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研究生:蔡孟昆
研究生(外文):Tsai, Meng-Kun
論文名稱:CD80與CD86於CD152仲介之移植耐受的角色
論文名稱(外文):The Role of B7 Ligands (CD80 and CD86) in CD152-mediated Allograft Tolerance
指導教授:何弘能何弘能引用關係
指導教授(外文):Ho, Hong-Nerng
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:92
語文別:英文
論文頁數:78
中文關鍵詞:CD80CD86C152移植耐受
外文關鍵詞:CD80CD86CD152Transplant tolerance
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CD80與CD86傳遞CD28/CD152共刺激訊息之調控機制至今不明。運用BALB/C→B10.A之低反應性心移植模型,我們探討CD80與CD86在CD152仲介之移植耐受的角色。
我們以免疫細胞化學染色(immunocytochemistry)與流式細胞儀(flow cytometry)分析心移植之浸潤細胞,進而分析阻斷CD80與CD86對超過100天長期接受移植小鼠(B10.A-100)之脾細胞的增殖(proliferation)與介白質-2(interleukin-2)之產生。並以即時定量聚合酶反應(Real Time Quantitative Polymerase Chain Reaction)與流式細胞儀分析B10.A-100脾臟T細胞之CD152表現。CD80與CD86在引導與維持移植耐受之角色也將在此模型中被檢視。
B10.A小鼠在無任何免疫抑制下可以接受BALB/c之心臟(11/22),而anti-CD152抗體卻可完全阻斷此移植耐受(10/10)。在100天的移植心臟中可以發現多數CD4+的T細胞表現CD152,此外anti-CD80或anti-CD86的抗體可以加強B10.A-100脾細胞之增殖與介白質-2的產生。而B10.A-100脾臟之T細胞對BALB/c的刺激確實是低反應性。
定量聚合酶反應發現,B10.A-100脾細胞在刺激24小時後,CD152/GAPDH之mRNA比值相對低(0.48 ± 0.14)。但anti-CD80、anti-CD86或anti-CD152抗體顯著使mRNA比值增加(比值分別是0.85 ± 0.18、1.41 ± 0.15及1.72 ± 0.21)。有趣的是,在anti-CD152存在下,加入anti-CD80或anti-CD86或兩者同時加入,卻明顯降低CD152/GAPDH之mRNA比值(分別是0.55 ± 0.22、0.51 ± 0.13及0.62 ± 0.13)。而且流式細胞儀發現刺激48小時後表現在細胞膜之CD152與mRNA比值相關。此外,阻斷CD80或CD86確實可以抑制透過注入B10.A-100老鼠之脾細胞所導致的移植耐受。同時,針對B10.A-100之移植心臟加以皮膚移植與抗CD80或CD86抗體時也可以使移植耐受的現象式微。
總而言之,CD80/CD86:CD28的作用對CD152的表現是必要的,同時CD152透過佔據CD80與CD86可以負面調控CD152之表現。CD152仲介之移植耐受是透過對CD152表現的負向回饋來維持。CD80與CD86兩者是導入與維持CD152仲介之移植耐受所必須。
The regulatory mechanism by which the B7 ligands (CD80 and CD86) direct the CD28/CD152 co-stimulatory pathways is unclear. A low-responding cardiac transplant model (BALB/c à B10.A) with possible long-term acceptance was employed to investigate the role of CD80 and CD86 in CD152-mediated allograft tolerance.
Immunocytochemical and flow cytometric analyses of the graft-infiltrating cells were conducted to characterize this transplant model. The influence of anti-CD80 and anti-CD86 treatments on the proliferation and interleukin (IL)-2 productions of the tolerant splenocytes (SC) was analyzed. Real time quantitative polymerase chain reaction (Q-PCR) and flow cytometric analyses of the splenic T cells from the B10.A recipients with 100-day grafts (B10.A-100) were performed to examine the regulatory mechanisms of CD152-mediated tolerance. The role of CD80 and CD86 in the induction and maintenance of the graft acceptance in this transplant model were also tested.
B10.A mice could accept the BALA/c cardiac allografts (11/22), and an anti-CD152 antibody blocked the graft acceptance (10/10). Immunocytochemical and flow cytometric analyses showed that CD152+ cells were predominant among the CD4+ cells infiltrating the 100-day grafts of the B10.A recipients (B10.A-100). Either anti-CD80 or anti-CD86 treatment significantly enhanced polyclonal proliferation and IL-2 production of the B10.A-100 SC. B10.A-100 splenic T cells were hyporesponsive to donor-specific stimulation, and either anti-CD80 or anti-CD86 treatment significantly enhanced the proliferation responses (p < 0.01).
Q-PCR revealed that the CD152/GAPDH mRNA expression ratio of the B10.A-100 splenocytes with 24 h stimulation was relatively low (0.48 + 0.14), and either anti-CD80, anti-CD86 or anti-CD152 treatment significantly increased the expression ratio (0.85 + 0.18, 1.41 + 0.15 and 1.72 + 0.21, respectively). Anti-CD80, anti-CD86 or combined anti-CD80 and anti-CD86 treatments in addition to anti-CD152 reduced the CD152 expression ratios (0.55 + 0.22, 0.51 + 0.13 and 0.62 + 0.13, respectively). The CD152 surface expression correlated with the mRNA expression as well as the proliferation responses of the B10.A-100 splenic T cells with or without CD152 blockade. Blockade of either CD80 or CD86 prohibited the tolerance transmitted by adoptive transfer, and anti-CD80 or anti-CD86 plus skin grafting undermined the established allograft tolerance.
CD80/CD86: CD28 engagement was essential to CD152 expression and at the same time negative regulated by CD152 through sequestration of CD80 and CD86. CD152-mediated allograft tolerance was maintained with a negative feedback system on CD152 expression. Both CD80 and CD86 were essential for the induction and maintenance of the CD152-mediated allograft tolerance.
Contents
Introduction..................................................1
Materials and methods.........................................7
Results......................................................14
I Creation of a murine cardiac transplant model with CD152-mediated tolerance...........................................15
II The CD152-mdiated suppression of polyclonal proliferation and IL-2 production of B10.A-100 splenocytes.................18
III The essential role of CD80 and CD86 in CD152-mediated regulation of T cell proliferation...........................20
IV A critical role of CD80 and CD86 in CD152 expression.....23
V Differential effects between CD28 and CD152 on surface expression of CD152..........................................25
VI The essential role of CD80 and CD86 in the induction and maintenance of CD152-mediated allograft tolerance............27
Discussion...................................................29
Conclusion...................................................37
Figures......................................................39
References...................................................59
Appendix.....................................................71
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