IDO酶抑制T细胞免疫与补体旁路途径相互活化环路缓解肝脏移植慢性排斥的机制研究

Chronic rejection(CR) remains an important cause of late liver allograft dysfunction and failure. The mechanisms of CR involved in both cellular immunity and humoral immunity (complement activation). Previous researchs revealed that T cell can active the alternative pathway of complement cascade by expressing properdin, meanwhile, complement can active T cell by combining with C3a/C5a receptors on antigen-presenting cell. Due to the present immunosuppressive agents are effective only on T-cell immunity, but not on complement activation, the treatment of choronic rejection is not as effective as expected. Studies showed that the murine conceptus is protected from maternal immunity by upregulating expressinon of indoleamine 2,3-dioxygenase (IDO），which can inhibit the alternative pathway of complement activation. In addition, Our proceeding study showed that IDO had a strong ability of anti-cellular immunity damage after liver transpplantation. Regarding the powerful anti-cellular imunity damage and anti complement activation, we hypothesized that IDO may provide liver graft dual protection from CR. In this proposal, we plan to further evaluate IDO as a managment of CR in protecting against the development of T cell immunity and inhibiting the feedback circuit between T cell and complement alternative pathway. we will make the rat liver transplantation administered with low dosage of cyclosporin A as CR model, using C1INH, CD46, CD59 to block three complement activation pathways respectively, and examine IDO enzyme activity in vivo with HPLC-microdialysis technique. There are three major aims for this proposal. The first aim will study the definitive complement activation pathway playing an important role in the liver CR. The second aim will determine the relationship between T cell and alternative pathway. The third aim will determine whether the non-viral gene system (Sleeping Beauty transposon/transposase) generates long term IDO expression and protection of rat liver grafts against CR. The proposal will be benefit to providing more understanding about the mechanism of CR and novel methods on the treatment of CR.

目前肝移植慢性排斥治疗主要依赖单方面的细胞免疫抑制剂，而忽略了体液免疫（补体活化）的作用，故疗效差。研究发现T细胞能表达P因子活化补体旁路,反之补体也可结合APC细胞C3a/C5a受体活化T细胞。故我们推测在慢排发生时，存在细胞免疫和补体旁路途径的相互活化环路。母胎排斥研究发现IDO酶能阻断T细胞活化补体旁路，我们前期揭示IDO酶尚具有抗肝移植T细胞免疫功能，因此IDO酶可能发挥既抑制T细胞免疫又阻断T细胞与补体相互活化环路的双重作用，从而缓解肝移植慢性排斥。本研究将以小剂量环孢素大鼠肝移植慢排模型，C1INH、CD46、CD59分别阻断补体活化不同位点，"微透析"监测IDO酶活性，探讨①肝移植慢排中补体活化的具体途径；②T细胞活化与补体旁路激活之间的关系；③"睡美人"转座子上调IDO基因表达抗T细胞免疫和补体旁路活化对慢排的双重保护机制。这将有助于肝移植慢排机制的理解和提供新的治疗思路。

目的：建立大鼠肝移植慢排模型，探讨补体激活旁路途径在肝移植慢排反应中的作用，研究IDO酶对缓解移植后慢排的保护机制，从而发现肝移植慢排治疗的新途径。.方法：以DA大鼠为供体，BN大鼠为受体，建立稳定的大鼠肝移植慢排模型。实验分为七组：SD→SD同品系肝移植 (A组)、DA→BN异品系小剂量环孢素A 处理肝移植组（B组）、DA→BN CD46阻断小剂量环孢素A 处理组 (C组)、DA→BN CD59 阻断小剂量环孢素A 处理组 (D组)、DA→BN C1INH 阻断小剂量环孢素A 处理组 (E组)、DA→BN上调IDO慢排组（F组）和DA→BN 1-MT抑制IDO酶慢排组(G组）。测定血ALT、TB水平评估术后肝功能情况，免疫组化观察各组移植后慢排发生情况和纤维化程度，荧光定量PCR和Western-blot观察补体激活后相关因子表达水平，免疫学检测观察各组大鼠局部肝组织炎症细胞和T细胞凋亡情况。.结果：1、与同品系肝移植组相比，异品系各组的慢排评分均显著升高（P<0.05）。其中B、E、G三组术后ALT、TB水平、CCR评分、淋巴细胞局部浸润和纤维化程度均显著高于C、D、F组（P<0.05）；2、与同品系肝移植组相比，异品系各组的内皮细胞被MAC活化后相关因子MCP-1、IL8、PDGF、bFGF水平和血清补体激活相关因子水平均显著升高（P<0.05）。且B、E、G三组的相关因子水平明显高于C、D、F组（P<0.05）。3、异品系慢排组中，B、D、E、G四组的T细胞凋亡指数明显高于C、F组（P<0.05）。.结论：通过CD46抑制补体激活旁路途径、CD59抑制补体激活终产物MAC形成和上调IDO酶表达可以有效缓解肝移植术后慢性排斥反应的发生，并且在补体激活促进慢排发生的机制中，经典途径和最终的MAC形成起到主要作用。上调表达IDO 酶可明显降低血清补体活化因子浓度，并且抑制内皮细胞被MAC活化后相关因子MCP-1、IL8、PDGF、bFGF水平的升高，从而发挥其对补体激活途径的抑制作用。上调IDO基因表达和CD46抑制补体旁路激活途径可以增强IDO酶活性，并促进T细胞凋亡，进而阻断补体激活旁路与细胞免疫的信号环路，从而对肝移植后慢排免疫反应起到保护作用。