摘要
目的:通过慢病毒转染UE7T-13细胞,构建Tet-on基因开关调控稳定表达肝细胞生长因子(hepatocyte growth factor,HGF)、成纤维细胞生长因子-4(fibroblast growth factor 4,FGF4)的人类骨髓间充质干细胞株.方法:全基因合成HGF和FGF4序列,通过酶切、重组分别构建包含目的基因HGF、FGF4的两个慢病毒载体plenti6.3/TO-HGF和plenti6.3/TO-FGF4,包装并计算其滴度.以U E7T-13细胞最适M O I值稀释慢病毒Lenti3.3/T R,并转染U E7T-13细胞,加入抗生素Zeocin筛选,获得带有Tet-on基因调控开关的UE7T-13-TR细胞株.采用已构建好的慢病毒plenti6.3/TO-HGF和plenti6.3/TOF G F4分别转染U E7T-13-T R细胞并经过抗生素Blasticidin筛选,构建Tet-on调控稳定表达HGF的UE7T-13-TR-HGF细胞株和Tet-on调控稳定表达FGF4的UE7T-13-TR-FGF4细胞株.通过Q-PCR、Western blot及ELISA在基因、蛋白及蛋白分泌水平检测目的基因HGF、FGF4的表达情况.结果:成功构建UE7T-13-TR-HGF和UE7T-13-TR-FGF4细胞株.经Q-PCR检测,当Tet存在时,HGF基因在UE7T-13-TR-HGF中的表达为无Tet时的78倍;FGF4基因则超过2万倍,且1μg/m L的Tet为较佳浓度.Western blot及ELISA结果在蛋白表达水平及蛋白分泌水平验证了以上结果,证明已合成的HGF及FGF4能成功分泌到细胞外.结论:通过慢病毒转染UE7T-13细胞,我们成功构建出Tet可调控稳定表达HGF、FGF4细胞株(UE7T-13-TR-HGF和UE7T-13-TRF G F4细胞株),为进一步的研究奠定了良好的实验基础.
AIM：To develop human bone marrow derived cells lines stably expressing Tet regulated hepatocyte growth factor（HGF） or fibroblast growth factor 4（FGF4） gene.METHODS：HGF and FGF4 genes were synthesized and then cloned into a lentiviral vector to result in plenti6.3/TO-HGF and plenti6.3/TO-FGF4,respectively.Lenti3.3/TR was transfected into UE7T-13 cells to develop a UE7T-13-TR cell line possessing Tet-on gene swift.Then,plenti6.3/TO-HGF and plenti6.3/TO-FGF4 were used to transfect UE7T-13-TR cell to result in UE7T-13-TR-HGF cell line that could stably express Tet regulated HGF and UE7T-13-TR-FGF4 cell line that could stably express Tet regulated FGF4.The expression of target genes was detected by Q-PCR,and the levels and secretion of proteins were detected by Western blot and ELISA.RESULTS：We successfully developed UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cell lines.Q-PCR analysis verified that the expression of the HGF gene in UE7T-13-TR-HGF in the presence of Tet was 78-fold higher than that in the absence of Tet,and the fold change for FGF4 was more than 20 thousand folds.Western blot and ELISA analyses verified that HGF and FGF4 proteins could be synthesized and secreted outside the cell membrane.CONCLUSION：We have successfully developed UE7T-13-TR-HGF and UE7T-13-TR-FGF4 cell lines through lentiviral transfection,which lays a foundation for further study.
出处
《世界华人消化杂志》
CAS
2015年第27期4317-4325,共9页
World Chinese Journal of Digestology
关键词
骨髓间充质干细胞
诱导分化
基因开关
人肝细胞
再生医学
Mesenchymal stem cells
Induced differentiation
Gene switch
Human liver cell
Regenerative medicine
