逆转录病毒载体介导骨髓基质细胞体外表达人凝血因子Ⅷ  

Human factor Ⅷ expression in retrovirus vector-transduced bone marrow stromal cells

作　　者：郭雪梅[1] 王鸿利[1] 储海燕[1] 王学锋[1] 遽斌[1] 尹俊[1] 康文英[1] 段宝华[1] 戚正武[2] 王振义[1] 

机构地区：[1]上海第二医科大学瑞金医院,上海血液学研究所,200025 [2]中国科学院上海生物化学研究所

出　　处：《中华血液学杂志》2001年第9期461-463,共3页Chinese Journal of Hematology

基　　金：上海市科委新药发展基金资助项目 ( 975 4190 0 1)

摘　　要：目的　探索骨髓基质细胞用于血友病A基因治疗的可能性。方法　采用一包含了B区缺失 (△ 76 0aa～ 16 39aa)的人凝血因子ⅧcDNA(FⅧ△BcDNA)的复制缺陷型重组逆转录病毒LNC FⅧ△B ,在低温 (32℃ )和离心等改进条件下转化体外分离培养的小鼠和兔骨髓基质细胞。分别采用一期法、ELISA法和半定量PCR方法检测细胞培养上清中人FⅧ的凝血活性 (FⅧ :C)和抗原含量 (FⅧ :Ag)以及骨髓基质细胞的基因转化效率。并对转化细胞表达的FⅧ蛋白进行Westernblot分析。结果　与标准方法相比 ,离心和 32℃转化使小鼠骨髓基质细胞的基因转染效率提高 5 0 %～ 80 %。转化后的小鼠和兔骨髓基质细胞分泌的人FⅧ :Ag分别为每 2 4h(5 5 6± 80 )ng/ 10 6细胞和每 2 4h(4 80± 5 6 )ng/ 10 6细胞 ,FⅧ :C分别为每 2 4h 2 .42U/ 10 6细胞和 1.8U/ 10 6细胞。Westernblot分析显示 ,骨髓基质细胞分泌缺失B区的FⅧ蛋白 (FⅧ △ 760～ 163 9aa)与正常人血浆中的野生型FⅧ相似 ,主要以重 轻链异二聚体形式存在。结论　转化后的骨髓基质细胞可以高效分泌人FⅧ ,结合离心和低温对方法的改进 。Objective To explore the feasibility of bone marrow stromal cells (BMSCs) used for gene the rapy of hemophilia A. Methods Murine and rabbit BMSCs were transduced with a recombinant replication incompetent retrovirus LNC FⅧ△B, which encloses a B domain deleted (760aa 1?639aa) human factor Ⅷ cDNA. An optimized protocol involving temperature declining (32?℃) and centrifugation was used. Antigen and coagulant activity of human FⅧ in the cell culture medium were measured by ELISA assay and one stage method, respectively. Semi quantitative PCR was performed for the transduction efficiencies. Western blot was used for FⅧ protein analysis. Results Transduction efficiencies were increased 5 to 8 fold by temperature decline and centrifugation, which resulted in high expression and secretion of FⅧ in transduced mouse[(556±80)ng/10 6cells/24hr] and rabbit [480±56)ng/10 6cells/24hr] BMSCs. Coagulant activities of human FⅧ secreted from transduced murine and rabbit BMSCs were 2.42?U/10 6 cells/24?hr and 1.8 U /10 6 cells/24?hr, respectively. Western blot analysis demonstrated that the B domain deleted FⅧ cDNA was secreted predominantly as heavy and light chain heterodimers that resemble the native forms in human plasma. Conclusions It was demonstrated that the BMSCs system has potential utility in the gene therapy for hemophilia A.

关 键 词：凝血因子Ⅷ 病毒载体 基因表达 逆转录病毒载体 骨髓基质细胞 血友病 基因治疗 

分 类 号：R554.1[医药卫生—血液循环系统疾病]