shRNA重组慢病毒载体在小鼠造血系统中的长期高效表达  

Long-term stable expression of shRNA recombinant lentiviral vectors in the hematopoietic system of mice

作　　者：黄馨[1] 石桂英[1] 陈显达[1] 鞠振宇[1] 

机构地区：[1]中国医学科学院实验动物研究所北京协和医学院比较医学中心卫生部人类疾病比较医学重点实验室,北京100021

出　　处：《军事医学科学院院刊》2010年第5期412-414,418,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家"十一五"新药专项支持(2009zx09501-026);国家自然科学基金面上项目(30771189)

摘　　要：目的构建在小鼠造血干细胞中高效表达的shRNA重组慢病毒载体并研究其在小鼠造血系统中的长期稳定表达。方法将p-CMV和重组并鉴定正确的p-SFFV慢病毒载体与辅助病毒载体PAX2和PMD2G混合后,采用磷酸钙方法共转染293T包装细胞,获得具有感染能力的成熟慢病毒感染经流式细胞仪分选并体外培养的小鼠骨髓造血干细胞,分析其表达效率后,选取感染效率较高的p-SFFV慢病毒载体感染小鼠骨髓造血干细胞,并通过骨髓移植技术输入放射线照射后的小鼠体内。在移植后4周和9周分别获取小鼠外周血样本,移植后16周获取小鼠骨髓样本,分析供体细胞在小鼠体内的表达。结果成功重组了p-SFFV慢病毒载体并在小鼠骨髓造血干细胞中高效表达(感染效率72.4%);骨髓移植实验表明,携带外源性shRNA的慢病毒载体可在小鼠造血系统内长期稳定表达(感染效率92%)。结论shRNA重组慢病毒载体在小鼠造血系统中可长期稳定表达。Objective To construct lentiviral short hairpin RNA（shRNA） expression vectors and have them expressed efficiently in the hematopoietic system of mice.Methods p-CMV and p-SFFV lentiviral vectors,PAX2 and PMD2G auxiliary vectors were mixed before 293T packaging cells were cotransfected by calcium phosphate to produce lentiviral vectors to infect HSCs of mice isolated by fluorescence-activated cell sorting（FACS）.The p-SFFV lentiviral vector which had higher transfection efficiency was used to infect HSCs of mice before infected cells were delivered into lethally irradiated mice.Four weeks and nine weeks after transplantation,the murine peripheral blood was harvested.Sixteen weeks after transplantation,the murine bone marrow was harvested.The proportion of donor cells was analyzed by FACS.Results The p-SFFV lentiviral vector was successfully restructured which could be effectively expressed in mouse bone marrow hematopoietic stem cells（infection efficiency was 72.4%）.The lentiviral vector carrying exogenous shRNA could be expressed in the murine hematopoietic system chronically and stably（infection efficiency was 92%）.Conclusion The recombinant shRNA lentiviral vector can be expressed in the murine hematopoietic system.

关 键 词：造血干细胞 慢病毒感染 骨髓移植 RNA干扰 

分 类 号：Q78[生物学—分子生物学]