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构建人pcDNA3.1-GCNF真核表达载体,并瞬时转染宫颈癌HeLa细胞,观察GCNF基因对HeLa细胞凋亡的影响。采用PCR的方法从pMD18-T-GCNF载体中扩增全长GCNF cDNA,将扩增产物克隆至真核表达载体pcDNA3.1(-),测序鉴定后,重组质粒pcDNA3.1-GCNF转染HeLa细胞,RT-PCR以及间接免疫荧光鉴定转染结果,并用Annexin V-PI双染流式细胞术分析细胞凋亡情况。结果显示,克隆的重组真核表达载体经序列测定分析后,证实pcDNA3.1-GCNF重组真核表达质粒构建成功,并获得瞬时转染pcDNA3.1-GCNF的HeLa细胞。流式细胞术检测:转染重组质粒组的细胞凋亡率(6.72%)低于转染空质粒组(9.78%)以及未转染组(9.34%),P<0.00313,转染重组质粒后细胞凋亡率降低。由此可知,pcDNA3.1-GCNF重组质粒瞬时转染子宫颈癌HeLa细胞后,GCNF基因可以抑制HeLa细胞凋亡。
The eukaryotic expression vector pcDNA3.1-GCNF was constructed and transiently transfected into cervical cancer HeLa cells to observe the effect of GCNF gene on the apoptosis of HeLa cells. The full length GCNF cDNA was amplified by PCR from pMD18-T-GCNF vector. The amplified product was cloned into the eukaryotic expression vector pcDNA3.1 (-). After sequencing, the recombinant plasmid pcDNA3.1-GCNF was transfected into HeLa The transfected cells were identified by RT-PCR and indirect immunofluorescence. Cell apoptosis was analyzed by Annexin V-PI double staining flow cytometry. The results showed that the cloned recombinant eukaryotic expression vector was sequenced and confirmed that the pcDNA3.1-GCNF recombinant eukaryotic expression plasmid was successfully constructed and the HeLa cells transiently transfected with pcDNA3.1-GCNF were obtained. Flow cytometry showed that the apoptotic rate (6.72%) in the transfected plasmid group was lower than that in the transfected plasmid group (9.78%) and the untransfected group (9.34%), P <0.00313. After transfected with the recombinant plasmid Apoptosis rate decreased. Thus, GCNF gene can inhibit the apoptosis of HeLa cells after transient transfection of HeLa cells with pcDNA3.1-GCNF recombinant plasmid.