稳定表达FLT3-ITD/FIV载体的急性髓细胞白血病细胞株的构建及鉴定
Construction and identification of FLT3-ITD/ FIV expression vector and acute myelogenous leukemia cell lines
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摘要: 构建稳定表达FLT3-ITD/FIV载体的急性髓细胞白血病细胞(AML)株，为研究FLT3-ITD在急性髓细胞白血病中的作用和以FLIT3为靶点的治疗提供一种新的体外模型。筛选临床上FLT3-ITD突变的AML患者，选取mutant/wt比值最高的FLT3-ITD突变作为克隆对象，PCR扩增FLT3基因全长。然后通过同源重组的方法构建FLT3-ITD/FIV慢病毒载体并包病毒，收集病毒液上清液进行K562白血病细胞株感染，通过流式细胞分选术筛选阳性转染细胞。收集阳性转染细胞做Western blot验证阳性质粒的表达情况。207名AML患者中有42例FLT3-ITD突变，成功扩增出了mutant/wt比值最高的FLT3-ITD突变患者的FLT3基因全长。双酶切FIV载体和模板DNA，连接以及感受态细胞转化筛选出阳性重组子，双酶切鉴定和测序结果验证。慢病毒成功感染K562白血病细胞株，用流式细胞分选术筛选出了稳定感染的细胞，且该细胞可以传代、扩增。同时，稳定感染细胞的Western blot鉴定也证实了阳性质粒的表达情况。通过筛选FLT3-ITD突变的AML患者，成功构建了稳定表达FLT3-ITD/FIV载体的白血病细胞株，为研究FLT3-ITD在AML发病中的作用以及针对FLT3-ITD为靶点的药物筛选治疗等提供了一个稳定的体外细胞模型。
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关键词:
- 急性髓细胞白血病(AML) /
- Fms样酪氨酸激酶-3 /
- 内部串联重复 /
- 细胞株
Abstract: The aim of this study was to construct the lentivirus expression vector of human FLT3-ITD mutation and to screen the AML cells for stable expression of FLT3-ITD. We screened the AML patient with FLT3-ITD mutation by peripheral blood DNA extraction and PCR, and then the ORF of FLT3-ITD was constructed using the method of homologous recombination, which used the BamH I and Not I double enzyme digestion of the ORF and the FIV vector. Results were confirmed by restriction enzyme identification and PCR. The constructed recombinant vector was co-transfected with VSVG and g/p into HEK293T cells to produce the lentiviral particles by transfection reagent. The lentiviral particles were transduced into K562 cells and the infection efficiency was measured by fluorescence microscope. The cells were sorted by the Flow Cytometer. The expression of FLT3 in the stable cell lines was detected by Western blot. There were 42 patients with FLT3-ITD mutation among the 207 patients we collected. We successfully amplified the ORF of FLT3 of the patient with highest mutant/wt ration. Then constructed the lentivirus expression vector FLT3-ITD/FIV and overexpressed it in K562 cell lines. We screened the cells expressed FLT3-ITD/FIV by fluorescence activated cell sorting, and Western Blot results proved the expression of FLT3-ITD. The over-expression 1entiviral vector of human FLT3-ITD/FIV has been successfully constructed and cell lines for stable expression of FLT3-ITD have been successfully established. This will shed a light on the future study of FLT3 function in AML and provide a new in vitro model for AML.-
Keywords:
- acute myeloid leukemia /
- FLT3-ITD /
- internal tanderm duplication /
- cell lines
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