腺病毒介导的B结构域缺失的人凝血因子Ⅷ在SD大鼠ADSC中的表达

Adenovirus-mediated Expression of B-domain-Deleted Human Coagulation FactorⅧin ADSC of SD Rats

目的:将一含B结构域缺失的人凝血因子Ⅷ的重组腺病毒载体体外转染SD大鼠脂肪干细胞(adipose tissue-derived stem cell,ADSC),为ADSC联合BDDhFⅧ基因治疗血友病A奠定实验基础。方法:取SD大鼠2侧腹股沟脂肪组织分离培养ADSC,传至第3代并进行鉴定;用重组腺病毒载体Ad-BDDhFⅧ-GFP体外感染第3代ADSC,实验分为Ad-BDDhFⅧ-GFP转染ADSC(A组)、Ad-GFP转染ADSC(B组)及未转染ADSC(C组)3组,采用CCK-8法检测3组感染后细胞增殖情况,ELISA法检测细胞上清中hFⅧ抗原表达水平,RT-PCR及Western blot分别检测感染后3组细胞内BDDhFⅧ基因和蛋白表达情况。结果:经鉴定分离培养的第3代细胞生长曲线呈倒"S"形;流式细胞术检测显示,第3代细胞CD29、CD90、CD44表达阳性、CD45表达阴性;经成脂及成骨诱导后可向成骨及成脂方向转化。CCK-8检测显示,转染后3组细胞的增殖未受影响。ELISA结果显示,转染后3组细胞在72 h A组hFⅧAg表达较B、C组明显升高(P<0.05)。RT-PCR结果显示,与A组相比,B、C组未见任何目的条带,无BDDhFⅧ目的基因表达;A组结果与扩增片段长度相符,有BDDhFⅧ目的基因表达。Western blot检测显示,A组hFⅧ蛋白表达水平较B、C组明显升高(P<0.05)。结论:重组腺病毒Ad-BDDhFⅧ-GFP在体外可高效转染大鼠ADSC,为血友病A的基因治疗奠定了实验基础。

Objective:To transinfect SD adipose tissue-derived stem cell(ADSC)in vitro with a recombinant adenoviral vector containing human B-domain-deleted FVIII(BDDhFⅧ),so as to lay the foundation for the treatment of hemophilia A by using ADSC combined with BDDhFⅧgene.Methods:ADSCs were isolated from the inguinal adipose tissue of SD rats and passed to third passage for identification.Third passage ADSCs were transfected in vitro with recombinant adenovirus vector Ad-BDDhFⅧ-GFP.The experiments were divided into Ad-BDDhFⅧ-GFP-transfected ADSCs group(A),Ad-GFP-transfected ADSC group(B),and untransfected ADSC group(C).CCK-8 method was used to detect the proliferation of transfected cells in three groups,and the expression level of hFⅧantigen in cell supernatant was detected by ELISA.RT-PCR and Western blot respectively were used to detect the mRNA and protein expression of BDDhFⅧin the three groups after transfection.Results:The growth curve of third passage cells isolated and cultured showed an inverted"S"shap;the flow cytometry detection showed the positive expression of CD29,CD90,CD44,and the negative expression of CD45 in third passage cells.After the adipogenic and osteogenic induction,the cells could transformed to adipogenic and osteogenic directions.CCK-8 detection showed that the proliferation of cells in 3 groups not was influenced.ELISA showed that the expression of hFⅧAg in group A was significantly higher than that in group B and C(P<0.05).RT-PCR showed that compared with group A,there was no target band in B and C groups,and BDDhFⅧgene was not expressed.The results in group A were consistent with the length of amplified fragments,and BDDhFⅧtarget gene was expressed.Western blot analysis showed that the expression of hFⅧprotein in group A was significantly higher than that in group B and C.(P<0.05).Conclusion:Recombinant adenovirus Ad-BDDhFⅧ-GFP can effectively transfect rat ADSC in vitro,which lays an experimental foundation for gene therapy of hemophilia A.