人Toll样受体3基因表达水平的实时荧光定量PCR测定

Detection of the expression level of Toll-like receptor3 by establishing real-time fluorescence quantitative method

目的建立检测人Toll样受体3（TLR3）mRNA表达水平的实时荧光定量逆转录PCR（FQ-RT-Pert）法，及探讨其在外周血单个核细胞（PBMC）中的表达水平。方法用Beacon Designer2．1软件设计TLR3基因特异性引物和荧光探针，逆转录扩增目的片段，构建重组体pMD18-T-TLR3作为定量标准品。在ABIPrism7000型荧光定量分析仪上，通过荧光强度达到一定阈值时的循环数来定量起始模板，建立标准曲线；并检测30名正常人及20例原发性胆汁性肝硬化（PBC）患者，20例慢性乙型肝炎肝硬化患者外周血PBMCTLR3基因的荧光强度，根据标准曲线及循环阈值，由软件自动计算样本中TLR3mRNA的起始拷贝数。结果FQ-RT-PCR检测TLR3基因的线性范围为10^2～10^8拷贝数／μl RNA（r=0．9974），内参β-actin的线性范围为10^3～10^8拷贝数／μl RNA（r=-0．9984）；高浓度样本批内和批间变异系数（CV）分别为6．7％和8．7％，低浓度样本批内及批间CV分别为12．3％和14．0％，30名健康人，20例PBC及20例慢性乙型肝炎肝硬化患者PBMC中均有TLR3 mRNA的表达，分别为3．46×10^2～4．51×10^3拷贝／μg RNA、4．92×10^2～1．42×10^4拷贝／μg RNA和2．58×10^2～7．17×10^3拷贝／μg RNA。结论成功建立检测TLR3 mRNA的FQ-RT-PCR方法，可作为临床和基础研究TLR3基因表达的工具。

Objective To establish a specific fluorescence quantitative method for determining the mRNA expression of Toll-like receptor3 （TLR3） in peripheral blood mononuclear cells （PBMCs）. Methods Using the Beacon Designer 2. 1 software, specific primers and Taqman-MGB probe were designed. The plasmid pMD18-T-TLR3 was constructed as calibrator and the amplified fragment was obtained by reversetranscript-PCR （RT-PCR）. RNA quantification based on cycle threshold values （Ct） was used to establish the standard curve. According to which, the TLR3 mRNA levels in 30 normal individuals, 20 patients with primary biliary cirrhosis （PBC） and 20 ones with chronic liver cirrhosis induced by HBV were calculated automatically by software after the fluorescence of PCR product was detected continuously during amplification. Results The linear detection range of the assay for TLR3 gene and [3-actin was 10^2-10^8（ r = -0. 9974） and 10^3 - 10^5（ r = -0. 9984） , respectively. The coefficient of variation of both intra-and interassay reproducibility for high concentration sample were 6. 7% and 8.7%, respectively, and those for low concentration sample were 12.3% and 14. 0%. The TLR3 mRNA expression level ranges from 3.46 × 10^2- 4. 51 × 10^3 copies/μg RNA, 4.92 × 10^2-1.42 × 10^4 copies/μg RNA and 2. 58 ×10^2-7. 17 × 10^3 copies/μg RNA for 30 healthy individuals, 20 PBC patients and 20 ones with chronic liver cirrhosis induced by HBV, respectively. Conclusion We have successfully set up a FQ-RT-PCR method for detecting TLR3 mRNA, which may be used as an excellent tool for the clinic and basic study on the expression of TLR3 gene.