Development of in Vitro Assay System for the Analysis of Transplanted Bone Marrow Cell Rejection

当造血细胞从亲本供体移植到其他异基因受体的F_1代时，除了同种抗原外，受体还通过识别造血细胞表面特异性表达的结构来排斥造血细胞。这种称为造血组织相容性（Hh）的特殊结构被类似于自然杀伤细胞的细胞识别。Hh抗原的分子结构、效应细胞的检测以及具有这些结构的细胞被排斥的机制仍然没有答案，因为大多数用于检查造血排斥的实验系统是体内测定。本课题的目的是建立一种能识别供者造血细胞Hh抗原而使供者造血细胞被清除的体外造血抵抗模型，并阐明排斥反应的机制。我们在去年的研究中已经发现：1）造血细胞的谱系和定向分化程度都不影响造血干细胞的分化， 关于我们 Hh抗原的表达。2)移植后3小时开始从宿主脾中消除移植的祖细胞，到24小时时，少于10%的祖细胞保持功能。3)排斥反应的严重程度不一定是由于宿主和供体之间同种特异性的差异。我们建立了造血抵抗的体外模型，并阐明了以下几点：1）通过尼龙毛柱和Percoll密度梯度后，介导造血祖细胞清除的效应细胞与NK细胞共富集，而与T细胞不共富集。2)用抗无唾液酸GM1对效应细胞进行细胞毒性处理可消除对造血集落生长的抑制。3）在广泛的分化和定型范围内的造血祖细胞在该体外试验中是敏感的。在宿主基质细胞存在下，观察到对供体集落生长的抑制而不是刺激。根据结果，我们得出结论，在体外观察到抑制供体造血集落生长的特异性与体内试验相似。然而，供体造血祖细胞的体外消除比体内测定系统中观察到的要弱得多。少

In addition to alloantigens, recipients reject the hemopoietic cells by recognition of surface structures expressed specifically on the hemopoietic cells, when the hemopoietic cells transplanted from a parental donor to F_1 hybrid of other allogeneic recipients. This specific structures called hemopoietic histocompatibility (Hh) are recognized by cells similar to natural killer cells. Molecular structures of Hh antigens, detection of effector cells and the mechanism by which cells bearing these structures are rejected are remained unanswered because most of the experimental system for examining hemopoietic rejection are in vivo assay. The purposes of this project is to make in vitro model of hemopoietic resistance in which donor hemopoietic cells could be eliminated by recognition of their Hh antigens and to clarify the rejection mechanism. We already detected the followings in lastyear : 1) Neither the lineage nor the degree of commitment or differentiation of hemopoietic cells affect … More their expression of Hh antigens. 2) Elimination of grafted progenitors from host spleen commences 3 hours after the transplantation and less than 10 % of progenitors are remained functional by 24 hour. 3) Severity of the rejection is not necessarily due to the difference of allospecificity between hosts and donors. Understanding these evidences, we developed in vitro model of hemopoietic resistance and clarified the followings : 1) Effector cells mediating elimination of hemopoietic progenitors were coenriched with NK activity but not with T cells after passing nylon wool column and applying Percoll density gradient. 2) Suppression of hemopoietic colony growth was abolished by the cytotoxic treatment of effector cells with anti-asial GM1.3) Hemopoietic progenitors in broad range of differentiation and commitment were susceptible in this in vitro assay. And 4) In the presence of host stromal cells, stimulation not but suppression of donor colony growth was observed. From the results, we concluded that suppression of donor hemopoietic colony growth was observed in vitro in the specificity similar to in vivo assay. However, the in vitro elimination of donor hemopoietic progenitors was much weaker than that observed in vivo assay system. Less