Myo9b在1型糖尿病遗传易感性中的作用及机制研究

ALR is a congenic strain for type 1 diabetes (T1D)-prone NOD mouse; they share 70% genome, but unlike NOD mice, ALR mice nevenr develop T1D. Dendritic cells (DC) originated from ALR mice show a tolergenic phenotype as manifested by the lower antigen uptake and allogenic stimulatory capability. Congenic mapping revealed that the unique ALR genome encodes protective loci on chromosome 8, which renders these mice highly resistant to the induction of β cell specific autoimmune response. Indeed, high throughput genomic sequencing and comparative analysis characterized an 11-AA (amino acid) deletion for the ALR Myo9b gene on chromosome 8 as compared with that of NOD and B6 genome. Importantly, polymorphisms derived from Myo9b have consistently been demonstrated to be associated with genetic susceptibility for autoimmune diseases such as inflammatory bowl disease (IBD) and Crohn’s disease. Particularly, there is evidence also suggesting that Myo9b confers genetic susceptibility for T1D, and DC deficienct in Myo9b manifest impaired capability for induction of adaptive immune response. We thus hypothesize based on these preliminary studies that Myo9b is a suscetible gene for T1D. To address this hypothesis, we will first conduct a case/control study in T1D patients to demonstrate that Myo9b is associated with T1D susceptibility. Targeted high throughput genomic sequencing will be next carried out in both T1D patients and controls to characterize functional Myo9b rare variants. Myo9b knockout model and DC specific ALR Myo9b transgenic model will be further employed in NOD mice to confirm that altered Myo9b function regulates the development of spontaneous T1D. The underlying mechanisms by which altered Myo9b function modulation of T1D susceptibility will be also investigated. Finally, we will explore the feasibility of Myo9b to be a target for T1D prevention and treatment. These studies will not only provide novel insight into the understanding of T1D pathogenesis, but also be important for developing genetic factor based effective strategies for T1D prevention and treatment.

ALR是人类1型糖尿病（T1D）模型NOD小鼠的近交系，拥有NOD 70%的基因组但从不罹患T1D，其DC呈免疫耐受表型以摄取抗原及激活T细胞活化能力低下为特征。基因组测序和比对分析发现ALR第8号染色体编码的Myo9b基因较NOD及B6小鼠缺失11个氨基酸。遗传研究揭示Myo9b多态性调控如IBD等自身免疫性疾病的易感性，并有研究显示Myo9b与T1D易感性相关。在发现DC缺失Myo9b不能激活T细胞诱发获得性免疫的基础上，申报者拟在T1D病人阐明Myo9b调控T1D易感性并测序鉴定其功能性突变体；在NOD小鼠验证Myo9b缺失或转基因表达ALR突变体可预防或延迟T1D的发生发展；解析Myo9b调控T1D发生发展的细胞及分子机制；探索以Myo9b为靶点预防和治疗T1D的可行性。上述研究不仅为阐明T1D发病机制提供新认识，而且为开发以遗传因子为基础的T1D防治策略提供实验依据。

我们前期在ALR小鼠中，发现其Myo9b突变型可显著拮抗小鼠1型糖尿病的发生。因此，我们首先在T1D病人中行Case/Control分析，经小样本的高通量测序及大样本SNP验证，揭示Myo9b的多态性位点RS64932023可介导T1D易感性，其Odds Ratio达6.39，p=1.90×10-4 ，从而在人体研究中阐明Myo9b介导T1D易感性。依据前期机制研究的初步结果，我们构建了NOD DC特异性Myo9b敲除小鼠与NOD ALR-Myo9b 敲入小鼠，并在这些小鼠模型中确证Myo9b调控T1D发生发展。在动物活体及细胞模型中，我们厘清Myo9b突变体可通过影响 RhoA-ROCK-LIMK-Cofilin 信号通路，调控 DC 功能的机制。我们还建立了巨噬细胞特异性缺失Myo9b的小鼠模型，发现其通过Myo9b/RhoA/PPARγ通路影响巨噬细胞吞噬的能力，从而造成巨噬细胞的表型改变，该机制也是Myo9b缺失小鼠显著拮抗1型糖尿病的重要机制。此外，我们还比较了ALR和NOD肠道菌群，在易感的NOD小鼠中缺乏普雷沃属（Prevotella）菌群，肠道的代谢产物缺乏丁酸。在小鼠饲养过程中将ALR小鼠的粪便移植至NOD或在饮水中增加丁酸，可显著降低T1D的发生率及发生时间，对血糖的控制也显明改善，其机制是ALR肠道菌群能增加肠道内丁酸含量，增加其Tuft细胞从而保护肠道的完整性，进而保护小鼠，使其免于罹患T1D。这些研究不仅为阐明T1D发病机制提供新认识，而且为开发以遗传因子为基础的T1D防治策略提供实验依据。.在此基础上，我们还制备了DC靶向性包裹Myo9b siRNA的纳米微粒，并验证其对DC功能的影响。此外，还建立了Myo9b表达报告小鼠用于Myo9b表达抑制剂的筛选，探索以Myo9b为靶点预防和治疗T1D的可行性。