人凝血因子ⅧcDNA在小鼠体内的转染与表达  

In vivo Transfection and Expression of Human Coagulant Factor Ⅷ cDNA in Mice

作　　者：康文英[1] 王鸿利[1] 王红 王学锋[1] 王从珠 傅启华[1] 丁秋兰[1] 武文漫[1] 方怡[1] 王振义[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,上海200025 [2]上海市医学检验重点实验室,上海200023

出　　处：《中国实验血液学杂志》2004年第2期188-193,共6页Journal of Experimental Hematology

基　　金：上海市科委科技发展基金资助项目 编号 0 14 1190 2 0

摘　　要：本研究观察逆转录病毒载体和聚酰胺 胺型 (PAMAM)树枝状聚合物介导的人凝血因子Ⅷ (FⅧ )基因在小鼠体内的转染和表达 ,并与体外转染方法作比较。应用含B区缺失 ( 76 0aa- 16 39aa)人FⅧcDNA(FⅧBDcDNA)的以逆转录病毒为框架的重组表达质粒载体 pLNC FⅧBD包装成为逆转录病毒LNC FⅧBD ,exvivo转染小鼠骨髓基质细胞 ,同时将 pLNC FⅧBD与PAMAM树枝状聚合物按照 1∶15混合以形成复合体PAMAM pLNC FⅧBD进行小鼠invivo转染。分别于注射后第 2 4 ,4 8小时 ,第 1,2 ,3,4周取血 ,分离血浆 ,采用一期法测定人FⅧ活性 (FⅧc) ,ELISA法测定人FⅧ抗原 (FⅧAg) ,并采用Bethesdainhibitorassay测定人FⅧ抗体 ;同时取脏器肝、脾、肺、肾提取RNA ,进行RT PCR ,观察各组织的转录 ,并用苏木素 伊红染色法观察各组织的形态学改变。结果表明 ,逆转录病毒转染人FⅧ基因的骨髓基质细胞输入体内后 ,可以在体内继续表达人FⅧ ,并且能有效地分泌至血液中。宿主小鼠体内可检测到的人FⅧ表达持续 3周以上 ,注射后第 2 4小时表达最高水平 ,人FⅧAg平均为 8.6ng/ml,此后人FⅧ抗体逐渐生成 ,人FⅧAg水平渐低 ,于注射后第 4周不再能测出人FⅧAg。注射复合体PAMAM pLNC FⅧBD在体内转染后 ,获得了短暂的人FⅧ生理水平的表达 ,在注射?The aim is to observe the expression of human factor Ⅷ gene in mice tranduced in vivo and ex vivo. The vector pLNC FⅧ BD was generated by cloning a B domain deleted (760aa-1639aa) FⅧ cDNA (FⅧBD cDNA) into retroviral vector pLNCX. 2×10 6 of mouse bone marrow stroma cells transduced by LNC FⅧ BD were infused into 4 week old BALB/c mice by tail vein injection. pLNC FⅧ BD was conjugated with PAMAM dendrimer to form complex PAMAM pLNC FⅧ BD, with which C57BL/6J were injected by tail vein (200 μl contained 15 μg/mouse) and sacrificed at days 1, 2, 7, 14, 21 and 28, respectively after injection. Tissue such as liver, spleen, lung and kindney were harvested, with which the transcription were detected by means of RT PCR. In addition, blood was collected to be measured human FⅧ Ag, human FⅧc and anti FⅧ of human inhibitors. The results showed that the highest level of human FⅧ in the recipient BALB/c mice was 8.6±1.44 ng/ml detected on the first day post injection; anti FⅧ antibodies were detected from the first week post injection, and then the level of FⅧ Ag decreased and cannot be measured on the fourth week. In the C57BL/6J mice physiological level of human FⅧ was expressed in plasma at 48 hours after injection and the average human FⅧc was 0.62 U/ml and the average human FⅧ Ag was 115.5 ng/ml, and gradually reduced later. Anti FⅧ of human inhibitors was not revealed all the time. Syngene image scanning demonstrated that the transcription of the human FⅧ BD cDNA occurred mainly in spleen and lung, and secondarily in liver and kidney. No side effects of PAMAM pLNC FⅧ BD were observed in mice tissue by pathological examination at 4 weeks. In conclusion, retrovirus transduced bone marrow stroma cells effectively produced human FⅧ after ex vivo transduction, but the development of anti FⅧ antibodies in recipient mice influenced the expression level. The human FⅧ gene can successfully be tranduced in vivo through injecting PAMAM pLNC FⅧ BD cDNA into

关 键 词：逆转录病毒 聚酰胺-胺型树枝状聚合物 凝血因子Ⅷ 基因治疗 

分 类 号：Q78[生物学—分子生物学] R544.1[医药卫生—心血管疾病]