PEX基因转染人脐带间充质干细胞的体外培养及鉴定

In Vitro Culture and Identificationg of Human Umbilical Cord Mesenchymal Stem Cells Modified by PEX Gene

目的构建PEX基因真核表达载体并成功转染人脐带间充质干细胞(HuMSCs),探讨PEX基因修饰HuMSCs的可行性。方法采用全贴壁细胞分离法培养HuMSCs,流式细胞技术鉴定其表面标记物;分子克隆技术构建PEX基因真核表达载体[PEX-pcDNA3.1(+)],重组质粒PEX-pcDNA3.1(+)转染HuMSCs,验证真核表达载体在HuMSCs中的表达。结果①P5代HuCMSCs表面分子标志物结果显示各代均表达间充质干细胞标志CD73、CD90和CD105,而不表达造血干细胞标志CD11b、CD19、CD34、CD45和组织相容抗原HLA-DR;②RT-PCR检测显示,目的基因PEX位于DNA Maker 500~750 bp的特异性条带;抽提质粒后双酶切鉴定显示,重组质粒PEX-pcDNA3.1(+)位于DNA Maker 5000 bp的特异性条带,且重组质粒PEX-pcDNA3.1(+)双酶切后位于DNA Maker 500~750 bp和5000 bp两条特异性条带;菌液测序结果证实,质粒载体PEX-pcDNA3.1(+)构建成功;重组质粒PEX-pcDNA3.1(+)在HuMSCs中高表达。结论构建的PEX-pcDNA3.1(+)真核表达载体可用于转染HuMSCs,为进一步探讨其在胶质瘤治疗中的作用奠定了基础。

Objective To explore the feasibility of modifying human umbilical cord mesenchymal stem cells(HuMSCs)with PEX gene by the construction of the eukaryotic expression vector of PEX gene and the transfection of EEV of PEX gene into the HuMSCs.Methods HuMSCs were cultured by adherent cell separation method,and their surface markers were identified by flow cytometry.The eukaryotic expression vector of PEX gene[PEX-pcDNA3.1(+)]was constructed by molecular cloning technique.The recombinant plasmid PEX-pcDNA3.1(+)was transfected into HuMSCs to verify the expression of eukaryotic expression vector in HuMSCs.Results①The results of P5 HuCMSCs surface molecular markers showed that all generations expressed mesenchymal stem cell markers CD73,CD90 and CD105,but did not express hematopoietic stem cell markers CD11b,CD19,CD34,CD45 and tissue-compatible antigen HLA-DR.②RT-PCR detection showed that the target gene PEX was located in the specific band of DNA Maker 500-750 bp.The recombinant plasmid PEX-pcDNA3.1(+)was located in the specific band of DNA Maker 5000 bp,and the recombinant plasmid PEX-pcDNA3.1(+)was located in the specific bands of DNA Maker 500-750 bp and 5000 bp after double enzyme digestion.The sequencing results confirmed that the plasmid vector PEX-pcDNA3.1(+)was successfully constructed,the recombinant plasmid PEX-pcDNA3.1(+)was highly expressed in HuMSCs.Conclusion The constructed PEX-pcDNA3.1(+)eukaryotic expression vector can be used to transfect HuMSCs,which lays a foundation for further exploring its role in the treatment of glioma.