基于条件性敲除的新型强直性脊柱炎小鼠模型的建立、发病机制和药物评价研究

This research plan is to establish a conditional knockout mouse model for ankylosing spondylitis (AS). Specifically, we have created mouse line in which SHP2 is knocked out in CD4-positive T cells (cSHP2-KO). The created conditional knockout mice will be used in five subaims. (1) The limb function and activities, spine deformation, immune-related inflammation in cSHP2-KO mice will be examined, which will be referenced to pathological characteristics of human patients with AS. (2) The transcriptional and translational profile of T cells and T-cell-receptor diversity distribution in cSHP2-KO mice will be determined by deep sequencing and T-cell repertoire sequencing technology. (3) The cartilage cell function and molecular mechanism of heterotopic ossification will be investigated. (4) The molecular diagnostic markers of AS will be identified by comparing AS-dependent molecular events between cSHP2-KO and the wild-type littermates. (5) cSHP2-KO mice will be used for drug evaluation of AS intervention. With the proposed AS conditional knockout mouse model, these five aims will unreveal the molecular events associated with pathogenesis of AS, which will be the basis for drug evaluation and screening for AS treatment.

为了在动物整体水平建立真实模拟强直性脊柱炎（简称AS）的疾病模型，本项目拟在前期研究基础上，以AS患者病理特征为对照，进一步比较CD4阳性T细胞上条件性敲除磷酸酶SHP2小鼠在肢体活动能力、脊柱关节变形及免疫炎症等指标上的相似度；利用T细胞免疫组库测序技术考察T细胞受体多样性的分布规律，并分析各T细胞亚群的活性和功能改变，探讨这些改变与AS模型发生发展的相关性；利用转录组和翻译组高通量测序技术，进一步将T细胞特定亚群和骨细胞上差异表达基因的主要生物学功能进行关联，探讨条件性敲除SHP2对异常骨质丢失和骨形成的影响及引起异位骨化的分子机制；借助临床AS患者血样和病理组织样本进一步明确SHP2及其下游相关信号通路与AS发生发展的相关性，提出早期诊断AS的分子标志物；基于本研究中的AS小鼠模型，建立药物评价平台。本项目对探索AS发生发展机制以及寻找新的药物作用靶标均具有十分重要的临床转化意义。

以CD4-Cre介导的磷酸酶SHP2条件性敲除小鼠中自发强直性脊柱炎样骨病变为基础，探索强直性脊柱炎中骨融合的过程及其分子机制，继而发现靶向治疗强直性脊柱炎的药物靶标和小分子候选药物。1）系统分析确认CD4-Cre;SHP2f/f小鼠在中轴关节自发产生的骨关节融合、骨形变和骨质疏松与临床强直性脊柱炎症状吻合，CD4-Cre;SHP2f/f小鼠可用做强直性脊柱炎病理模型；2）发现CD4-Cre;SHP2f/f小鼠T细胞免疫与骨病变同时发生，并不是导致强直样骨病变的诱因；3）通过谱系追踪发现CD4-Cre介导了肥大型软骨细胞中SHP2敲除，并导致软骨细胞过度增殖分化，延迟骨骺线闭合；4）首次发现并证实骨骺线闭合延迟导致骨关节中不均衡骨生长是强直性脊柱炎中骨关节融合的直接原因；5）在强直样骨病变中不均衡骨生长引起骨变形和骨质疏松等并发症；6）基于代谢组学发现SHP2缺失的软骨细胞中嘌呤代谢上调并通过旁分泌形式促进强直样骨病变，靶向软骨细胞可作为强直性脊柱炎治疗的新途径。发表标注81673436资助SCI文章12篇（IF大于10的2篇），中文核心期刊1篇。申请国家发明专利3项，获授权1项。