Sabek Omaima, Dorak M Tevfik, Kotb Malak, Gaber A Osama, Gaber Lillian
Department of Surgery, College of Medicine, University of Tennessee, Memphis 38163, USA.
Transplantation. 2002 Sep 15;74(5):701-7. doi: 10.1097/00007890-200209150-00019.
The quest for noninvasive methods to diagnose rejection in solid-organ transplants has been rejuvenated by recent observations that specific cytotoxic T-cell markers are up-regulated during rejection.
We developed a one-step real-time polymerase chain reaction (PCR) method allowing reliable detection of the expression of several T-cell genes within a relatively short period of time. The assay is highly sensitive and reproducible with a wide dynamic range allowing accurate quantification of target mRNA in as little as 3 pg total RNA. The utility of this assay in detecting renal allograft rejection was evaluated. Peripheral blood mononuclear cells were collected from 27 patients undergoing kidney allograft biopsies for renal dysfunction after transplantation. Expression of the T-cell activation markers, granzyme B, perforin, and HLA-DRA, was quantified and correlated to the histopathologic changes in the renal biopsies.
In cases with allograft rejection (n=8), peripheral lymphocyte expression was increased for granzyme B (P <0.001) and perforin (P <0.08) compared with cases without rejection (n=19). Granzyme B mRNA up-regulation showed the highest specificity for detecting rejection (95%). Moreover, HLA-DRA mRNA was significantly up-regulated (P <0.0016) and had the highest sensitivity (88%) detecting rejection. The up-regulation of both granzyme B and HLA-DRA was most specific in detecting rejection, P<0.001.
These data demonstrate that a rapid test of target gene up-regulation using real-time PCR can be used as an aid in the diagnosis of kidney allograft rejection. This is also the first report on the possible utility of HLA-DRA mRNA up-regulation as a marker for kidney transplant rejection.
近期观察发现，在排斥反应期间特定细胞毒性T细胞标志物会上调，这使得寻找用于诊断实体器官移植排斥反应的非侵入性方法的研究重新活跃起来。
我们开发了一种一步法实时聚合酶链反应（PCR）方法，能够在相对较短的时间内可靠地检测几种T细胞基因的表达。该检测方法高度灵敏且可重复，动态范围广，能够在仅3 pg总RNA中准确定量目标mRNA。评估了该检测方法在检测肾移植排斥反应中的效用。从27例移植后出现肾功能障碍并接受肾移植活检的患者中收集外周血单个核细胞。对T细胞活化标志物颗粒酶B、穿孔素和HLA - DRA的表达进行定量，并与肾活检中的组织病理学变化相关联。
在发生移植排斥反应的病例（n = 8）中，与未发生排斥反应的病例（n = 19）相比，颗粒酶B（P < 0.001）和穿孔素（P < 0.08）的外周淋巴细胞表达增加。颗粒酶B mRNA上调在检测排斥反应方面显示出最高的特异性（95%）。此外，HLA - DRA mRNA显著上调（P < 0.0016），在检测排斥反应方面具有最高的敏感性（88%）。颗粒酶B和HLA - DRA的上调在检测排斥反应方面最具特异性，P < 0.001。
这些数据表明，使用实时PCR快速检测靶基因上调可辅助诊断肾移植排斥反应。这也是关于HLA - DRA mRNA上调作为肾移植排斥反应标志物的可能效用的首次报道。
