携带肾上腺髓质素基因腺病毒体外转染骨髓间充质干细胞及其蛋白的表达

Adenovirus carrying adrenomedullin gene for transfection of bone marrow mesenchymal stem cells in vitro and their protein expression

背景:肾上腺髓质素具有促进骨髓间充质干细胞增殖、生长及减少凋亡等作用。目的:观察携带肾上腺髓质素基因的腺病毒体外转染骨髓间充质干细胞及其蛋白的表达。方法:培养293A细胞,并扩增携带肾上腺髓质素基因的腺病毒(Ad-ADM),实验分为对照组、空载体组和Ad-ADM转染组,3组分别于细胞长满至50%～60%时加入等量的无血清L-DMEM、Ad-lacZ和Ad-ADM,噬斑法检测扩增后腺病毒的滴度;贴壁法体外分离、扩增培养大鼠骨髓间充质干细胞;Ad-GFP体外感染骨髓间充质干细胞后检测转染效率;RT-PCR检测Ad-ADM转染后肾上腺髓质素mRNA的表达。结果与结论:病毒扩增后噬斑试验测定Ad-ADM的病毒滴度为1.1×109pfu/mL。Ad-ADM感染骨髓间充质干细胞后,肾上腺髓质素mRNA的表达明显增加,对照组、空载体组未见ADMmRNA表达;量化结果表明其表达量从第1天开始逐渐增加,第7天达高峰,在11d时仍可检测其表达。提示腺病毒对骨髓间充质干细胞有较高的转染效率。感染Ad-ADM后,骨髓间充质干细胞可有效表达肾上腺髓质素。

BACKGROUND：Adrenomedullin is one of potential growth factor that can promote cellular differentiation,growth and reduce apoptosis.OBJECTIVE：To investigate the possibility of bone marrow mesenchymal stem cells transfected by adenovirus vector carrying adrenomedullin（Ad-ADM） ,and to determine the protein expression of adrenomedullin following transfection.METHODS：The 293A cells were cultivated and the Ad-ADM was amplified.The experiment was divided into three groups：control,blank vector and Ad-ADM transfection.Cells in three groups were cultured with serum-free L-DMEM,Ad-lacZ and Ad-ADM,respectively after they reached 50%-60% confluence.The virus titer of amplified Ad-ADM was detected by plaque methods.Bone marrow mesenchymal stem cells were isolated and expanded using the preplating methods.The efficiency of Ad-GFP transfected mesenchymal stem cells in vitro was determined.Adrenomedullin mRNA expression was detected with reverse transcription-polymerase chain reaction following transfection.RESULTS AND CONCLUSION：The virus titer of amplified Ad-ADM was 1.1×109 pfu/mL by plaque methods.After bone marrow mesenchymal stem cells were infected with Ad-ADM,the expression of adrenomedullin mRNA was significantly increased.The expression of adrenomedullin mRNA was not observed in the control group and empty vector group.Quantitative results showed that the expression gradually increased from day 1,reached the peak at day 7,and was still detectable at day 11.The Ad-ADM can successfully transfect bone marrow mesenchymal stem cells.Following transfection,bone marrow mesenchymal stem cells highly express adrenomedullin.