持续机械应力刺激对内淋巴囊上皮细胞免疫功能影响的研究

Mechanical stress on the tissue can lead to the change of cells morphology, proliferation, differentiation and function. microRNA-mediated epigenetics changes play an important role in this process. We found that under continuous mechanical stress, the NF-κB signaling pathway of endolymphatic sac epithelial cells was activated, which increased expression of inflammatory factors related, While miR-23b, miR-24-1, miR-27b expression regulated accordingly. However, after adding LPS, the expression of miR-23b, miR-27b is inhibited. Therefore, we hypothesized that miR-23b, miR-24-1, miR-27b are involved in the immune function regulation in endolymphatic sac epithelial cells under continuous mechanical stress. At the same time there is a correlation between the factors regulating. Our current study based on results in previous research is intended to further explore the immune regulation mechanism of miR-23b~27b~24-1 cluster in endolymphatic sac epithelial cells under continuous mechanical stress, from the perspective of microRNA-mediated epigenetics. Implementation of this project will provide a new theoretical basis for study of the pathogenesis of membranous labyrinth edema, and a new target from the microRNA pathway for the treatment of inner ear disorders such as Meniere's.

机械应力作用于组织细胞，会导致细胞形态、增殖、分化及功能改变，其中microRNA介导的表观遗传学变化在这一过程中起到重要作用。我们在前期研究中发现：持续机械应力作用下，内淋巴囊上皮细胞NF-κB被激活，相关炎性因子表达升高；同时miR-23b，miR-24-1，miR-27b表达亦相应上调；但加入LPS后，能够抑制miR-23b，miR-27b的表达。因此我们推测miR-23b，miR-24-1，miR-27b参与了持续机械应力刺激对内淋巴囊上皮细胞免疫功能的调控，同时各因子间存在相互反馈调节。本课题组拟在此基础上，从microRNA介导的表观遗传学角度出发，进一步研究持续机械应力作用下，miR-23b~27b~24-1 cluster 对内淋巴囊上皮细胞免疫功能调控机制。本课题的实施将为膜迷路水肿发病机制研究提供新的理论依据，为梅尼埃等内耳疾病的治疗从microRNA途径提供新的靶点。

机械应力作用于组织细胞，会导致细胞形态、增殖、分化及功能改变，其中microRNA介导的表观遗传学变化在这一过程中起到重要作用。我们在前期研究中发现：持续机械应力作用下，内淋巴囊上皮细胞NF-kB被激活，相关炎症因子表达升高;同时miR-23b，miR-24-1.miR-27b表达亦相应上调；但加入LPS后，能够抑制miR-23b，miR-27b的表达。因此我们推测miR-23b，miR-24-1，miR-27b参与了持续应力刺激对内淋巴囊上皮细胞免疫功能调控，同时各因子间存在相互反馈调节。本课题拟在此基础上，从microRNA介导的表观遗传学角度出发，进一步持续机械应力作用下，miR-23b，miR-27b，miR-24-1 cluster 对内淋巴囊上皮细胞免疫功能调控机制。在这四年之间，我么们1、首先明确了目标基因miR-23b，miR-24-1和miR-27b在小鼠内淋巴囊上皮细胞的表达，2、构建miR-23b，miR-24-1和miR-27b的干扰及mimic病毒载体，建立稳定表达细胞株。检测内淋巴囊上皮细胞协同刺激分子CD80、CD86、CTL-4、ICAM-1表达的改变及炎性细胞因子IL-2、IL-6的分泌，3、利用CRISPR/CAS9方法构建miR-23b~27b~24-1 cluster基因敲除小鼠，进行在体实验；观察内淋巴囊上皮细胞协同刺激分子CD80、CD86、CTLA-4、ICAM-1表达的改变及炎性细胞因子IL-2、IL-6的分泌谱变化，4、观察持续性机械压力对miR-23b~27b~24-1 cluster表达的影响，5、应用microRNA芯片、综合生物信息学分析，观察miR-23b~27b~24-1 cluster与NF-kB信号通路之间的相互反馈机制。