聚合酶链反应方法检测外周血淋巴细胞HLA-A mRNA方法学的建立及在肾移植中的初步应用  

Development and primary application of the real-time fluorescent quantitative RT-polymerase chain reaction for the detection of HLA-A mRNA expression on host peripheral blood lymphocyte in renal transplantation

作　　者：李晓丽[1] 邹雄[1] 杨晓静[1] 张义[1] 庄学伟[1] 单宁宁[1] 王洪春[1] 赵胜梅[1] 

机构地区：[1]山东大学齐鲁医院检验科,济南250012

出　　处：《中华检验医学杂志》2006年第12期1074-1078,共5页Chinese Journal of Laboratory Medicine

摘　　要：目的通过建立检测宿主外周血淋巴细胞(PBL)表面HLA-AmRNA表达程度的实时荧光定量逆转录聚合酶链反应(RT-FQ-PCR)方法,检测肾移植患者移植后外周血淋巴细胞表面HLA-AmRNA的表达程度,探讨其在诊断移植后早期排斥反应的发生的价值及机体免疫状态的关系。方法以6-磷酸葡萄糖脱氢酶(G6PDH)作内参照,用Taqman探针荧光定量技术,建立检测PBLHLA-AmRNA的RT-FQ-PCR方法;并检测35例随访移植患者的不同时间的60个标本和60名正常人的阈循环数(Ct),然后用REST软件分析PBLHLA-AmRNA的表达水平。结果正常人和51个移植标本的PBLHLA-AmRNA对G6PDH的相对表达量分别为0·036±0·012和0·026±0·015(双侧t=1·981,P<0·05),提示服用免疫抑制药(目前临床用药剂量均偏大)后患者PBLHLA-AmRNA表达减弱,机体免疫状态低,无排斥反应发生但易导致感染、肿瘤等并发症的产生;其中9个移植标本的PBLHLA-AmRNA对G6PDH的相对表达量为0·042±0·017(双侧t=2·002,P<0·05),提示较其他51例移植标本患者PBLHLA-AmRNA表达增强的患者,经病例回访查询发现该5例移植患者在这期间有排斥反应(根据临床表现、肾功能检查、影像学及核医学检查的临床排斥反应期)的发生,其中1例在移植后6个不同时间的标本显示2次有增高,每次经治疗后均降低。结论建立的宿主PBLHLA-AmRNART-FQ-PCR检测方法简便、特异、重复性好、可信度高,可用于肾移植后PBLHLA-AmRNA表达水平的检测,评估机体的免疫状态,预测排斥反应的发生。Objective To develop a real-time fluorescent quantitative RT-PCR method for detection of HLA-A mRNA expression levels on host peripheral blood lymphocyte （PBL）. Methods Using Taqman probe , with G6PDH as reference, a real-time fluorescent quantitative RT-PCR method was established. HLA-A mRNA expression levels of 60 samples of 35 renal transplantation patients in different time and 60 normal samples were analyzed by REST software and the SPSS11.5 statistics software. Results The relative expression ratio of HLA-A mRNA in normal and renal transplantation samples were 0. 036 ± 0. 012 and 0. 026 ± 0. 015 with G6PDH as reference（ double t = 1. 981, P 〈 0.05 ） , respectively, which indicated immunity level of kidney transplantation patients was down-regulated. The relative expression ratio of 9 of 60 kidney transplantation samples were 0. 042 ± 0. 017 （ double t = 2. 002, P 〈 0.05 ）, which is higher than normal. Among them, according to clinical diagnosis, 5 consecutive-monitored patients have the rejection response, 1 of 5 patients has twice rejection in 6 different period, given more immuno-suppressant , it was down-regulated each time. Conclusion The real-time fluorescent quantitative RT-PCR method for HLA-A mRNA quantification was simple, specific, reproductive and reliable, and could be used to study HLA-A mRNA expression level after kidney transplantation for evaluating host immunity condition and forecasting the rejection response.

关 键 词：淋巴细胞 HLA-A抗原 肾移植 聚合酶链反应 移植物排斥 RNA 信使 

分 类 号：R699.2[医药卫生—泌尿科学] R450[医药卫生—外科学]