慢病毒介导SOX9基因转染骨髓间充质细胞中的基因表达

摘要

Objective:To construct one lentiviral vector containing mouse SRY-related high mobility group-box gene 9 (SOX9) and transfect the murine bone mesenchymal stem cells (mBMSCs) in vitro and observe the expression of target gene.Methods:RNA from the vectors containing mouse SOX9 gene were extracted and SOX9 genes were amplified by reverse transcription-Polymerase Chain Reaction (RT-PCR).The SOX9 genes were connected into lentiviral vectors pGC-FU.Then pGC-FU-SOX9 transduced into 293T cells to produce recombinant lentivirus called as Lenti-SOX9-EGFP.mBMSCs were transfected.The expression of target gene was detected by immunofluorescence,RT-PCR and Western Blot.Results:LentiSOX9-EGFP was recombined successfully and transduced efficiently into mBMSCs.The expression of SOX9 gene was confirmed by RT-PCR and Western Blot.Conclusion:Lentiviral vector of mouse SOX9 gene can transfect successfully into mBM-SCs.Meanwhile,SOX9 gene may be expressed in mBMSCs.This will provide the target cells for the following study about SOX9 gene repairing cartilage injury.%目的:构建携带小鼠SOX9基因的慢病毒载体,体外转染小鼠骨髓间充质细胞,观察小鼠SOX9基因在小鼠间充质细胞中的表达.方法:从含有小鼠SOX9基因的质粒提取总RNA,利用RT-PCR方法扩增目的基因.连接目的基因与经Age-Ⅰ酶切线性化的慢病毒载体,转化感受态的大肠杆菌对质粒进行扩增,筛出阳性转化子,经293T细胞包装,收集病毒后,通过基因测序和限制性核酸内切酶酶切的方法对质粒进行鉴定.Lenti-SOX9-EGFP体外转染小鼠骨髓间充质细胞,利用倒置荧光显微镜观察转染是否成功,并通过流式细胞仪测定转染效率.同时利用RT-PCR和Western Blot检测小鼠SOX9基因的表达.结果:成功构建了携带SOX9基因慢病毒载体,Lenti-SOX9-EGFP能高效转染小鼠骨髓间充质细胞.RT-PCR和Western Blot检测显示经SOX9基因转染的小鼠骨髓间充质细胞表达目的基因产物.结论:利用慢病毒介导SOX9基因成功地转染小鼠骨髓间充质细胞,而且SOX9基因在小鼠骨髓间充质细胞中得到表达,这为SOX9修复软骨损伤的进一步研究奠定了基础.