供体心脏低剂量γ射线辐射抑制心脏移植排异反应的功能与机制研究

Cardiac transplantation is the most effective remedies to save the life for patients with end stage heart failure, while allograft rejection is a major challenge for the survival of grafts and clinical outcome of recipients. Immunosuppressant is the mainly treatment to avoid rejection; however, continuous use may result in severe side effects and deteriorate long-term prognosis. The local metabolism of vitamin D3 has been widely confirmed to regulate immune reaction in autoimmune disease as well as allograft transplantation. Our recent study found that γ-irradiation of isolated hearts can induce cardiac endothelial cells (CECs) to highly express CYP27B1, an enzyme to synthase the most physiologically active 1,25(OH)2VD3; furthermore, γ-irradiation induced CECs could also inhibit inflammatory reaction of macrophage when co-cultured in vitro. Thus, we proposed that γ-irradiation of isolated hearts could induced significant expression of CYP27B1 in CECs, and then aggregated high concentration of 1,25(OH)2VD3 in local cardiac tissue, which could suppress immunological rejection through inhibition of inflammation and induction of immune tolerance. Based on this hypothesis, we plan to develop a heart transplantation mouse model using wild type or endothelial cells (ECs)-specific CYP27B1 knockout donor heart to observe the influence of isolated heart γ-irradiation on allograft rejection. Further, a series of in-vitro studies based on isolated CECs will also be conducted to illustrate the related immunoregulation mechanisms as well as signal pathways. Our study aims at establish an original strategy to suppress allograft immune rejection, which can render the allografts to survive, or at least reduce the need of anti-rejection drugs.

心脏移植是治疗终末期心衰的有效途径，然而排异反应是导致器官失活并影响移植患者预后的重要因素。免疫抑制剂作为抗排异首选，但具有副作用，影响患者长期预后。VitD3局部代谢可以调节免疫炎症反应，诱导移植免疫耐受预防器官移植排异反应，但效用有限。我们的前期研究发现：离体心脏低剂量γ射线辐射可以有效促进心脏内皮细胞表达CYP27B1（1,25(OH)2VD3合成酶），分离培养该内皮细胞可以抑制共培养的巨噬细胞炎症反应。因此，我们提出假设：低剂量γ射线照射离体供体心脏可诱导心脏内皮细胞表达CYP27B1，促进局部合成高活性的1,25(OH)2VD3，通过抑制免疫炎症、诱导免疫耐受，进而抑制排异反应。因此，本课题组拟构建正常及内皮细胞特异性敲除CYP27B1EC-/-小鼠同种异体心脏移植模型，观察γ射线辐射对移植排异免疫反应的影响；同时利用体外分离培养心脏内皮细胞模型，阐明相关免疫调节机制。

终末期心衰目前最为行之有效的治疗方式是进行同种异体心脏移植，然而免疫排异反应是导致移植器官失活并影响患者预后的重要原因。目前临床常用免疫抑制药物来控制排异反应，然而此类药物长期使用会带来一系列副作用，例如感染，肾衰，肿瘤等。因此，寻找新兴的治疗方式控制免疫排异反应以替代或者辅助减量免疫抑制药物是心脏移植领域的重要发展方向。本项目在执行期间揭示了1,25(OH)2VD3对心脏移植免疫反应的调控关系。首先发现γ射线辐射处理小鼠离体心脏可上调血管内皮细胞CYP27B1表达，提高内皮细胞1,25(OH)2VD3合成能力，抑制巨噬细胞生成TNF-α及Th2型免疫反应，从而诱导免疫耐受。构建CYP27B1敲除小鼠作为受体探究1,25(OH)2VD3对同种异体心脏移植物的影响，相对于野生型对照，CYP27B1敲除小鼠的心脏移植物具有更高的血栓形成风险，并利用单细胞转录组测序技术探究CYP27B1在免疫排斥反应介导的血栓形成中的具体作用。.构建了急性排斥小鼠心脏移植模型,并通过单细胞转录组测序技术解析急性排斥反应机制及新的干预靶点。结果表明急性排斥组相比缺血再灌注损伤组，具有更多的单核巨噬细胞浸润、更高的T细胞激活水平、更强的树突状细胞抗原提呈能力，同时发现Hif1α调控的糖酵解水平在急性排斥中明显升高。利用Hif1α抑制剂干预急性排斥小鼠心脏移植模型发现Hif1α抑制剂干预可明显降低急性排斥反应中的炎症细胞浸润。为了推进研究结果的临床转化，构建了同种异体猪心脏移植模型。使用单细胞测序技术揭示猪心脏移植急性排斥反应的动态演变过程，通过差异分析鉴定了在各个时期富集的细胞类型及基因表达特征，为机制探索及药物靶点开发提供了良好的参比数据。.该项目累计资助发表高水平 SCI 论文 2 篇，分别发表于Circulation、Cardiovasc Res，累计影响因子31.771分。