SYBR green实时荧光定量PCR检测VEGF mRNA方法建立.docVIP

SYBR green实时荧光定量PCR检测VEGF mRNA方法建立[摘要]目的：建立SYBR green 实时荧光定量PCR 检测VEGF mRNA 水平的方法，并初步应用于美容相关疾患。方法：构建内参GAPDH 质粒，倍比稀释VEGF 和GAPDH 标准品，建立PCR 标准曲线，同时绘制熔解曲线，后应用SYBR Green 实时荧光定量PCR 检测正常皮肤、烧伤瘢痕、恶性黑素瘤细胞株A2058和WM793 cDNA的VEGF mRNA 水平。结果：VEGF、GAPDH的标准曲线，符合线性要求，相关系数r均为1，熔解曲线峰值单一，产物特异度好，四组样本的SYBR green 荧光定量PCR结果提示瘢痕组织、A2058、WM793 的VEGF mRNA 水平明显高于正常皮肤。结论：本研究成功建立了VEGF mRNA 水平的SYBR green 荧光定量PCR 检测方法。 [关键词]血管内皮生长因子；瘢痕疙瘩；血管瘤；恶性黑色素瘤；SYBR green实时荧光定量PCR [中图分类号]R826.8[文献标识码]A[文章编号]1008-6455（2008）06-1025-03 Establishment of VEGF mRNA detection by using SYBR green real-time fluorescent quantitative PCR LUAN Qi,SUN Jing,SUN Lin-chao,LI Chun-ying,GAO Tian-wen (Institute of Dermatology of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi, China) Abstract:ObjectiveTo establish a method of SYBR green real-time quantitative PCR for detecting mRNA levels of VEGF, and apply the method to cosmetic diseases preliminarily.MethodsThe recombinant plasmid of GAPDH was constructed as standard control. Standard curves of VEGF and GAPDH were established by using a series dilution of quantified plasmids and the specificity of PCR products was analysed by their melting curves. VEGF mRNA levels of normal skin, burning scar, A2058 and WM793 melanoma cell lines were measured by SYBR green real-time quantitative PCR.ResultsTwo standard curves of VEGF and GAPDH had a linear feature and their R values were both 1. In addition, the melting curve analysis showed a single peak. The results of four groups showed that the VEGF mRNA levels were higher in burning scar tissue, A2058 cells and WM793 cells than that in normal skin tissue.ConclusionThe method of detecting VEGF mRNA levels by SYBR green real-time quantitative PCR was established successfully in this study. Key words: vascular endothelial growth factor(VEGF); keloid; hemangioma; m alignant melanoma; SYBR green real-time quantitative PCR 血管内皮生长因子（Vascular endothelial growth factor,VEGF）作为一类功能多