The study elucidating the molecular mechanisms underlying transplant vasculopathy

为了确定大鼠慢性排斥模型中再移植心脏浸润细胞的起源，我们尝试建立PCR原位杂交技术，使用雄性特异性基因SRY和雄性优势重复DNA序列RN91ES8作为靶点。尽管我们对固定时间、蛋白酶处理条件、PCR条件或启动子和探针的选择进行了细致的滴定，但我们仍无法检测到组织中的特定信号。然后，我们开始用RT-PCR技术建立大鼠细胞因子mRNA的半定量。我们现在几乎已经成功地量化了移植心脏中表达的大约40种不同的大鼠细胞因子mrna。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。一方面尝试建立分子技术，另一方面利用常规技术研究疾病的病理生理。发表表中列出的研究表明，移植后最初5天内的关键免疫反应决定了移植血管病变的发生，CD4+或CD8+ T细胞和巨噬细胞在关键免疫反应中都是必不可少的。此外，将同种异体移植物再移植到F1动物或裸鼠体内的实验表明，在移植后的初始阶段，T细胞的同种免疫反应对于疾病的进展是必不可少的。我们正在将本研究中建立的分子技术应用于这种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.