基因重组腺病毒载体与慢病毒载体转染兔骨髓间充质干细胞的对比

Comparison of lentiviral vector and adenoviral vector mediated gene transfer into rabbit bone marrow mesenchymal stem cells

背景:腺病毒载体和慢病毒载体均是较好的组织工程基因载体,两者在介导骨形态发生蛋白2转染兔骨髓间充质干细胞中的差异尚待探究。目的:比较腺病毒和慢病毒载体介导EGFP/骨形态发生蛋白2转染体外培养兔骨髓间充质干细胞的转导效率、持续时间及外源基因表达差异。方法:将第5代兔骨髓间充质干细胞分3组培养,A组以腺病毒载体介导EGFP/骨形态发生蛋白2(Ad-EGFP/BMP-2)转染细胞,B组以慢病毒载体介导EGFP/骨形态发生蛋白2(Lenti-EGFP/BMP-2)转染细胞,C组为未进行转染。转染24 h、48 h、72 h、1周、3周,检测EGFP基因表达;转染72 h后,免疫组织化学观察细胞骨形态发生蛋白2的表达;转染后72 h、1周、3周,Western blot检测骨形态发生蛋白2蛋白表达。结果与结论:(1)转染24 h后,A、B组可见EGFP表达,A组明显强于B组;转染48 h后,A、B组荧光进一步增强;转染72 h后,A组荧光强度近高峰,B组荧光持续增强。转染1周后,A组荧光强度开始下降,B组荧光强度仍然增强。转染3周后,A组荧光强度明显下降甚至消失,B组荧光强度有所下降,但仍然保持一定的表达。C组在各时间点均无EGFP表达;(2)A、B组胞浆均呈骨形态发生蛋白2阳性表达,C组呈阴性表达;(3)转染72 h后,A组骨形态发生蛋白2蛋白表达量强于B组;转染1周后,A组表达下降,B组表达增强并强于A组;转染3周后,A组表达微弱,B组持续表达且明显强于A组;(4)结果表明,相对腺病毒载体,慢病毒载体介导EGFP/骨形态发生蛋白2基因转染兔骨髓间充质干细胞在表达持续时间上具有一定的优势。

BACKGROUND：Both lentiviral vector and adenoviral vector are considered as good vectors for gene mediation,and their differences in transferring bone morphogenetic protein 2（BMP-2）into rabbit bone marrow mesenchymal stem cells（BMSCs）are unclear.OBJECTIVE：To compare the duration,efficiency and the deviation of exogenous gene expression after rabbit BMSCs transfection using lentiviral vector and adenoviral vector which are used to mediate enhanced green fluorescent proteins（EGFP）and BMP-2.METHODS：Rabbit BMSCs at passage 5 were exposed to Ad-EGFP-BMP-2（group A）or Lenti-EGFP-BMP-2（group B）with multiplicity of infection of 100,as transfection groups.And in control group（group C）,the same quality of culture medium was required equivalent to the groups A and B.The expression of EGFP was observed by inverted fluorescence microscope at various time intervals.And the expression of exogenous gene BMP 2 in cells was detected and analyzed by immunohistochemical staining at 72 hours after transfection as well as by western blot at 72 hours,1,3 weeks after transfection.RESULTS AND CONCLUSION：The intense green fluorescence emerged under the microscope at 24-48 hours after transfection in group A,which was stronger than group B,reached the peak at 72 hours,and then decreased at 1 week until disappearance at 3 weeks.No EGFP expression was detected in group C.High expression of BMP-2 was found in group A but was dramatically downregulated after 1 week.Group B showed the high expression of EGFP/BMP-2 persisted for a longer period after transfected that even lasted for 3 weeks.Overall,the lentiviral vector and adenoviral vector can efficiently transfect rabbit BMSCs and stably express the target gene of EGFP/BMP-2.Under the same MOI,compared to the adenoviral vector,transfection of lentiviral vector to rabbit BMSCs is more effectively and expression of EGFP/BMP-2 can be persistent in a longer term.