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

采用卵转移技术对B6、bm1、bm12背景的绿色荧光蛋白转基因小鼠进行清洗，获得特异性无病原体小鼠。首先，通过皮肤移植测试GFP是否作为移植抗原，我们发现它仅作为非常弱的移植抗原（即平均生存期为60天）。因此，在接下来的研究中，我们将利用转基因GFP作为在体内追踪异体细胞的标记。其次，通过非转基因小鼠植皮得到以下结果：A: 1 - 1000万个（B6×bm1）F1脾细胞静脉致敏B6宿主，可以剂量依赖性地延长I类mhc异种bm1皮肤移植物的存活时间。B：静脉致敏B6宿主，即使是1亿个（B6×bm12） F1脾细胞，对II类mhc异源bm12皮肤移植物的存活也没有显著影响。第三，使用gfp阳性的B6、（B6×bm1） F1或（B6×bm12） F1脾细胞在体内追踪注入B6宿主的I类或II类mhc异体细胞的命运。每只小鼠静脉注射1000万个脾细胞，注射后1、2、4、8周用流式细胞仪分析宿主脾细胞。研究结果如下：c：注射后1周，B6细胞占宿主脾细胞的1-2%，注射后在宿主体内检测到的B6细胞比例与注射前成正比。注射后4周检出细胞数逐渐减少至0.2-0.3%，8周后检出细胞数消失。腐烂的速度似乎不受注射细胞类型的影响。D：注射后1周和2周检测到I类mhc异类（B6×bm1） F1细胞，4周及以后无法检测到。检测到的细胞数量不断少于注射的B6细胞，但衰减的速度似乎不受检测细胞类型的影响。E：注射后1、2、4、8周，注射的II类mhc异类（B6×bm12） F1细胞的总数与（B6×bm1） F1细胞的总数大致相似。然而，II类不同mhc的B细胞比脾脏中其他类型的细胞消失得更快。综上所述，尽管由于小鼠清理过程的延迟，我们无法完成所有计划的研究，但我们可以从新方法中获得一些有趣的结果，这些结果有可能阐明尚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，在其他研究领域也被证明是有用的，使用这些方法的三篇论文发表在表格的另一边。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