摘要
目的:通过检测17-AAG对人RPE细胞不同处理条件下mRNA的SYBR Green实时PCR方法,观察17-AAG对人RPE细胞中PDGFR和EGFR基因表达的调控。方法:体外培养人RPE细胞株。当加入17-AAG并作用0,2,5,10μmol/L及0,16,24,48h后,收集细胞,抽提RNA进行反转录。同时根据PDGFR、EGFR的基因序列,设计PDGFR、EGFR的引物,利用ABI7300PCR仪和SYBR Green,建立实时荧光PCR反应条件,通过比较Ct值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明了PCR反应的特异性;相对定量结果显示,17-AAG可以下调PDGFR基因的表达(P<0.05),上调EGFR基因表达(P<0.05)。结论:SYBR Green实时荧光PCR可特异、准确地分析RPE细胞相关增殖基因表达差异,为系统研究17-AAG治疗PVR的复杂调控机制创造有力条件。
AIM： To establish a reliable SYBR Green real time PCR method for detecting the effect of 17-AAG on the expression of genes in human RPE cell. METHODS： RPE cells were cultured and incubated with 17-AAG for 16,24 and 48 hours, then collected for RNA extraction and reverse transcription. The primers were designed for the primer of PDGFR, EGFR based on their gene sequence. After optimization of PCR parameter, real time PCR was carried out on an ABI 7300 PCR Detection System with fluorescence dye SYBR Green . The relative quantification analysis was performed with comparative threshold cycle method. RESULTS： Melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification productS. Exposure to 17-AAG could down regulated the expression of PDGFR （P 〈 0.05） , but up regulated the expression of EGFR （P 〈 0.05） . CONCLUSION： The developed real time PCR for detecting the expression of genes related to RPE proliferation with high specificity and good quality is suitable to investigate the expression of genes and its regulation mechanism in development and diseases.
出处
《国际眼科杂志》
CAS
2008年第12期2431-2434,共4页
International Eye Science
