聚酰胺-胺型树枝状聚合物介导人凝血因子Ⅷ的体外高效稳定表达  

Effective and stable in vitro expression of human coagulation factor Ⅷ by retrovirus-based plasmid vector coupled with polyamidoamine dendrimer

作　　者：康文英[1] 王鸿利[1] 王学锋[1] 王红 王从珠 傅启华[1] 丁秋兰[1] 武文漫[1] 方怡[1] 段宝华[1] 

机构地区：[1]上海第二医科大学附属瑞金医院上海血液学研究所,200025 [2]上海市医学检验重点实验室

出　　处：《中华血液学杂志》2003年第9期464-466,共3页Chinese Journal of Hematology

基　　金：上海市科委科技发展基金资助项目

摘　　要：目的　应用纳米材料聚酰胺 胺型 (PAMAM)树枝状聚合物 逆病毒基础的质粒载体复合体 ,在表达水平、持续时间和细胞毒性三方面进行血友病A基因治疗的体外研究。方法　PAMAM树枝状聚合物与含B区缺失 (76 0aa～ 16 39aa)人FⅧcDNA(FⅧBDcDNA)的以逆病毒为框架的重组表达质粒载体pLNC FⅧBD形成复合体后 ,转染NIH3T3细胞系 ,于转染后第 2 ,5 ,10 ,15 ,30d留取细胞培养上清 ,分别采用一期法和ELISA法检测其中人FⅧ的凝血活性 (FⅧ∶C)和抗原含量 (FⅧ∶Ag) ,并应用RT PCR方法检测细胞中FⅧBDcDNA的转录 ,以细胞生长抑制率来表示PAMAM的细胞毒性。结果　PAMAM载体介导的人FⅧ的体外表达可持续 30d ,2 4h内每ml细胞上清中 10 6 细胞表达FⅧ∶C平均为 0 .92 9U ,FⅧ∶Ag平均为 0 .188μg ,期间FⅧ表达未见降低 ;PAMAM的细胞生长抑制率为 5 .32 %。 结论　PAMAM能够有效介导逆病毒基础的质粒载体pLNC FⅧBD转染靶细胞 ,并维持高效稳定表达 ,且PAMAM的细胞毒性较低。Objective To demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine(PAMAM) dendrimer in stable gene expression of FⅧ in vitro and to study the cytotoxicity of PAMAM. Methods The retrovirus-based plasmid vector pLNC-FⅧ BD was generated by cloning a B-domain-deleted (760aa～1639aa) FⅧ cDNA(FⅧBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FⅧ BD transfer FⅧ BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FⅧ in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FⅧ BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM. Results Human FⅧ was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FⅧ in these 30 days was 0.929 U/ml, and the FⅧ antigen was 0.188 μg/ml by 10 6 cells in 24 hours, respectively. The level of FⅧ didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%. Conclusion PAMAM could effectively transfer pLNC-FⅧ BD into NIH3T3 cells and FⅧ could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.

关 键 词：纳米材料 聚酰胺-胺型树枝状聚合物 人凝血因子Ⅷ 基因表达 细胞毒性 血友病 基因治疗 

分 类 号：R554.1[医药卫生—血液循环系统疾病] R394[医药卫生—内科学]