实时定量PCR检测GDNF基因转染大鼠BMSCs后的转录水平

Determination of the transcription level of GDNF gene in rat bone marrow mesenchymal stem cells after transfection by quantitative real-time PCR

目的了解外源性胶质细胞源性神经营养因子(GDNF)基因在大鼠骨髓间充质干细胞(BMSCs)中的转录水平。方法体外分离培养大鼠BMSCs,将其随机分为GDNF基因转染组、空质粒转染组和未转染组3组;GDNF基因转染组和空质粒转染组分别用GDNF基因和空质粒重组腺病毒感染BMSCs,并按感染后培养时间的不同(3、6、9和12天)又分为4个亚组。采用实时定量PCR技术检测GDNF基因在BMSCs中的转录水平。结果空质粒转染组和未转染组无GDNFmRNA的表达,GDNF基因转染组在转染后3～12天的GDNFmRNA相对表达量为0.00751±0.00067。结论腺病毒介导的基因转染技术将GDNF基因转染至骨髓间充质干细胞中,有外源性基因GDNFmRNA的表达,这为进一步研究以GDNF基因转染的BMSCs治疗中枢神经系统疾病奠定了基础。

Objective To study the transcription level of exogenetic glial cell line-derived neurotrophic factor （GDNF） in rat bone marrow mesenchyrnal stem cells （BMSCs）. Methyls The rat BMSCs were isolated and cultured in vitro, and randomly divided into three groups： GDNF-transfected group, blank plasmid-transfected group and non-transfected group. GDNF-transfected group and blank plasmid-transfected group were infected by the recombinant adenovirus of GDNF gene and blank plasmid, respectively. Each of the groups was divided again into 4 subgroups according to the cultured time （3d, 6d, 9d and 12d） after infection. The transcription level of GDNF gene in BMSCs was tested by quantitative real-time PCR. Results Blank plasmid-transfected group and non-transfected group had no expression of GDNF mRNA, and the relative level of GDNF mRNA transcription was 0. 00751±0. 00067 in GDNF-transfected group during the following 3-12 days after transfection. Conclusion Gene transfection technoqie mediated by adenoviral vector can transfect GDNF gene into BMSCs with the expression of exogenetic GDNF mRNA. BMSCs transfected with GDNF gene will be used for further research on therapy for the disease of central nervous system.