慢病毒-SOX9基因载体的构建及其在小鼠骨髓间充质干细胞中过表达的研究  

Construction of SOX9 lentiviral vector and over-expression in mouse bone marrow mesenehymal stem cells

作　　者：白洁玉[1] 梁大川[1] 杨自权[1] 

机构地区：[1]山西医科大学第二临床医学院骨科,太原030001

出　　处：《中国医疗前沿》2012年第9期9-11,2,共4页China Healthcare Innovation

基　　金：国家自然科学基金(编号:30973048)

摘　　要：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 Construct SOX9 lentiviral vectors and transduce them into mBMSCs to acquire therapeutic seed cells for gene therapy of articular cartilage injuries.Methods Mouse SOX9 cDNA（NM_011448.4） was obtained from Gene bank and amplified by RT-PCR.SOX9 was linked to the lentiviral vector for constructing Lenti-SOX9EGFP vector.After the viral titer determination by real time quantitative PCR,Lenti-SOX9-EGFP vectors were transduced into mouse bone mesenehymal stem cells（mBMSCs）.The expression of SOX9 in mBMSCs was confirmed by immunofluorescence（80% approximately infected） and Western blotting.Results PCR（445bp）,DNA sequencing and Western blotting（83KDr） demonstrated that SOX9 lentiviral vectors were constructed successfully.Real time quantitative PCR determined that the viral titer is 2×108TU/ml.Immunofluorescence confirmed that 80% of SOX9 transduced cells could show green fluorescence.Western blotting demonstrated that SOX9 gene was stably expressed in SOX9 transduced mBMSCs.Conclusion We obtained the SOX9 expressed stably mBMSCs and these cells may be applied for gene therapy of articular cartilage injuries.

关 键 词：骨髓间充质干细胞 SOX9 慢病毒 软骨分化 

分 类 号：R392.1[医药卫生—免疫学]