敲减SOX9基因对骨髓间充质干细胞表达的影响  

Gene expression of bone mesenehymal stem cells transduced by the lentiviral vector of SOX9 gene knockdown

作　　者：梁大川[1] 白洁玉[1] 杜少华[1] 程鹏[1] 王震[1] 康宁[1] 杨自权[1] 

机构地区：[1]山西医科大学第二医院骨科骨与软组织损伤修复山西省重点实验室,山西太原030001

出　　处：《中国骨伤》2013年第9期760-763,共4页China Journal of Orthopaedics and Traumatology

基　　金：国家自然科学基金资助项目(编号:30973048);国际科技合作项目(编号:2010DFA32450);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)~~

摘　　要：目的:构建小鼠S0X9基因干扰的慢病毒载体,体外转染小鼠骨髓间充质干细胞,观察小鼠SOX9基因在小鼠间充质干细胞中的表达影响。方法:针对小鼠SOX9基因序列,设计RNA干扰靶点序列,合成含干扰序列的双链DNAoligo连入酶切后的RNA干扰载体上,构建Lenti-SOX9-siRNA-EGFP载体,鉴定扩增。利用SOX9基因沉默载体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Lenti-SOX9-siRNA-EGFP,SOX9基因沉默慢病毒载体,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基因表达沉默。结论:利用慢病毒结合RNA干扰技术敲减小鼠SOX9基因成功地转染小鼠骨髓间充质干细胞,且SOX9基因在小鼠骨髓间充质干细胞中发生了沉默,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 silencing group-box gene 9 （SOX9） and to transfect murine bone mesenehymal stem cells（mBMSCs） in vitro and observe the expressi on of target gene.Methods：RNA inteference target sequence was designed in connectin with mice SOX9 genese quence.The double strands DNAoligo containing interference sequence were synthesized and cloned into len tivirus vector.The siRNA lentiviral vector with SOX9 gene silencing was constructed and identified,which was transfected into rat bone mesenehymal stem cells.The expression of target gene was detected by immuno fluorescence,RT-PCR and Western blot.Results：Lenti-SOX9-siRNA-EGFP was recombined successfully and tra nsduced efficiently into mBMSCs.The expression of SOX9 gene silencing was confirmed by RT-PCR and Western blot.Conclusion：Mouse SOX9 gene silencing by RNA interference and Lentiviral vector can transfected successfully into mBMSCs.Meanwhile,SOX9 gene may be silenced in SOX9 transduced mBMSCs.This will provide targ et cells for the following study about SOX9 gene respairing cartilage injury.

关 键 词：SOX9 慢病毒属 转染 骨髓间充质干细胞细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]