五种急性髓细胞白血病细胞株FAM样酪氨酸激酶3表达及其短状串联重复突变检测

Expression of Fams-like tyrosine kinase 3 in five cell lines with acute myelocytic leukemia and detection of internal tandem duplication mutation

目的:探讨不同急性髓细胞白血病细胞(AML)FAM样酪氨酸激酶3(FLT3)表达状况,筛选FLT3过表达细胞,并检测其编码FLT3近膜(JM)区的基因是否存在短状串联重复(ITD)突变。方法:选择THP-1、K562、HL-60、Dami、Meg-01细胞,以含内参GAPDH对照的半定量RT-PCR检测其FLT3mRNA的表达,以流式细胞仪检测细胞膜表面FLT3蛋白的表达;提基因组DNA,PCR扩增其JM区,对有FLT3过表达的细胞,PCR扩增产物连T-载体后测序。结果:5种AML细胞中仅有THP-1、HL-60存在FLT3基因的表达,其mRNA表达相对于GAPDH的含量分别是0.83±0.07、0.48±0.05,2者比较差异有统计学意义(t=7.047,P<0.01);细胞膜表面FLT3蛋白表达率分别是(43.55±4.44)%、(27.57±3.42)%,2者比较差异有统计学意义(t=8.029,P<0.001)。2种细胞经FLT3-JM区PCR产物测序均不存在ITD突变。结论:THP-1、HL-60是FLT3野生型过表达的AML细胞株。

Aim ： To explore expression of Fams-like tyrosine kinase 3 （FLT3） in five cell lines with acute myelocytic leukemia in order to screen FLT3 over-expression cell lines, and investigate mutation of FLT3 internal tandem duplication （FLT3-ITD） in juxtamembrane domain （JM）. Methods： For THP-1, K562, HL-60, Dami, and Meg-O1 cells, semi-quantitative RT-PCR was used for the detection of FLT3 mRNA, while flow eytometry was used for FLT3 protein. Genomic DNAs from these cells were employed to amplify the sequences of JM domain, and their PCR fragments were ligated into T- vector and sequenced. Results： Just THP-1 ,HL-60 were detected with FLT3 expression, which related contents to GAPDH in mRNA level were 0.83 ± 0.07, 0.48± 0.05, respectively （ t = 7. 047, P 〈 0.01 ）. The expressional rates of FLT3 protein in cell membrane were （43.55 ± 4.44） % , （27.57 ± 3.42 ） % , respectively （ t = 8. 029, P 〈 0.001 ）. According to results of sequence, there weren＇t ITD mutation in the two cells. Conclusion： THP-1 and HL-60 are AML cell lines with wild-type FLT3 receptor but over-express it in mRNA and protein levels.