人源化小鼠中非补体C3依赖的人红细胞排斥机制研究和新型红细胞人源化小鼠模型的开发

Humanized mice attract lots of attentions in bio-medical fields, because they could represent human physiological and pathological features directly and are considered be to an ideal tool to simulate complex human biological processes or substitute human beings for pre-clinical tests. The humanized mouse models made by human hematopoietic stem cell transplantation have been broadly used in the fields including allo/xeno transplantation, infectious diseases, cancer therapy and so on. However, it was less commonly applied in human red blood cell (RBC) relevant study due to the lack of human RBC reconstitution found in their blood. Our previous work demonstrated that robust rejection mediated by mouse macrophages to human RBCs is the key reason lead to the deficiency of mature human RBC development in humanized mice. Recently, our team further indicated that NOD/SCID mouse macrophages rejecte human RBCs through both mouse complement C3 dependent and independent approaches. Moreover, we made the humanized mice with human RBCs reconstitution by combinatory treatment of Clodronate and Cobra Venom Factor. While due to the severe toxicity of Clodronate, the experimental time window of those animals was too narrow for many studies. Therefore, in this proposal we planned to deeply investigate the mechanism how NOD/SCID mouse macrophages reject human RBCs through the C3 independent way, and develop a novel humanized mouse model with high level of human RBC reconstitution that can be used for long term investigations of human RBC relevant issues or diseases in vivo.

人源化小鼠是近年来广受关注的动物模型。它能够直接反应人类的生理、病理学特征，被认为是研究复杂生物医学问题的理想工具之一。通过人造血干细胞移植构建的人源化小鼠模型已广泛应用于移植、感染、肿瘤等领域。但由于其没有成熟人红细胞重建，它在人红细胞相关研究领域的应用较少。我们早期研究表明小鼠巨噬细胞的排斥作用是导致其人红细胞重建缺陷的关键原因。我们近期工作进一步揭示NOD/SCID小鼠巨噬细胞可以通过补体C3依赖和非依赖两个途径清除人红细胞；并建立了基于Clodronate和Cobra Venom Factor联合处理的红细胞人源化小鼠模型。然而，Clodronate的毒副较大，该模型的应用时间窗口仅为3周左右。因此，本项目拟继续深入研究NOD/SCID小鼠中补体C3非依赖的人红细胞排斥机制，并开发具有高水平人红细胞长期重建的新型人源化小鼠模型，为人红细胞及其相关疾病的体内研究提供理想实验平台。

具有人红细胞重建的人源化小鼠模型人红细胞相关疾病的病理机制和治疗研究十分重要。然而，目前仍然无法实现人源化小鼠重高水平人红细胞长期重建，制约了该模型的应用。课题组前期研究揭示小鼠巨噬细胞(MΦ)对人红细胞强烈的排斥作用是导致人源化小鼠中人红细胞重建失败的关键原因，并发现小鼠补体C3可通过对人红细胞的调理作用加速其在小鼠体内的清除。然而，在小鼠补体C3耗竭小鼠体内，人红细胞仍然会被排斥，提示仍然有其他机制介导了人红细胞被小鼠巨噬细胞排斥。本项目旨在进一步研究人源化小鼠中人红细胞的缺失机制，并在此基础上优化红细胞人源化小鼠模型的效果。在本项目的支持下取得了以下成果: 1)在体外体系中，发现小鼠巨噬细胞可在没有小鼠补体C3的参与下，通过其表面Receptor-X特异性识别人红细胞并促进免疫排斥；2）揭示红细胞表面Y分子（Receptor-X配体）的构象变化导致人红细胞（而非小鼠红细胞）被小鼠巨噬细胞排斥；3）构建Receptor-X基因敲除小鼠，证明Receptor-X直接介导人红细胞排斥，为红细胞人源化小鼠模型的优化奠定基础；4）揭示人红系细胞内Z基因参与了人红细胞被小鼠巨噬细胞Receptor-X的识别。此外，在本项目的资助下，课题组还将提出了人源化小鼠模型优化构建方案，并将其拓展到人流感病毒、SARS-Cov-2等人呼吸系统病毒感染；提出白血病动员联合异体淋巴细胞回输的新型抗白血病治疗策略。