基于高通量测序技术研究痰湿体质NKT细胞受体库特征谱及其circRNA调控机制

Phlegm-wet constitution is characterized by “phlegm-wet gathering” state which is susceptible to hyperlipidemia,diabetes,atherosclerosis and such other immune inflammatory diseases. The NKT cell receptor repertoire is the sum of the NKT cell diversity expanding clones reflecting its characteristics of immune antigen recognition and immunity capability. Circular RNA(circRNAs) is a new type of non-coding RNA which regulates immune activity and metabolism of glucose and lipid. Our previous studies indicated that NKT cells can promote atherosclerosis progression which is a phlegm-wet prone disease. However, the immune-modulatory effect of NKT cells in phlegm-wet constitution and its mechanism have not yet been clarified. We speculate that NKT cells express characteristic antigen receptor repertoire in the phlegm-wet constitution modulating the body's immunity, which regulated by circRNAs. To verify our hypothesis, we will utilize immune-magnetic bead sorting, flow cytometry, Arm-PCR and RNA-Seq sequencing to analyze NKT cell receptor repertoire and circRNA expression profiles in phlegm-wet constitution; construct the NKT cell receptor repertoire-circRNA regulatory networks by bioinformatics analysis; establish animal model and research on the mechanism of that by circRNA.This research can provide a new scientific basis for the molecular diagnosis of phlegm-wet constitution and the pathogenesis of this physique predisposed disease.

痰湿体质是“痰湿停聚”为特征的体质状态，其易患高脂血症、糖尿病和动脉粥样硬化等免疫炎性疾病。NKT细胞受体库是NKT细胞表达多样性受体克隆的总和，反映其免疫抗原识别特征和能力；环状RNA(circRNAs) 是一类新型非编码RNA，能调节糖脂代谢和免疫细胞活性。我们前期研究提示NKT细胞可促进痰湿质易患疾病动脉粥样硬化发生，但NKT细胞对痰湿体质的免疫调节作用及其机制尚未明确。我们推测“痰湿体质的NKT细胞表达特征性受体库而调节机体免疫，circRNAs调控其受体库表达”。为验证该假说，我们拟采用免疫磁珠分选、流式细胞、Arm-PCR和RNA-Seq基因测序分析痰湿体质NKT细胞受体库和circRNAs表达谱；生物信息学分析NKT受体库特征谱及其circRNAs调控网络；建立动物模型，筛选circRNA进行调控机制研究。本研究可为痰湿体质分子诊断及该体质易患疾病的发病机制提供新的科学依据。

中医体质学将人群分为9种体质，不同体质人群易患特定疾病。痰湿体质人群其易患高脂血症、糖尿病和动脉粥样硬化等代谢免疫疾病，机体免疫功能紊乱是导致其易患疾病的重要病机。NKT/T细胞在介导固有免疫和应答性免疫过程中发挥关键作用，NKT/T细胞受体库是其表达多样性受体克隆的总和，反映机体淋巴细胞免疫抗原识别的多样性和特征性。TCRβ链V-D-J基因重排组合是受体产生CDR3氨基酸序列克隆受体库的分子基础，研究报道circRNAs可调节糖脂代谢和免疫细胞活性。本项目通过招募平和体质和痰湿体质人群，流式细胞分析发现痰湿体质人群外周血中NKT、CD8+T细胞亚群和血清IL-5、IL-10、IL-6和IL-12P70水平明显高于平和体质组。免疫磁珠分选NKT/T细胞后，利用高通量基因测序技术分析NKT/T细胞受体的TCRβ链V-D-J基因重排组合，发现痰湿体质人群TCRβ链的TRBV6-5_TRBJ2-1_TRBD2、TRBV7-2_TRBJ2-5_TRBD2和TRBV4-1_TRBJ1-5_TRBD 2等6种V-D-J重排组合表达上调和12种CDR3氨基酸序列克隆型特异性高表达，而其免疫组库多样性指数D50值较平和体质组小。CicrRNA测序发现痰湿体质组有41个circRNAs表达与平和体质组间存在显著差异，通过差异circRNAs的host基因GO富集和KEGG分析，提示circRNA hsa-PIP4K2A_0001、hsa-HCLS1_0002和hsa-DOCK8_0007可能是参与痰湿体质细胞免疫过程的重要调控分子。整合糖脂代谢指标和免疫细胞因子指标，构建了痰湿体质人群的客观血清分子诊断模型，其ROC曲线下面积达高0.911。本项目为揭示痰湿体质机体免疫学特征谱提供了重要科学依据，对及早诊断和防治该体质及其易患疾病发生具有重要意义。