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目的构建慢病毒-SOX9基因载体并转染小鼠骨髓间充质干细胞以得到用于基因治疗软骨损伤的种子细胞。方法自Gene bank中获得小鼠SOX9的cDNA序列(NM_011448.4),设计两端引物,并在其两端分别引入Xhol和BamHI酶切位点序列,用RT-PCR法扩增出SOX9cDNA,连入慢病毒表达载体,构建慢病毒-SOX9-EGFP(Lenti-SOX9-EGFP)重组体。通过Real time定量PCR法测定滴度后使用Lenti-SOX9-EGFP转染小鼠骨髓间充质干细胞(mBMSCs)。采用免疫荧光和Westernblotting鉴定SOX9在mBMSCs中的表达。结果 PCR(445bp)、DNA测序、Western blotting(83KDr)检测证明Lenti-SOX9-EGFP载体构建成功。经Real time定量PCR法测定的病毒滴度为2×108TU/ml。Lenti-SOX9-EGFP转染mBMSCs 72h后经免疫荧光检测约80%的mBMSCs可表达SOX9。Western blotting也证明了SOX9在经SOX9基因转染的mBMSCs内稳定表达。结论获得了稳定表达SOX9的mBMSCs,为基因治疗关节软骨损伤奠定了基础。
Objective To construct lentivirus-SOX9 gene vector and transfect mouse bone marrow mesenchymal stem cells to obtain seed cells for gene therapy of cartilage injury. Methods The cDNA sequence of mouse SOX9 (NM_011448.4) was obtained from Gene bank. The two-terminal primers were designed and Xhol and BamHI restriction sites were introduced into both ends respectively. SOX9 cDNA was amplified by RT-PCR Into lentiviral expression vector to construct lentivirus-SOX9-EGFP (Lenti-SOX9-EGFP) recombinant. Real-time quantitative PCR was used to determine the titer of mouse bone marrow mesenchymal stem cells (mBMSCs) transfected with Lenti-SOX9-EGFP. The expression of SOX9 in mBMSCs was identified by immunofluorescence and Western blotting. Results PCR (445bp), DNA sequencing and Western blotting (83KDr) showed that Lenti-SOX9-EGFP vector was successfully constructed. The virus titer determined by Real time quantitative PCR was 2 × 108TU / ml. Approximately 80% of mBMSCs can express SOX9 by immunofluorescence after 72 hours of transfection of mBMSCs with Lenti-SOX9-EGFP. Western blotting also demonstrated that SOX9 is stably expressed in mBMSCs transfected with SOX9 gene. Conclusion The mBMSCs stably expressing SOX9 were obtained, which laid the foundation for the gene therapy of articular cartilage injury.