转染鼠脂肪干细胞用于血友病A基因治疗的可能性实验

Possibility of the transfected adipose tissue-derived stromal cells in the gene therapy of hemophilia A

目的:探索表达载体含有B结构域缺失的人凝血因子ⅧcDNA的重组质粒能够成功转染脂肪干细胞作为血友病A基因治疗靶细胞的可能性。方法:实验于2004-01/06在武汉大学医学院病理生理学实验室完成。先将重组质粒表达载体含有B结构域缺失的人凝血因子ⅧcDNA的重组质粒扩增、纯化和鉴定。取大鼠的腹腔和肾后脂肪组织,分离出脂肪干细胞。当培养细胞生长至80%融合时,将重组质粒表达载体和阳离子脂质体Lipofectamine转染试剂以0.5∶1,1∶1,1.5∶13种不同的质量比混合后转染大鼠的脂肪干细胞。培养36h后分别采用比色法和半定量多聚酶链式反应方法检测培养细胞上清中人凝血因子Ⅷ的凝血活性和脂肪干细胞的外源基因转化效率。结果:①转化效率结果:质量比为1.5∶1时转染效率比质量比为0.5∶1时约高5%,质量比为1∶1时转染效率最高,约比质量比为0.5∶1时高50%。②转化36h后检测脂肪干细胞上清中的人凝血因子Ⅷ活性的结果显示:表达载体与阳离子脂质体质量比为0.5∶1时,人凝血因子Ⅷ活性为0.2%;表达载体与阳离子脂质体质量比为1∶1时,人凝血因子Ⅷ活性为1.6%;表达载体与阳离子脂质体质量比为1.5∶1时,人凝血因子Ⅷ活性为0.4%。没有表达载体转染的脂肪干细胞上清中人凝血因子Ⅷ活性为0;正常人凝血因子Ⅷ活性为50%～180%。结论:外源基因含有B结构域缺失的人凝血因子ⅧcDNA的重组质粒能成功转染鼠脂肪干细胞,而且转化后的细胞能够表达人凝血因子Ⅷ,但表达的人凝血因子Ⅷ活性不高,这可能与本次实验中质粒的纯度不高、转染效率不高和其他原因有关。

AIM:To investigate the feasibility of adipose tissue-derived stromal cells(ADSCs) which were successfully transfected by expression vector of recombinant plasmid pRC/RSV-FⅧ BD cDNA used as the target cells for gene therapy of hemophilia A.METHODS:The experiment was finished in the laboratory for pathophysiology,Medical College of Wuhan University from January 2004 to June 2004.At first,recombinant plasmid pRC/RSV FⅧ BD was proliferated,purified and identified.ADSCs were separated from rat abdominal cavity and postrenal adipose tissues.ADSCs were transfected with recombinant plasmid pRC/RSV FⅧ BD and lipofectamine(transfection reagent) at different ratio(W/W 0.5∶ 1,1∶ 1,1.5∶ 1) when they grew in a confluence rate of 80% . After 36 hour culture,the coagulant activity of human FⅧ in the supernatant and transfer efficiency of exogenous gene in ADSCs were measured by chromometry and semi quantitative polymerase chain reaction respectively.RESULTS:① Transfer efficiency was 5% higher when W/W was 1.5∶ 1 than when W/W was 0.5∶ 1,and 50% higher when W/W was 1∶ 1 than when W/W was 0.5∶ 1,so it was the highest when W/W was 1∶ 1.② Coagulant activity of human FⅧ in the supernatant 36 hours after ADSCs were transferred was 0.2% when W/W of recombinant plasmid and lipofectamine was 0.5∶ 1,1.6% when W/W of recombinant plasmid and lipofectamine was 1∶ 1 and 0.4% when W/W of recombinant plasmid and lipofectamine was 1.5∶ 1.Coagulant activity of human FⅧ was 0 when ADSCs were not transferred,and it was between 50% and 180% of normal human FⅧ .CONCLUSION:Exogene(pRC/RSV FⅧ BD) can be transfected successfully into ADSCs,and human FⅧ can be expressed in the transfected ADSCs with a low coagulant activity,which may be associated with the low purity and low transfer efficiency of plasmid in our experiment,or other reasons.