人肠系膜动脉平滑肌sGCα和sGCβ基因表达水平检测方法的建立

ESTABLISHMENT OF METHOD DETECTING GENE EXPRESSION LEVEL OF sGCα AND sGCβ IN HUMAN MESENTERIC ARTERY SMOOTH MUSCLE

目的:建立实时荧光定量PCR检测人肠系膜动脉平滑肌sGCα和sGCβ基因表达水平的检测方法,并构建相应质粒标准品,为进一步研究基因功能奠定基础。方法:提取人肠系膜动脉组织总RNA,采用RT-PCR方法获取sGCα、sGCβ、GAPDH基因片段,与pMD-18T vector连接,并转化到大肠杆菌DH5α细胞,通过PCR和测序鉴定重组质粒。提取含目的基因的质粒作为标准品,采用SYBR GreenⅠ法检测人肠系膜动脉平滑肌sGCα和sGCβ基因表达和构建标准曲线。结果:人肠系膜动脉平滑肌存在sGCα和sGCβ基因表达,质粒标准品经过PCR鉴定并测序,与数据库公布的序列完全一致,标准曲线线性关系好,相关系数R2>0.99。结论:建立了稳定的定量检测方法,并成功用于检测人肠系膜动脉平滑肌sGCα和sGCβ基因表达,构建质粒标准品能用于后续实验。

Objective：To establish a real-time PCR technique for detection of sGCα and sGCβ gene expression in human mesenteric artery smooth muscle,and to construct a DNA plasmid as the standard control for further studying the function of sGCα and sGCβ.Methods：Total RNA was extracted from human mesenteric artery smooth muscle,cDNA fragments of sGCα,sGCβ,GAPDH gene were aquired.The cDNA fragments were inserted into pMD 18-T vector,which were transformed into Escherichia coli DH5α.Their specificity was tested by direct PCR and sequencing method.The plasmids including sGCα,sGCβ,GAPDH gene were used as standards for standard curves constructing and detecting of the sGCα and sGCβ gene expression with SYBR GreenⅠ.Results：There was sGCα and sGCβ gene expression in human mesenteric artery smooth muscle.The sequences of the plasmids including the target genes were consistent with the Pubmed database completely.The standard curve has good linear correlation with R20.99.Conclusion：A good quantitative method is established for detecting sGCα and sGCβ gene expression in human mesenteric artery smooth muscle is established.The constructed plasmid standards can be used for further research.