【摘要】 目的:构建人FGFR2IIIc重组慢病毒表达系统,感染并筛选获得大鼠肌原细胞L6表达人FGFR2IIIc的重组细胞株,初步研究FGFR2IIIc对L6细胞的作用。方法:从人胎盘组织中获得FGFR2IIIc基因,并克隆到Gateway慢病毒系统的入门载体pENTR-11,采用LR重组酶将重组的pENTR-FGFR2IIIc和表达载体pLenti6/V5-DEST进行重组反应得到pLenti6/V5-DEST-FGFR2IIIc。将该重组表达载体和Viral Packaging Mix包装质粒通过阳离子脂质体共转染293FT细胞,待细胞完全裂解后收集重组慢病毒颗粒上清液;取适量上清液感染L6细胞,杀稻瘟毒素筛选2～4周,挑单克隆细胞进一步扩大培养并建立重组细胞株。RT-PCR、间接免疫荧光和Western blot鉴定重组细胞株中目的基因FGFR2IIIc的表达,bFGF和硫酸乙酰肝素共同诱导各重组L6细胞株后流式细胞术分析细胞周期,形态观察检测成肌分化水平,并通过相关信号通路抑制剂分析与各信号通路的关系。结果:构建了含人FGFR2IIIc基因的重组慢病毒表达载体,获得的重组慢病毒颗粒能高效感染L6细胞,RT-PCR和间接免疫荧光实验说明正确表达目的基因;流式细胞术结果表明L6中高表达FGFR2IIIc的重组细胞株的G1/G0期的细胞增多;各重组细胞株分别加入Erk1/2和p38通路抑制剂抑制诱导2天,发现重组细胞株发生不同的形态变化。结论:通过慢病毒表达系统,成功构建高表达FGFR2IIIc的重组L6细胞株,FGFR2IIIc通过Erk1/2和p38通路影响了L6细胞的形态发生,该结果为下一步在大鼠L6细胞中研究FGFR2IIIc基因与成肌分化功能相关性奠定基础。更多还原

【Abstract】 Objective:To establish the recombinant L6 myoblasts with exogenous FGFR2IIIc by lentivirus and to distinguish their characters.Methods: FGFR2IIIc gene was obtained by RT-PCR from human placental and was cloned into pENTA-11.Recombinant expression vector pLenti6/V5-DEST-FGFR2-IIIc was obtained by using pENTA-FGFR2IIIc and pLenti6/V5-DEST plasmids with LR recombinase.Then such vector and Viral Packaging Mix vectors were co-transfected into 293FT cells to gain recombinant lentiviral particles.After infected by Lentivirus,stable L6 cell monoclones were selected by blasticidin for 2~4 weeks.The recombinant cell lines with exogenous FGFR2IIIc were identified by RT-PCR,indirect immunofluorescence and Western blot.Co-stimulated by both bFGF and HSPG,cell cycles of recombinant L6 cells were evaluated by flow cytometry.Their myogenic differentiation was observed with or without inhibitors of ERK and p38 signal pathway.Results: The vector pLenti6/V5-DEST-FGFR2-IIIc was reconstructed to gain lentiviral particles to establish recombinant L6 myoblasts with exogenous FGFR2IIIc.The recombinant L6-FGFR2IIIc group show an increase in the percentage of cells in the G1/G0 phase compared to the L6-vector group.Phosphorylated Erk1/2 inhibition with PD98059 leads to some cells myogenesis;And phosphorylated p38 inhibition with SB203580 leads to cells apoptosis.Conclusions: FGFR2IIIc can inhibit L6 myoblasts proliferation and result in the tendency of myogenic differentiation.更多还原