c-kit突变型人胃肠间质瘤永生化细胞系的建立与鉴定

Established immortalized c-kit mutanted human gastrointestinal stromal tumors cell line express the hTERT

目的通过在c-kit突变型人胃肠间质瘤原代细胞中过表达人端粒酶逆转录酶(h TERT),建立c-kit突变型人胃肠间质瘤永生化细胞系。方法利用聚合酶链反应(PCR)技术获得h TERT目的基因片段,测序正确后,体外构建端粒酶亚单位(h TERT)逆转录病毒表达载体,转染c-kit突变型胃肠间质瘤原代细胞,通过RT-PCR及western bloth技术检测h TERT在原代细胞内的表达变化,选取h TERT表达升高的细胞,利用有限稀释法获得高表达h TERT的单克隆细胞株。结果实验中成功获得h TERT目的基因片段,获得的细胞株与原代细胞相比h TERT表达上调,且体外传代50次后依然保持很好的细胞形态及活力。结论成功获得c-kit突变型人胃肠间质瘤永生化细胞系,为进一步研究c-kit突变对胃肠间质瘤的影响奠定基础。

Objective To establish immortalized c-kit mutant human gastrointestinal stromal tumors cell Line by overexpressing the human telomerase reverse transcriptase（ hTERT） in c-kit mutant human gastrointestinal stromal tumors primary cells.Methods The hTERT gene fragments was gotten by PCR and sequenced correctly,then construct the retroviral expression vectors of telomerase subunit（ hTERT） in vitro,and transfect the c-kit mutant GIST primary cells,detect the expression changes of hTERT by RT-PCR and western blot between the primary cells and the transfect cells,select upregulate hTERT cells.The limiting dilution method was used to obtain monoclonal cell lines with high expression of hTERT.Results hTERT up-expression in the obtained cell lines can be passaged more than 50 times with good cell morphology and viability,compared to the primary cells.Conclusion This method can successfully constructed the immortalized c-kit mutant human gastrointestinal stromal tumors cell lines,which is foundational for further study of c-kit mutation on the impact of gastrointestinal stromal tumor.