CXCR4基因慢病毒载体及其过表达骨髓间充质干细胞株的建立  

Construction of Lentivirus Vectorcarrying CXCR4 Gene and Bone Mesenchymal Stem Cells Over-expressing CXCR4 Gene

作　　者：修光辉[1] 孙洁[1] 尹云玉 周娟 陈献忠 凌斌[1] Xiu Guanghui;Sun Jie;Yin Yunyu(Intensive Care Unit,the No.4 Hospital Affiliated to Kunming Medical University,Kunming 650021,China)

机构地区：[1]昆明医科大学第四附属医院重症医学科,昆明650021

出　　处：《华中科技大学学报（医学版）》2019年第2期177-182,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81360289);云南省科技厅-昆明医科大学联合专项重点项目[No.2017FE468(-180)];面上项目(No.2012FB076);云南省高层次卫生计生技术人才培养项目(H-2017060);昆明医科大学研究生创新基金项目(No.2018S048)

摘　　要：目的构建表达大鼠CXCR4基因的慢病毒载体,建立CXCR4基因过表达骨髓间充质干细胞株。方法 PCR克隆CXCR4基因,将其连接到慢病毒表达载体PGMLV-6751上,转染293细胞,将构建的重组慢病毒载体与包装质粒混合物共转染293T细胞,收集病毒悬液,测定滴度,转染SD大鼠骨髓间充质干细胞,以嘌呤霉素进行筛选,定量PCR和Western blot检测转染后细胞目的基因mRNA和蛋白的表达水平。结果成功构建了表达CXCR4基因的慢病毒载体,病毒滴度为2×10^(8 )TU/mL,建立了CXCR4基因过表达的大鼠骨髓间充质干细胞株,经定量PCR和Western blot检测,过表达细胞株CXCR4基因的mRNA和蛋白表达水平均较对照组明显升高(均P<0.05)。结论成功构建携带CXCR4基因的慢病毒载体及CXCR4基因过表达的骨髓间充质干细胞株。Objective To construct the lentivirus vector carrying rat CXCR4 gene and the bone mesenchymal stem cells over-expressing CXCR4 gene. Methods CXCR4 gene was cloned by polymerase chain reaction and inserted to lentivirus vector named PGMLV-6751 which expressed Zs-Green1,to transfect 293 cells.The 293T cells were transfected with recombinant lentivirus vector and packaging plasmid mixture,and then the virus suspension was collected and the vector titer detected.The lentivirus vectors containing CXCR4 gene were transfected into the bone marrow mesenchymal stem cells from sprague-dawley rats,and then screened by Puromycin medium.The mRNA and protein expression levels of CXCR4 were detected by quantitative PCR(Q-PCR)and Western blotting,respectively. Results The recombinant lentivirus vector carrying rat CXCR4 gene was successfully constructed and its titer was 2×10^8 TU/mL.The bone mesenchymal stem cell line which over-expressed CXCR4 gene was confirmed.Q-PCR and Western blotting suggested that the mRNA and protein expression levels of CXCR4 gene were significantly higher in CXCR4 gene overexpreesion group than in the control groups(P<0.05). Conclusion The recombinant lentivirus vector carrying CXCR4 gene and bone marrow mesenchymal stem cells over-expressing CXCR4 gene are successfully constructed.

关 键 词：CXCR4 慢病毒载体 骨髓间充质干细胞 大鼠 

分 类 号：R[医药卫生]