RT-PCR法测定脂肪组织解偶联蛋白mRNA表达水平的探讨

QUANTIFICATION OF UNCOUPLING PROTEIN mRNA EXPRESSION LEVEL IN ADIPOSE TISSUE BY RTPCR ASSAY

目的：建立解偶联蛋白（ＵＣＰ）ｍＲＮＡ表达水平的竞争性ＲＴ－ＰＣＲ测定法；并应用该方法测定ＵＣＰｍＲＮＡ在成人腹膜内与腹膜外脂肪组织的表达水平。方法：应用剪接重叠延伸ＰＣＲ（ＳＯＥ－ＰＣＲ）合成中央缺失型ＵＣＰ竞争者ＲＮＡ。ＵＣＰｍＲ－ＮＡ与作为内设标准的ＵＣＰ竞争者ＲＮＡ用相同的引物在同一反应体系中进行逆转录反应和ＰＣＲ扩增。通过比较ＵＣＰｍＲ－ＮＡ产物和ＵＣＰ竞争者ＲＮＡ产物的放射性信号强度求取ＵＣＰｍＲＮＡ表达水平。结果：腹膜内与腹膜外脂肪组织样品中均检测到ＵＣＰｍＲＮＡ；ＵＣＰｍＲＮＡ表达水平腹膜内脂肪组织为（６．８５３±４．２０７）ａｍｏｌ／ｆｍｏｌβ－ａｃｔｉｎ，腹膜外脂肪组织为（０．５２１±０．４９７）ａｍｏｌ／ｆｍｏｌβ－ａｃｔｉｎ（ｘ±ｓ）（Ｐ＜０．００１）。结论：本竞争性ＲＴ－ＰＣＲ方法测定脂肪组织ＵＣＰｍＲＮＡ表达水平灵敏、准确；ＵＣＰｍＲＮＡ在成人腹膜内与腹膜外脂肪组织均有表达，并且表达水平腹膜内脂肪组织高于腹膜外脂肪组织。

Objective: To establish a competitive reverse transcriptionPCR(cRTPCR) assay for quantification of uncoupling protein (UCP) mRNA and determine UCP mRNA expression levels in intra-and extraperitoneal adipose tissues of adult humans Methods: Splice overlap extensionPCR (SOEPCR) was used for synthesis of a competitor fragment that contained a deletion at its centerUCP mRNA and UCP competitor RNA which was used as an internal standard were reverse transcribed and coamplified in one reaction in which the same primers were usedThe signal intensities of UCP mRNA products were compared with the signal intensities of UCP competitor RNA products to quanlify UCP mRNA abundance Results: UCP mRNA was detected in all intraand extraperitoneal adipose tissue samples studied; UCP mRNA expression levels in intra and extraperitoneal adipose tissues were (68534207) amol/fmol actin and (05210497) amol/fmol actin respectively (P<0001) Conclusion: The cRTPCR assay is a sensitive and accurate method for quantification of UCP mRNA expression level in adipose tissue;UCP mRNA expresses in both of intraand extraperitoneal adipose tissues and UCP mRNA abundance is higher in intraperitoneal than in the extraperitoneal tissue