CAJ | 학술논문

目的构建人凝血因子VII(FVII)与免疫球蛋白Fc片段融合基因(mFVII/Fc)的慢病毒表达载体,并检测其在人骨髓间充质干细胞(hBMSCs)中的表达情况,获取mFVII/Fc稳定表达的干细胞载体.方法体外克隆人凝血因子VII,用点突变技术在基因水平将341部位Lys突变为Ala后,利用DNA连接酶与免疫球蛋白IgG1 Fc片段的基因融合,经酶切整合,测序鉴定后转染人胚肾293T细胞包装为重组mFVII/Fc慢病毒载体,鉴定载体构建成功后,测定慢病毒滴度.确定转染第三代hBMSCs的最佳MOI值后批量转染,荧光显微镜下观察GFP的表达情况,RT-PCR、ELISA分别在不同时间点对mFVII/Fc的mRNA及蛋白表达进行相对定量分析.结果所构建融合基因与GenBank ID,AF272774对比,除同义变异外完全相符;包装的慢病毒滴度为2×108TU/ml;hBM-SCs鉴定结果为CD+29(98.08%)、CD+44(97.63%)、CD34(0.31%)、CD+45(0.58%);转染hBMSCs 72 h后的效率为(84±3)%,经RT-PCR及ELISA证实转染mFVII/Fc基因的hBMSCs有大量mRNA转录和蛋白表达水平.结论实验成功获得了融合基因的hBMSCs稳定遗传表达载体,为靶向前列腺癌组织因子研究及肿瘤基因治疗研究提供了实验依据.
목적 구건인응혈인자VII(FVII)여면역구단백Fc편단융합기인(mFVII/Fc)적만병독표체재체,병검측기재인골수간충질간세포(hBMSCs)중적표체정황,획취mFVII/Fc은정표체적간세포재체.방법 체외극륭인응혈인자VII,용점돌변기술재기인수평장341부위Lys돌변위Ala후,이용DNA련접매여면역구단백IgG1 Fc편단적기인융합,경매절정합,측서감정후전염인배신293T세포포장위중조mFVII/Fc만병독재체,감정재체구건성공후,측정만병독적도.학정전염제삼대hBMSCs적최가MOI치후비량전염,형광현미경하관찰GFP적표체정황,RT-PCR、ELISA분별재불동시간점대mFVII/Fc적mRNA급단백표체진행상대정량분석.결과 소구건융합기인여GenBank ID,AF272774대비,제동의변이외완전상부;포장적만병독적도위2×108TU/ml;hBM-SCs감정결과위CD+29(98.08%)、CD+44(97.63%)、CD34(0.31%)、CD+45(0.58%);전염hBMSCs 72 h후적효솔위(84±3)%,경RT-PCR급ELISA증실전염mFVII/Fc기인적hBMSCs유대량mRNA전록화단백표체수평.결론 실험성공획득료융합기인적hBMSCs은정유전표체재체,위파향전렬선암조직인자연구급종류기인치료연구제공료실험의거.
Objective To construct a recombinant Ientiviral vector of mFVII/Fc and investigate its transfective efficiency into human bone mesenchymal stem cells (hBMSCs),and to detect the expression of mFVII/Fc fusion gene in vitro. Methods Coagulation factor VII (FVII) was cloned in vitro,with a point mutation from Lys to Ala in the position of 341 in the gene level.The cDNA fragments of mutational FVII (mFVII) and those of IgG1Fc were fused together with DNA ligase.After digestion,integration and sequencing,the fusion DNA was identified and transfected human embryonic kidney 293T cell packaging for re-mFVII/Fc lentiviral vector.After successful identification of vectors,detect the Ientiviral titer determination,bulk transfer after the determination of best MOI value of the third generation of hBMSCs,obseve the GFP expression with fluorescence microscope,have relative quantitative analyse of mRNA and protein expression of mFVII/Fc with RT-PCR and ELISA at different time points. Results In contrast with GenBank ID: AF 272774,the fusion gene matches exactly except the synonymous mutation,and the titer of packaging lentivirus was 2×108 TU/ml.Analyzed by Flow cytometry, indentification results of hBMSCs were as follows,CD+29(98.08%),CD+44 (97.63%),CD+34(0.31%) and CD+45(0.58%),respectively.The transfection efficiency of hBMSCs after 72 hours was (84±3)%,and the hBMSCs with mFVII/FC transfcetion have a large number of mRNA transcription and protein expression levels. Conclusions In this experiment we obtained a stable genetic vector with hBMSCs fusion gene expression successfully,which lay a foundation for the tissue factor study of prostate cancer targeting therapy and cancer gene therapy research.