慢病毒介导的HIF-1α mODD在肿瘤细胞中的高表达

Lentiviral Vector Mediated High Expression of HIF-1α mODD in Tumor Cells

目的在肿瘤细胞中利用重组慢病毒介导缺氧诱导因子-1α突变体(HIF-1α mODD)在正常氧压下高表达,以模拟肿瘤缺氧微环境下肿瘤细胞中HIF-1α的高表达,为研究HIF-1介导的缺氧信号通路及其在肿瘤细胞中的作用提供实验模型。方法将含HIF-1αmODD基因片段的慢病毒表达质粒pWPI GW/HIF-1α mODD,与pVSVG及pSPAX质粒共转染人胚肾293T细胞,包装制备重组慢病毒,重组病毒经过纯化后直接感染GLC、H157和YTMLC等肿瘤细胞,并在正常氧压条件下培养,通过RT-PCR、免疫印迹等方法检测HIF-1α mODD在细胞中的表达水平。结果重组慢病毒介导HIF-1α mODD在GLC、H157和YTMLC细胞内获得高表达。结论成功模拟了肿瘤细胞在缺氧微环境下高表达HIF-1α的情况,为体外研究HIF-1α在肿瘤血管新生及肿瘤生长中的作用提供途径。

Objective To induce high expression of HIF -1α mODD in tumor cells mediated by lentiviral vector in normoxia condi- tion, and simulate high expression of HIF -1αin tumor cells under hypoxia microenvironment in vivo. Methods The packing cell line （human embryonic kidney 293T cells） was eotransfected with pWPI GW/HIF -1α mODD expression vector, pVSVG and pSPAX. The recombinant lentivirus was packaged and amplified, followed by purification, infection of GLC, H157 and YTMLC cells. The expression level of HIF -1α mODD gene was analyzed by RT - PCR and Western blotting. Results RT - PCR and Western blotting results demonstrated that HIF -1αmODD was highly expressed in GLC, H157 and YTMLC cells. Conclusion A model of high expression of HIF -1α in tumor cells hypoxia microenvironment was successfully created. It will provide a method to investigate the role of HIF -1αin tumor angiogenesis and growth in vitro.