敲除Per1基因影响变应性鼻炎FcεRIβ合成干扰肥大细胞脱颗粒的研究

The finding that immune mechanism plays key roles in type-I allergy is of milestone significance. However there are other mechanisms, one of which is neuro-immune-endocrine regulation. As a central circadian clock, SCN plays fundamental roles in the development of allergic rhinitis through HPA axis. Studies indicate that the clock genes in the mast cells in peripheral tissues control the synthesis of FcεR1β, a ligand for IgE antibody, according to the message from SCN. Such a clock gene- involved degranulation in mast cell shows obvious circadian pattern. Based on these preliminary study, a hypothesis in the present study was proposed about the clock gene knockout in mast cells and its effect on the non-synchronous signal transmission of FcεR1βsynthesis so as to regulate the circadian cyle of mast cell degranulation. We will firstly make a clock gene knockout cell model in mast cells through so called CRISPR gene edit technique in an animal model. Degranulation level test in mast cells will be followed before and after such a gene knockout trial in comparison with normal control cells. Such a study will shed light to the final elucidation of the mechanisms in relation to mast cell functions and will provide new strategy for the management of exacerbation during the night or early morning as well as the seasonal attacks of upper airway allergy.

免疫机制的发现是变应性鼻炎（AR）具有里程碑意义的事件。研究提示在免疫机制之外还有其它的机制，如神经内分泌机制，其中由视交叉上核(SCN)调控的、HPA轴参与的作用机制，在AR发病中发挥作用。肥大细胞（MC）接受SCN指令，在其内在生物钟基因（如Per1 基因）的调控下，通过调控FcεRIβ的合成，实现其与对应IgE抗体结合的同步化。该过程具有明显的昼夜节律。我们据此假设，敲除Per1 基因将影响FcεRIβ合成，使FcεRIβ合成的信号及其与IgE结合的信号发生错时传导或“脱节”，进而影响MC脱颗粒的昼夜节律。我们将在AR动物通过基因编辑技术——CRISPR，对MC核内主要生物钟基因Per1 进行靶向敲除，检测敲除前后MC脱颗粒水平。如果课题取得预期结果，FcεRIβ合成信号错时传导影响MC脱颗粒的理论将对AR夜间或晨间加重、季节性发作等依靠现有策略和方法无法解决的问题，提供新的思路。

变应性鼻炎（allergic rhinitis, AR）的主要症状呈现~24h昼夜节律性波动，多在夜间至晨起加重，表现为时相反应。研究表明，生物钟（circadian clock）影响鼻黏膜的周期性活动，调控AR的时相反应。但具体机制不清。. 基于前期研究基础和文献追踪，本课题通过构建肾上腺切除（adrenalectomy, ADX）小鼠、正常AR小鼠、生物节律紊乱（circadian rhythm disruption, CRD）小鼠，依次进行相关实验探究。. 研究结果示：（1）空白对照组、AR组、双侧ADX/AR组、单侧ADX/AR组小鼠Th2细胞表达率，AR组显著高于其他3组；双侧ADX/AR组较单侧ADX/AR组表达率稍降低。各组小鼠Th1细胞表达率，两两组间比较无统计学差异。各组小鼠脾脏Treg细胞含量与空白对照组相比，其他3组该指标均显著降低；与AR组相比，双侧ADX/AR组、单侧ADX/AR组Treg细胞比例增多，前者有统计学差异，后者无统计学差异。（2）AR组小鼠鼻症状评分在ZT4时显著高于ZT16时，伴鼻黏膜和PBMCs中Per2 mRNA含量显著上调；AR组小鼠PBMCs中Th2正相关转录因子Gata3 mRNA含量和Th17正相关转录因子Rorgt mRNA含量在ZT4时显著高于ZT16时，而Th2和Th17负相关转录因子E4bp4 mRNA含量在ZT4时显著低于ZT16时。（3）CRD导致小鼠昼伏夜出行为逐渐减弱，加重AR小鼠的鼻过敏症状，伴随明显的焦虑样反应；CRD+AR组小鼠Th2细胞、Th17细胞及其细胞因子的比例显著高于AR组，而CRD+AR组小鼠Th1细胞及其细胞因子、Treg细胞的比例显著低于AR组，两两比较有统计学差异。. 研究表明：（1）单侧或双侧ADX导致HPA轴功能缺陷，可引发内源性糖皮质激素紊乱，进一步影响Th1/Th2免疫平衡以及Treg细胞反应，对AR发病有重要调控作用。（2）时钟基因Per2可能具有抗炎的作用，其潜在机制在于周期性调控Th2和Th17相关转录因子Gata3和Rorgt的免疫反应。（3）生物节律在AR发病中具有重要的调控作用。本研究提示人们应该尽可能保持规律的生活作息，减少现代社会快节奏带来的CRD，降低AR等变应性疾病的发生风险。