小鼠造血干细胞的分离、培养及重组慢病毒载体介导的离体hFⅨ基因表达

造血干细胞是基因治疗理想的靶细胞之一,尤其适用于遗传性血液病.而重组慢病毒载体能高效感染造血干细胞,成为造血干细胞途径基因治疗的理想载体.从小鼠骨髓细胞中分离出单个核细胞(MNCs)进行体外悬浮培养,并用免疫磁珠法分离得到高纯度的小鼠Lin-CD117+造血干细胞(HSCs).体外悬浮培养期间,添加细胞因子的造血干细胞的细胞数和集落数逐渐增加,而未添加细胞因子的对照组的细胞数量无明显增加,细胞集落递减.用磷酸钙介导的共转染法制备了携带FⅨ基因的FUXW重组慢病毒,用慢病毒载体分别感染从ICR小鼠和C57小鼠中分离得到的MNCs,7d后测得细胞上清中hFⅨ的表达量分别为41.7±4.2和34.5±6.6ng/mL,而慢病毒感染C57小鼠造血干细胞,添加细胞因子组上清中hFⅨ的表达量为46.6±5.7ng/mL,不添加细胞因子组为33.3±4.8ng/mL.实验结果表明,重组FUXW慢病毒载体可有效感染小鼠单个核细胞和Lin-CD117+造血干细胞,添加细胞因子可提高转移基因的表达量.

Isolation and Cultivation of Murine Hematopoietic Stem Cells and Expression of hFIX Mediated by Recombinant Lentiviral Vectors in vitro

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy of genetic disorders of the blood system.Recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for its high efficiency of infection.Murine mononuclear cells (MNCs) were isolated from bone marrow and in suspension culture,then high purity Lin~-CD117~+ HSCs were isolated by immunomagnetic beads.The numbers of cell and colony increased in HSCs supplied with cytokines contrast to control group after culturing.FUXW recombinant lentiviral vectors produced by calcium phosphate-mediated transient cotransfection infected MNCs of ICR and C57 mice.The hF expressions were 41.74.2 and 34.56.6 ng/mL in supernatant in 7 d for ICR and C57 mice,respectively.While the hF expressions of HSCs infected with FUXW recombinant lentiviral vectors were 46.65.7 (with cytokines) and 33.34.8 ng/mL (without cytokines) in supernatant in 7 d,respectively.Results indicated that recombinant lentiviral vectors can infected with murine MNCs and Lin~-CD117~+ HSCs efficiently.The expression of transgene can be improved by supplication with cytokines.