摘要
目的:建立检测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Akt,Raf,Hsp90,Hsp70基因表达的调控。方法:体外培养人RPE细胞株。当加入处理因素17-AAG并作用不同浓度及时间梯度后,收集细胞,抽提RNA进行反转录。同时根据Akt,Raf,Hsp90,Hsp70的基因序列,设计Akt1,Raf1,Hsp90,Hsp70的引物,利用ABI 7300PCR仪和SYBR Green,建立实时荧光PCR反应条件,通过比较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明了PCR反应的特异性;相对定量结果显示,17-AAG可以下调Akt及Raf基因的表达,上调Hsp90及Hsp70基因表达。结论:应用SYBR Green实时荧光PCR可以特异、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 Akt, Raf, Hsp90, Hsp70 genes in human RPE cell. METHODS: RPE cells were cultured in vitro and incubated with 17-AAG for 16, 24 and 48 hours, then collected for RNA extraction and reverse transcription. The primers were designed for Aktl, Raf1, Hsp90, and Hsp70 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 Akt and Raf ( P 〈 0.05), but up regulated the expression of Hsp90 and Hsp70. CONCLUSION: The developed real-time PCR assay 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9期1792-1794,共3页
International Eye Science