B7-H1共刺激通路在大鼠部分肝移植急性排斥反应中的免疫调节机制

BN-Lewis rat liver transplantation is a model of spontaneous liver graft acceptance. However, an acute lethal rejection of liver graft was triggered in this model when the graft weight to recipient liver weight (GW/RLW) was below 30% after 70% hepatectomy, implicating that partial liver transplantation could result in a higher allogeneic immune response. Recent studis showed that B7-H1 molecule plays a pivotal role in maintaining the spontaneous liver graft acceptance. Knockout of B7-H1 caused an acute rejection in liver graft. The expression level of B7-H1 was regulated by the level of IL-6, IL-10, IFN-gamma and the activation of MAPK and STAT3 signal pathway. We hypothesize that GW/RLW<30% BN-Lewis partial liver transplantation breaks the spontaneous liver graft acceptance by decreasing the expression of B7-H1. In this project, we first plan to observe the influence of transplantation damage and hepatic regeneration on B7-H1 expression. Second, we plan to investigate the regeneration rate of partial live graft having different GW/RLW under allogeneic and syngeneic condition, respectively. Furthermore, we try to figure out the relevance between hepatic regeneration and B7-H1 expression. Last but not least, we want to use Pioglitazone, Lipopolysaccride and Mesalamine to regulate the expression of B7-H1 in BN-Lewis partial liver transplantation, expecting a corresponding changes in the allogeneic immune response. Addtionally, this project could help to reveal a new potential application of these drugs.

BN-Lewis大鼠全肝移植是自发免疫接受模型。当供肝行肝切除使供肝/受肝肝重（GW/RLW）<30%时，自发免疫接受被打破，发生急性排斥反应。提示部分肝移植可以调高免疫反应。B7-H1分子可通过诱导T细胞凋亡来维持自发性免疫接受。B7-H1的基因敲除会导致排斥反应的发生。而B7-H1受到IL-6、IL-10、IFN-gamma、MAPK信号途径和STAT3分子的调节。因此我们推测GW/RLW<30%的BN-Lewis部分肝移植是通过调低B7-H1的表达来打破耐受。本课题首先观察在部分肝移植中不同的肝再生、移植损伤对与B7-H1分子表达的影响，其次分析在同种同基因和同种异基因下不同GW/RLW对于肝再生率的影响，继而探讨不同肝再生率与B7-H1分子表达的相关性。以明确部分肝移植对于B7-H1的影响。最后使用匹格列酮、LPS和美沙拉嗪来干预B7-H1的表达，期望能调节排斥反应的强弱

本研究建立了不同于利用离体肝切除法的在体70%的肝切除术的大鼠部分肝移植模型。改进了基于大鼠血管解剖的70%肝切除显微外科技术和大鼠肝动脉重建的显微吻合技术。另外还比较了 BN-Lewis全肝移植、GW/RLW>33%部分肝移植（PLTx）和GW/RLW<30%PLTx生存时间、黄疸、体重曲线、移植肝Banff-RAI、移植排斥相关的细胞因子、B7-H1分子、Caspase3等指标。发现全肝移植和GW/RLW>33%PLTx术后均能免疫接受，而GW/RLW<30%受鼠在22天死于急性排斥反应。相较于全肝移植和GW/RLW>33%PLTx，GW/RLW<30%的受鼠在术后第7天肝酶和穿孔素、颗粒酶B、IL-2和CD3 的mRNA水平均升高。移植肝的干扰素γmRNA也低于免疫接受的受鼠。移植物中B7-H1的mRNA及蛋白均低表达。且移植物内的caspase3蛋白也低于全肝组。提示GW/RLW<30%可能是通过降低干扰素γ，低表达B7-H1，减少凋亡来增强排斥反应。另外还通过给GW/RLW <30%的BN→Lewis大鼠PLTx受鼠皮下注射奥曲肽50ug/kg q12h来观察奥曲肽对于移植物功能及免疫排斥反应的影响。结果显示奥曲肽组和对照组受鼠均于术后14天内死亡，生存率无差异，肝组织见大量淋巴细胞浸润。在术后第4天和第9天奥曲肽组的Ki67+细胞明显减少。奥曲肽组第4天血清ALT和AST水平高于对照组，其第9天的血清白蛋白较术后第4天降低，而对照组未见差异。另外奥曲肽还使术后第4天的肝移植物IL-6和CXCL9的mRNA水平升高。在术后第9天，两组移植肝均被大量淋巴细胞浸润。其总胆红素水平分别高于术后第4天的对照组和奥曲肽组。在术后第9天的奥曲肽组的穿孔素mRNA水平明显高于对照组，但B7-H1的mRNA水平在两组间没有差异结果，提示在GW/RLW <30%的PLTx术后注射奥曲肽会明显抑制移植物肝再生，导致移植物非特异性损伤，伴随IL-6和CXCL9的升高，可能会引起移植物特异性排斥反应。在国际合作中，同合作者发现在BN-Lew全肝移植中术前注射LPS导致术后50%的生存率，而术后立即注射LPS未导致Lewis受鼠死亡。提示基因变化导致对于LPS的炎症刺激反应不同。