The study elucidating the molecular mechanisms underlying transplant vasculopathy

在我们的大鼠慢性排斥模型中，为了确定再移植心脏中浸润性细胞的来源，我们尝试建立了以男性特异基因SRY和男性显性重复DNA序列RN91ES8为靶点的PCR原位杂交技术。尽管仔细地滴定固定时间、酶处理条件、聚合酶链式反应条件或选择引物和探针，我们还是不可能在组织中检测到特定的信号。然后，利用RT-PCR技术建立了大鼠细胞因子mRNA的半定量检测方法。我们现在已经成功地定量了大约40种不同的细胞因子mRNAs在移植心脏中的表达。目前，我们正在研究大鼠心脏再移植模型中移植血管病变的分子机制。一方面，我们试图建立分子技术，另一方面，我们已经使用常规技术研究了疾病的病理生理学。这些研究表明，移植后5天内的关键免疫反应决定了移植血管病变的发生，表明CD_4或CD_8 T细胞和巨噬细胞是关键免疫反应所必需的。此外，将同种异体移植物重新移植到F1动物或裸鼠体内的实验表明，移植后初期T细胞的同种免疫反应对于疾病的发展是必不可少的。我们正在应用这项研究中建立的分子技术来研究这一独特的移植血管病变模型，以阐明该疾病的分子机制。

To determine the origin of infiltrating cells in a retransplnted heart in our rat chronic rejection model, we have tried to establish the PCR in situ hybridization technique using a male specific gene, SRY,and a male dominant repetitive DNA sequence, RN91ES8, as targets. Despite the meticulous titration of fixation time, conditions of protease treatment, PCR conditions or the selection of ptimers and probes, it as not possible for us to detect a specific signal in the tissue. Then, we moved to the establishment of semi-quantitation of rat cytokine mRNA by RT-PCR techniques. We have now almost succeeded to quantitate about 40 different rat cytokine mRNAs expressed in transplanted heart. We are studying the molecular mechanisms underlying transplant vasculopathy in our rat heart-retransplantation model right now.Trying to establish the molecular techniques on the one hand, we have studied the pathophysiology of the disease using conventional techniques on the other. The studies listed in the publication table revealed that the critical immune responses within the first 5 days after transplantation determined the occurrence of transplant vasculopathy and showed that either CD4+ or CD8+ T cells and macrophages were essential for the critical immune responses. Moreover, the experiments retransplantting an allograft into a F1 animal or a nude rat indicated that the alloimmune responses by T cells after the initial period from the transplantation are dispensable for the progression of the disease. We are applying the molecular techniques established in this study on this unique model of transplant vasculopathy to elucidate the molecular mechanisms of the disease.