实时荧光PCR检测γ-干扰素对大鼠神经干细胞versican基因表达的调控

Detecting the Regulation of IFN-γ on the Expression of Versican Isoforms in Rat Neural Stem Cells by SYBR Green Real-time PCR

目的建立检测硫酸软骨素蛋白多糖versican不同亚型mRNA的SYBR Green实时PCR方法,观察炎症因子γ-干扰素对大鼠神经干细胞中不同versican亚型基因表达的调控。方法分离孕大鼠胚胎的神经干细胞,在含有营养因子的基质中进行体外培养。当加入处理因素γ-干扰素并作用48h后,收集细胞,抽提RNA进行反转录。同时根据versican不同亚型的基因序列,设计特异性不同的引物,利用ABI7900PCR仪和SYBR Green,建立优化的实时荧光PCR反应条件,通过比较Ct(△△Ct)进行基因表达的相对定量分析。结果熔解曲线分析和琼脂糖凝胶电泳结果证实了PCR反应的特异性;相对定量结果显示,γ-干扰素可以上调versican V2基因的表达,但对versican V1的表达无影响。结论应用SYBR Green实时荧光PCR可以特异、准确、快速地分析versican不同亚型的基因表达差异,为系统研究其复杂的调控机制创造了有力条件。

Objective To establish a reliable SYBR Green real-time PCR method for detecting the effect of IFN-γ on the expression pattern of versican isoforms in rat neural stem cell（NSCs）. Methods The NSCs were isolated from embryonic rat and cultured in growth medium supplement with EGF and bFGF in vitro. For cytokine stimulation,the cells were incu-bated with IFN-γ for 48 hours ,then collected for RNA extraction and reverse transcription. The primers were designed for different versican isoforms based on their gene sequence. After optimization of PCR parameter,real-time PCR was carried out on an ABI7900 PCR Detection System with fluorescence dye SYBR Green. The relative quantification analysis was performed with comparative threshold cycle （△△Ct） method. Results Melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification products. Exposure to 100U/ml IFN-γ could upregulated the expression of versican V2 by 2.9 folds （P〈0.01）, but had no effect on versican V1. Conclusion The developed real-time PCR assay for detecting the expression of versican isoforms,with high specificity and good quality,is suitable to investigate the expression pattern of versican isoforms and its regulation mechanism in development and diseases.