CAJ | 학술논문

目的：构建 hi FGF2( high molecular weight isoform fi-broblast growth factor-2,hi FGF2)真核表达载体,并观察其过表达后对细胞凋亡的影响。方法设计合成hi FGF2 cDNA模板引物,Nhel和Hind III双酶切pDsRed1-N1质粒,T4 DNA连接酶重组hi FGF2质粒,PCR扩增目的基因,琼脂糖凝胶电泳检测及测序鉴定。将重组 hi FGF2质粒瞬时转染HEK293细胞,荧光倒置显微镜检测转染效率。 Annexin V-FITC/PI 双染法流式细胞仪检测细胞凋亡。结果 hi FGF2真核表达载体符合设计要求,瞬时转染HEK293细胞的转染率达70%以上。过表达hi FGF2,HEK293细胞FITC/PI双染阳性率达(29．12±2．81)%,与正常组、转染空载体组差别有显著性(P<0．01或P<0.05)。结论成功构建hi FGF2真核表达载体,过表达hi FGF2导致细胞凋亡。
목적：구건 hi FGF2( high molecular weight isoform fi-broblast growth factor-2,hi FGF2)진핵표체재체,병관찰기과표체후대세포조망적영향。방법설계합성hi FGF2 cDNA모판인물,Nhel화Hind III쌍매절pDsRed1-N1질립,T4 DNA련접매중조hi FGF2질립,PCR확증목적기인,경지당응효전영검측급측서감정。장중조 hi FGF2질립순시전염HEK293세포,형광도치현미경검측전염효솔。 Annexin V-FITC/PI 쌍염법류식세포의검측세포조망。결과 hi FGF2진핵표체재체부합설계요구,순시전염HEK293세포적전염솔체70%이상。과표체hi FGF2,HEK293세포FITC/PI쌍염양성솔체(29．12±2．81)%,여정상조、전염공재체조차별유현저성(P<0．01혹P<0.05)。결론성공구건hi FGF2진핵표체재체,과표체hi FGF2도치세포조망。
Aim To construct eukaryotic expressing plasmid of hi FGF2 ( high molecular weight isoform fi-broblast growth factor-2,hi FGF2) gene and to investi-gate its effect on apoptosis after its overexpression in HEK293 cells. Methods The DNA template primer was designed and synthesized. The pDsRed1-N1 plas-mids were digested by the restriction enzymes of Nhel and Hind III. The hi FGF2 was ligated with linearized pDsRed1-N1 by T4 DNA Ligase. The recombinant plasmid was identified by endonuclease digestion and sequenced. The recombinant hi FGF2 plasmid was transient transfected into HEK293 cells by Lipofectami-neTM 2000 Reagent. The transfection efficiency was de-tected by fluorescence inversion microscope. The cell apoptosis was detected by Annexin V-FITC/PI apopto-sis detection kit with flow cytometry analysis. Results The pDsRed1-N1 eukaryotic expression vector was consistent with the design. The recombinant hi FGF2 plasmid was transfected in HEK293 cells. The trans-fection rate was more than 70%. The FITC/PI dyeing rate in hi-FGF2 over-expression HEK297 cells was a-bout ( 29. 12 ± 2. 81 )%. Conclusions pDsRed1-N1 eukaryotic expression vector is successfully constructed and transfected into HEK293 cells. Over-expression of hi FGF2 induces cell apoptosis.