人同源盒基因HOXB4慢病毒载体的构建及其在人脐带间充质干细胞中的表达(英文)  

Construction of Lentivirus Vector Containing Human Homeobox Gene HOXB4 and Its Expression in Human Umbilical Cord Mesenchymal Stem Cells

作　　者：乔艳[1] 赵春亭[1] 刘竹珍[1] 冯献启[1] 王丽[2] 刘世海[3] 

机构地区：[1]青岛大学医学院附属医院血液科,山东青岛266003 [2]青岛大学医学院附属医院中美干细胞中心,山东青岛266003 [3]青岛大学医学院附属医院中心实验室,山东青岛266003

出　　处：《中国实验血液学杂志》2012年第3期703-709,共7页Journal of Experimental Hematology

基　　金：山东省自然基金项目,编号ZR2009CM056

摘　　要：本研究构建人同源盒基因(homeobox gene)HOXB4的慢病毒载体,探讨慢病毒介导的HOXB4感染人脐带间充质干细胞后的变化。利用PCR扩增获得HOXB4,并克隆入慢病毒穿梭载体lenti-shuttle;运用4质粒慢病毒包装系统转染HEK293T细胞,48 h收获慢病毒lenti-HOXB4,并测定慢病毒滴度;将lenti-HOXB4感染人脐带间充质干细胞,在倒置荧光显微镜下观察细胞感染效果,确定最佳的病毒感染复数(MOI);同时,利用RT-PCR、免疫荧光染色、CCK-8、流式细胞术检测HOXB4的表达情况及对细胞生长的影响。结果表明:利用四质粒共转染293T细胞,成功获得lenti-HOXB4,测定的病毒滴度为3×108TU/ml;当MOI为20时,lenti-HOXB4对人脐带间充质干细胞具有较高的转染效率,达80%以上;感染lenti-HOXB4的人脐带间充质干细胞,在mRNA和蛋白水平上均检测到了目的基因的表达,其能促进脐带间充质干细胞的增殖;流式细胞术检测结果显示,HOXB4基因并未明显影响间充质干细胞的表面标志。结论:成功获得带有HOXB4基因的慢病毒并实现在脐带间充质干细胞中表达,HOXB4基因能促进脐带间充质干细胞的大量增殖,并未明显影响脐带间充质干细胞表面标志的表达。This study was purposed to construct lentivirus vector containing human homeobox gene HOXB4 and explore changes of human umbilical cord mesenchymal stem cells（HUCMSC） after infected with HOXB4 mediated by lentivirus.PCR amplification was performed to obtain HOXB4,which was cloned in lenti-shuttle vector.Four-plasmid lentivirus packaging system was used to transfect HEK293T cells.After 48 h,lentivirus Lenti-HOXB4 was harvested and lentivirus titer was determined.Lenti-HOXB4 was used to infect HUCMSC.The infected cells were observed under inverted fluorescence microscope to determine the optimal multiplicity of infection（MOI）.Meanwhile,RT-PCR,immune fluorescence staining,CCK-8 and flow cytometry（FCM） were used to determine the expression of HOXB4 and its effect on cell growth.The results indicated that lenti-HOXB4 was successfully obtained by co-transfecting the 293T cells with four plasmids.The determined virus titer was 3×108 TU/ml;when MOI was 20.Lenti-HOXB4 had a high transfection rate in HUCMSC,over 80%.In HUCMSC infected with lenti-HOXB4,the expression of target gene could be detected both at mRNA and protein levels.It could promote the proliferation of HUCMSC.FCM results indicated HOXB4 gene did not significantly influence the surface marker of HUCMSC.It is concluded that HOXB4 gene can promote the high proliferation of HUCMSC and does not significantly influence the expression of the surface marker of HUCMSC.

关 键 词：HOXB4 人脐带间充质干细胞 慢病毒载体 

分 类 号：Q78[生物学—分子生物学]