Experimental study for the immunological tolerance using orthotopic liver transplantation using mice

小鼠原位肝移植免疫耐受的实验研究

• 批准号: 12671183
• 负责人: UYAMA Ichiro
• 金额: $ 1.98万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671183/
• 关键词: orthotopic mouse liver transplantation; immunological tolerance; differential display; DNA tip; flowcytometry; RT-PCR; soluble MHC class I; differential display

In mouse liver transplantation(OMLT), immunological tolerance is induced spontaneously across MHC barrier. To identify specific protein and gene for inducing tolerance, differential display and DNA tip analysis performed using mouse liver that acquired tolerance. As the result, 40kDa and 75kDa protein was detected. On the other hand, several genes specific for tolerance are identified using DNA tip, for example secretory leukocyte protease inhibitor, lymphotoxin B, rpt-1, CD24, which are related to immunological response.Flowcytemetry revealed the existence of the double-positive cells which expressed both MHC class I antigen for donor and recipient in the grafted liver. Analysis of surface antigens showed the double-positive cells possessed the characteristics of the premature dendritic cells of the recipient origin. RT-PCR using specific primers for donor and recipient antigen demonstrated that it was constantly generated in the grafted liver. These data suggested that the double-positive cells were possibly the recipient type premature dendritic cells which trapped the donor type soluble MHC class I antigen to their surface.And to make the soluble MHC class I antigen, we constructed the cellular surface domain, α1, α2, α3, and β2 microgloblin using pichia vector, and transformed to Pichia pastories. Now the protein has been purified, after that, it will be administrated naive mice and examine the ability to induce tolerance.

在小鼠肝移植（OMLT）中，免疫耐受是通过MHC屏障自发诱导的。为了鉴定诱导耐受的特异性蛋白和基因，利用获得耐受的小鼠肝脏进行差异显示和DNA末端分析。结果，检测到40 kDa和75 kDa蛋白。另一方面，用DNA探针技术鉴定了与免疫应答相关的分泌性白细胞蛋白酶抑制因子、光敏素B、rpt-1、CD 24等免疫耐受特异性基因，流式细胞术显示移植肝中存在同时表达供、受体MHC Ⅰ类抗原的双阳性细胞。表面抗原分析表明，双阳性细胞具有受体来源的未成熟树突状细胞的特征。使用供体和受体抗原的特异性引物进行RT-PCR表明，它在移植肝脏中不断产生。为制备可溶性MHC Ⅰ类抗原，我们利用毕赤酵母表达载体构建了细胞表面结构域、α1、α2、α3和β2微球蛋白，并将其转化到毕赤酵母中。目前该蛋白已被纯化，之后，将其施用给幼稚小鼠并检测诱导耐受的能力。

项目成果

Pan TL, Lin CL, Chen CL, Lin YC, Gojo S, Lee TH, Wang YH, Lord R, Lai CY, Tsu LW, Tseng HP, Wu ML, Iwashita Y. Kitano S. Chiang KC, Hashimoto T, Sugioka A, Goto S: "Identification of the indoleamine 2,3-dioxygenase nucleotide sequence in a rat liver trans

潘 TL、林 CL、陈 CL、林 YC、Gojo S、Lee TH、王 YH、Lord R、Lai CY、Tsu LW、Tseng HP、Wu ML、Iwashita Y. Kitano S.Chiang KC、Hashimoto T、Sugioka A

Sugioka A, Morita M, Fujita J, Hasumi A, Shiroishi T: "Graft acceptance and tolerance induction in mouse liver transplantation using wild mice."Transplantation Proceedings. 33 (1-2). 137-139 (2001)

Sugioka A、Morita M、Fujita J、Hasumi A、Shiroishi T：“使用野生小鼠进行小鼠肝移植中的移植物接受和耐受性诱导。”移植论文集。

Adachi K, Tamura A, Sugioka A, Morita M, Yan H, Li XK, Kitazawa Y, Amemiya H, Suzuki S, Miyata M, Kimura H: "Evidence of regulatory T lymphocytes that constitute peripheral blood microchimerism following rat liver transplantation."Transplantation Proceedi

Adachi K、Tamura A、Sugioka A、Morita M、Yan H、Li XK、Kitazawa Y、Amemiya H、Suzuki S、Miyata M、Kimura H：“大鼠肝移植后调节性 T 淋巴细胞构成外周血微嵌合的证据。”

A.Sugioka: "Graft Acceptance and Tolerance Induction in Mouse Liver Transplantation Using Wild Mice"Transplantation Proceedings. 33(1-2). 137-139 (2001)

A.Sugioka：“使用野生小鼠进行小鼠肝脏移植中的移植物接受和耐受性诱导”移植论文集。

Adachi K.: "Evidence of regulatory T lymphocytes that constitute peripheral blood microchmerism following rat liver transplantation"Transplantation Proceedings. 32(7). 2297-2299 (2000)

Adachi K.：“大鼠肝移植后构成外周血微化学的调节性 T 淋巴细胞的证据”移植论文集。