A study for the in vivo mechanism of transplantation tolerance using GFP transgenic mice

采用卵移植技术将B6背景的绿色荧光蛋白（GFP）转基因小鼠、bm 1或bm 12小鼠净化，获得特异性无病原小鼠。首先，通过皮肤移植测试GFP是否充当移植抗原，并且我们发现它仅充当非常弱的移植抗原（即：平均存活>60天）。因此，在以下研究中，GFP转基因被用作标记物以在体内追踪同种异体细胞。第二，通过使用非转基因小鼠的皮肤移植获得了以下发现：A：通过静脉内预致敏B6宿主1至100万个（B6× bm 1）F1脾细胞，I类MHC不同的bm 1皮肤移植物的存活时间呈剂量依赖性延长。静脉注射B6宿主的1亿个（B6× bm 12）F1脾细胞对II类MHC的存活没有任何显著影响。第三，I类或II类MHC-不同的同种异体细胞的命运， 关于我们 用GFP阳性的B6、（B6× bm 1）F1或（B6× bm 12）F1脾细胞在体内追踪B6宿主的感染。静脉内注射来自每只小鼠的一千万个脾细胞，并在注射后1、2、4或8周用FACS分析宿主脾细胞。通过研究获得了以下结果：C：注射的B6细胞在1周时由1-2%的宿主脾细胞组成，并且在宿主中检测到的注射细胞的分数与注射前的那些成比例。检测到的注射细胞数在4周时逐渐下降至0.2-0.3%，并且在注射后8周变得不可检测。D：注射的I类MHC不同（B6× bm 1）F1细胞在注射后1周和2周检测到，在4周及以后变得不可检测。检测到的细胞数量始终小于注射的B6细胞，但衰减速度似乎不受检测到的细胞类型的影响。E：注射后1、2、4或8周，注射的II类MHC不同的（B6 × bm 12）F1细胞的总数与注射的（B6× bm 1）F1细胞的总数大致相似。然而，II类MHC-不同的B细胞比脾中包括的其他类型的细胞消失得更快。总之，尽管由于小鼠清理过程的延迟，我们不能进行所有计划的研究，但我们可以从新的方法中获得一些有趣的结果，该方法有可能阐明未发现的移植耐受机制。此外，本研究中建立的其他分子生物学方法（如定量RT-PCR）被证明在其他研究领域也很有用，使用这些方法的三篇论文已发表，如表格的另一侧所列。少

The Green Fluorescent Protein (GFP) transgenic mice with B6 background, bm1, or bm12 mice were cleaned up by egg-transfer technique and specific pathogen-free mice were generated. First, whether the GFP acts as transplantation antigen was tested by skin-grafting and we found it only worked as very weak transplantation antigens (i.e.: mean survival>60 days). Thus, GFP transgene are used as marker to track allogeneic cells in vivo in the following studies. Second, the following findings were made by the skin-grafting using non transgenic mice.A : The survival of class I MHC-disparate bm1 skin grafts were dose-dependently prolonged by intravenous presensitization of B6 host with one to ten million of(B6×bm1)F1 spleen cells.B : Intravenous presensitization of B6 host with even a hundred million of (B6×bm12) F1 spleen cells did not have any significant effect on the survival of class II MHC-disparate bm12 skin grafts.Third, the fate of class I or class II MHC-disparate allogeneic cells inje … More cted into B6 host was tracked in vivo using GFP-positive B6, (B6×bm1) F1, or (B6×bm12) F1 spleen cells. Ten million spleen cells from each mice were intravenously injected and the host spleen cells were analyzed with FACS 1, 2, 4, or 8 weeks after the injection. The following results were achieved by the investigation.C : The injected B6 cells consisted of 1-2% of host spleen cells at 1 weeks and the fraction of the injected cells detected in hosts were proportional to those before the injection. The number of injected cells detected gradually decreased to 0.2-0.3% by 4 weeks and became undetectable 8 weeks after the injection. The speed of decay seemed not to be affected by the type of cells injected.D : The class I MHC-disparate (B6×bm1) F1 cells injected were detected at 1 and 2 weeks after the injection and became undetectable at 4 weeks and thereafter. The numbers of cells detected were constantly smaller than those of injected B6 cells but the speed of decay seemed unaffected by the type of cells detected.E : The total number of class II MHC-disparate (B6×bm12) F1 cells injected was roughly similar to those of (B6×bm1) F1 cells at 1, 2, 4, or 8 weeks after the injection. However, class II MHC-disparate B cells were disappeared more quickly than the other types of cells included in spleen.In summary, although we could not perform all the studies planned because of the delay in cleaning-up process of the mice, we could get some interesting results from the novel method which has the potential to clarify the undiscovered mechanisms of transplantation tolerance. In addition, the other molecular biological methods established in this study such as quantitative RT-PCR proved to be useful in the other fields of investigation and the three papers using those methods were published as listed on the other side of the form. Less