PD-L1基因实时荧光定量RT-PCR法的建立及在大鼠肝移植中的应用

Establishment of real-time quantitative RT-PCR assays for detecting PD-L1 of liver transplantation in rat

目的建立real-time R T-qPCR(实时荧光定量R T-PCR)检测大鼠PD-L1的方法,应用该方法检测大鼠移植肝中PD-L1 mR NA的水平。方法两袖套法复制大鼠肝移植耐受组(BN→BN)及排斥组(LEW→BN)模型,术后7 d处死受体,检测肝功能ALT和AST水平,HE染色病理分析排斥程度,Trizol法提取移植肝总RNA并反转录为cDNA;选β-actin作为内参,复制SYBR GreenⅠreal-time RT-qPCR检测法。利用该方法检测移植肝中PD-L1和β-actin的初始模板量,PD-L1/β-actin计算PD-L1 mRNA的相对表达量。结果异基因LEW→BN组出现急性排斥反应,ALT、AST排斥组均高于耐受组(均P<0.05)。RAI评分排斥组高于耐受组(均P<0.05)。PD-L1扩增效率为98.8%,相关系数为0.996;溶解曲线为特异单峰;变异系数小于2.0%;移植肝中PD-L1 mRNA相对表达为耐受组([0.95±0.10)×10-2]高于排斥组([0.81±0.09)×10-2],差异有统计学意义(t=2.62,P=0.026)。结论成功建立了大鼠源PD-L1的real-time R T-qPCR检测方法,耐受组PD-L1的高表达提示其可能有利于大鼠移植肝的存活,为研究肝移植免疫耐受奠定了理论基础。

[Objective] To establish a real time quantitative RT-PCR detection method of PD-L1 and detect the PD-L1 mRNA level of liver transplantation in rats. [Methods] Establish orthotopic liver transplantation （OTC） tolerance （BN→BN） and rejection （LEW→BN） models in rats by two cuff technique. On the 7th day, kill the recipients and detect the level of ALT and AST. Evaluate the degree of rejection by HE stain- ing. Extract total RNA with Trizol and reverse transcribe it to cDNA. Ues 13-actin as internal control and build applications SYBR Green I real-time RT-qPCR assay. Detect PD-L1 and [3-actin of initial template amount in OTC model by this method and calculate the relative expression of PD-L1 by PD-L1/β-actin. [Results] Acute rejection happen in allogeneic LEW →BN group. Rejection group were higher than the tolerance group of ALT, AST （all P〈0.05）. The tolerance group＇s RAI score is higher than rejection group （P〈 0.05）. The amplification efficiency of PD-L1 is 98.8%. Correlation coefficient is 0.996. Melting curve is specific single peak. Coefficient of variation is less than 2.0%. The relative expression of PD-L1 mRNA in trans-planted liver, tolerance group [（0.95 ± 0.10）×10^-2] is higher than the rejection group [（0.81 ± 0.09）× 10^-2] and the difference is significant （t=2.62, P=0.026）. [Conclusion] The establishment of the PD-LI in rats source real-time RT-qPCR detection method is successful. High expression of PD-L1 of tolerance group suggests that it may benefit the survival of rat liver graft. It lays theoretical basis for the study of liver transplan- tation tolerance.