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

目的 建立real-time RT-qPCR(实时荧光定量RT-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 mRNA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7d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 Green I real-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 RT-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.

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

【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 staining.Extract total RNA with Trizol and reverse transcribe it to cDNA.Ues β-actin as internal control and build applications SYBR Green Ⅰ real-time RT-qPCR assay.Detect PD-L1 and β-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 transplanted 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-L1 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 transplantation tolerance.