c-kit-D816V稳定表达细胞系的构建及药物筛选

Construction of stable cell line expressing c-kit-D816V and drug screening

目的建立过表达人干细胞因子(stem cell factor,SCF)受体c-kit基因突变小鼠红白血病HCD57细胞株,以应用于抗癌小分子药物筛选。方法以带有人c-kit-D816V基因突变质粒为模板,设计并合成引物,PCR扩增c-kit基因,逆转录病毒载体用限制性内切酶EcoRⅠ进行单酶切,采用同源重组法构建pMSCV-c-kit-D816V表达载体。采用酶切和基因测序对重组质粒进行鉴定。利用Lipofectamine 3000将重组质粒和辅助质粒共转染GP2-293细胞进行病毒包装。在荧光显微镜下观察转染效果,收集、浓缩病毒上清液。将制备好的逆转录病毒感染HCD57细胞,采用甲基纤维素半固体培养基筛选单克隆稳定转染细胞株,采用流式细胞术鉴定c-kit基因表达。采用CCK-8法进行小分子靶向药物筛选实验。结果酶切鉴定结果显示,电泳后重组质粒单酶切后可见约为693、2250和6488 bp大小的3条DNA条带,与载体及c-kit基因片段大小相符;DNA测序结果显示,c-kit基因成功插入到逆转录病毒表达载体中。流式细胞术检测结果显示,感染后的HCD57单克隆细胞株高表达C-kit蛋白。CCK-8细胞增殖实验显示,伊马替尼对HCD57-c-kit-D816V细胞的生长无明显抑制作用,帕博西尼、达沙替尼、艾德拉尼均对HCD57-c-kit-D816V细胞抑制作用明显。结论逆转录病毒载体介导C-kit蛋白稳定表达的HCD57细胞系构建成功,药物初筛实验成功,可用于抗癌小分子药物筛选实验。

Objective To establish a mouse erythroleukemia HCD57 cell line over expressing human stem cell factor(SCF)receptor c-kit gene for the screening of anti-cancer drugs with small molecule.Methods A plasmid with a mutated human c-kit-D816V gene was used as a template to design and synthesize primers for PCR amplification of the c-kit gene.and the retroviral vector was cleaved by restriction endonuclease EcoRⅠ.pMSCV-c-kit-D816 V expression vector was constructed by homologous recombination.The recombinant plasmid was identified by restriction enzyme digestion and Sanger sequencing.The recombinant plasmid and helper plasmid were co-transfected with GP2-293 cells for vial packaging using Lipofectamine 3000.The transfection efficiency was observed under a fluorescence microscope.Then the supernatant containing viruses was collected,concentrated,and used to infect HCD57 cells.The stable transfected cell lines were screened using methylcellulose semisolid medium,and the expression of c-kit gene was identified by flow cytometry.The screening experiment of small molecular targeted drugs was carried out by CCK-8 method.Results The results showed that three DNA bands of approximately 693,2250 and 6488 bp in size were visible after single digestion of the recombinant plasmid by electrophoresis,which were consistent with the size of the vector and the c-kit gene fragment;Sanger sequencing results showed a successfully insertion of c-kit gene into the retroviral expression vector.Flow cytometry analysis was used to detect that the HCD57 monoclonal cell lines expression high level of C-kit protein.CCK-8 assay showed that imatinib fail to inhibit the growth of HCD57-c-kit-D816 V cells while Pabosinib,Dasatinib and Adranil significantly inhibited the growth of HCD57-c-kit-D816 V cells.Conclusion The HCD57 with stable expression of C-kit was successfully constructed,which was validated in a preliminary drug screening assay.Andcan be used in the large-scale screening of small molecular anticancer drugs.