摘要
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。
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：Lenti-SOX9-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.
出处
《中国骨伤》
CAS
2013年第7期597-600,共4页
China Journal of Orthopaedics and Traumatology
基金
国际科技合作项目(编号:2010DFA32450)
国家自然科学基金资助项目(编号:30973048)
国家人事部及山西省人事厅留学回国人员科技活动择优资助项目
山西省留学基金项目(编号:107)
山西省自然科学基金资助项目(编号:2010011050-6)~~
