摘要
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.
出处
《中国组织工程研究》
CAS
北大核心
2017年第9期1340-1345,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金资助项目(31160199)
广西区自然科学基金资助项目(2014GXNSFAA118263)~~