Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display.
Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display.
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慢性心脏排斥：通过差异 mRNA 显示鉴定移植心脏中五个上调基因。
DOI：
10.1073/pnas.91.14.6463
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发表时间：
1994
影响因子：
11.1
通讯作者：
Russell,ME
中科院分区：
文献类型：
--
作者：
Utans,U;Liang,P;Wyner,LR;Karnovsky,MJ;Russell,ME
移植动脉硬化是慢性排斥反应的主要表现，在同种异体(Lewis到F344)大鼠心脏移植后发生，而不是同基因(Lewis到Lewis)大鼠心脏移植后发生。为了确定与慢性心脏排斥反应相关的转录调节介质，我们采用了体内移植标本的差异mRNA展示技术。将四例早期出现慢性排斥反应的同种异体心脏的基因转录模式与两例接受相同手术但组织学正常的同种异体心脏进行了比较。共鉴定出12条差异表达的cDNA条带。我们提高了从单个展示带中分离出一个或多个同种异体移植物特异性cDNA的可能性，方法是首先使用回收的和重新扩增的PCR产物作为RNA印迹分析的探针。然后将从单个条带中克隆的cDNA片段用于第二次RNA印迹分析，这允许将特定的mRNA转录本与cDNA克隆相关联。5个克隆产生了与时间相关的同种异体移植物特异性杂交。序列分析表明，其中两个cDNA对应于未知基因，而另外三个代表以前与慢性排斥无关的已知基因。后者包括半乳糖/N-乙酰半乳糖胺的巨噬细胞凝集素(一种细胞表面受体)、核P1基因(酵母复制蛋白的同源物)和泛素样基因。我们的差异显示技术的应用使我们能够在保护疾病过程环境的活体条件下直接识别潜在的介体，包括对炎症反应至关重要的渗透细胞群。
Transplant arteriosclerosis, the major manifestation of chronic rejection, develops after allogeneic (Lewis to F344) but not syngeneic (Lewis to Lewis) rat cardiac transplantation. To identify transcriptionally regulated mediators associated with chronic cardiac rejection, we adapted the differential mRNA display technique for in vivo transplant specimens. Gene transcript patterns in four allogeneic hearts showing early signs of chronic rejection were compared with those in two syngeneic hearts exposed to the same surgical procedure but histologically normal. Twelve differentially expressed cDNA bands were identified. We improved the probability of isolating one or more allograft-specific cDNAs from a single display band by first using recovered and reamplified PCR products as probes in RNA blot analysis. cDNA fragments cloned from individual bands were then used in a second RNA blot analysis, which allowed for the correlation of specific mRNA transcripts with cDNA clones. Five cDNA clones produced time-dependent, allograft-specific hybridization. Sequence analysis demonstrated that two of these cDNAs corresponded to unknown genes, whereas the other three represented known genes not previously associated with chronic rejection. The latter group included the macrophage lectin specific for galactose/N-acetylgalactosamine (a cell-surface receptor), the nuclear P1 gene (a homologue of a yeast replication protein), and a ubiquitin-like gene. Our application of the differential display technique allowed the direct identification of potential mediators under in vivo conditions that preserve the environment of the disease process--including infiltrating cell populations critical to the inflammatory response.