旨在构建编码大鼠铁蛋白重链多肽1(ferritin heavy chain 1,Fth1)的慢病毒,并检测其在大鼠骨髓间充质干细胞(RMSC)中的表达.PCR扩增大鼠Fth1基因,克隆至pLenti-GFP-C慢病毒表达载体,包装慢病毒,感染RMSC细胞.荧光显微镜观察细胞内GFP荧光情况,Western blot检测Fth1的表达情况,WST-1试剂检测Fth1慢病毒感染组(RMSC-Fth1)和空载体组(RMSC-GFP)的细胞增殖活性.DNA测序结果表明,pLenti-GFP-C-Fth1慢病毒载体构建成功.包装慢病毒感染RMSC细胞后,荧光显微镜下可见明显绿色荧光,表明感染成功.Western blot结果显示,实验组细胞裂解液中可检测到特异的Fth1表达条带.细胞增殖实验显示,与空载体组相比,过表达Fth1并不影响RMSC细胞生长.成功构建并包装携带大鼠Fth1基因的慢病毒,其能够成功感染RMSC细胞并表达Fth1蛋白,且对细胞增殖无明显影响.%Aims are to construct a lentivirus encoding rat ferritin heavy chain 1 (Fth1) and to detect its expression in rat bone mesenchymal stem cells (RMSCs).The rat Fthl gene was amplified by PCR and cloned into the lentivirus expression vector pLenti-GFP-C.The lentivirus carrying pLenti-GFP-C-Fth1 or empty vector was then packaged in HEK 293T cells,by which the RMSCs were infected.GFP expression was observed under a fluorescence microscope.Fthl expression was detected by Western blot.WST-1 reagent was used to analyze the proliferation of infected cells (RMSC-Fth1) and empty vector (RMSC-GFP).Results of DNA sequencing showed that the pLenti-GFP-C-Fth1 lentivirus expression vector was successfully constructed.After RMSCs were infected by the packaged lentivirus,GFP was observed under a fluorescence microscope,indicating the infection was achieved.A specific band of Fth1 in the lysate of Fth1-lentivirus infected cells (RMSC-Fth1) was detected by Western blot.Cell proliferation assay showed that the growth of RMSC was not affected by overexpressing Fth1 compared with RMSC-GFP.As conclusion,the recombinant lentivirus vector carrying rat Fth1 gene was successfully constructed,infected RMSC as expected,and expressed FTH1 protein even without significant impacts on the cell proliferation.
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