNA1对其有较强的抑制作用,转染72小时蛋白抑制率达(79.67±0.66)%。结论:FLT3-shRNA1具有较好的FLT3基因靶向抑制作用,可作为进一步研究该基因作用机制及靶向治疗可能性的工具。
FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase receptor that continues to activate in most acute myeloid leukemia (AML) patients and is associated with poor prognosis. To investigate the silencing effect of three kinds of FLT3 targeting short hairpin RNA (shRNA) on acute myeloid leukemia cell line THP-1, three FLT3 targeting shRNAs (shRNA1, shRNA2 and shRNA3) were designed and synthesized in vitro. The expression of FLT3 mRNA was detected by RT-PCR. The expression of FLT3 protein was detected by flow cytometry and immunofluorescence assay. The results showed that: shRNA1, shRNA3 can significantly down-regulate the expression of FLT3mRNA, which shRNA1 inhibition is stronger. 48 hours after transfection with 25nmol / L shRNA1, the inhibitory rate of FLT3mRNA was (72.95 ± 2.07)% and the effect was up to 72 hours. ShRNA1 at a concentration of 5nmol / L and above has a down-regulation effect on FLT3mRNA, and the effect exists in a dose-effect relationship. The inhibition rate of 15 nmol / L shRNA 1 was (67.53 ± 0.66)%. FLT3 protein is located on the cell membrane, shRNA1 has a strong inhibitory effect, 72 hours after transfection of protein inhibition rate (79.67 ± 0.66)%. Conclusion: FLT3-shRNA1 has a good target inhibition of FLT3 gene, which can be used as a tool to further investigate the mechanism of action of this gene and the possibility of targeted therapy.