慢性阻塞性涎腺炎中NF-κB活化及炎性microRNA对其调控研究

The nuclear factor-κB (NF-κB), as a important transcription factor, plays critical roles in regulating the expression of genes whose products are involved in inflammation and the immune response. Therefore the aim of this research is to investigate the role of axis of NF-κB in the pathology of chronic obstructive sialadenitis (COS) and study regulating effects of microRNA (miR) on the signal axis.The thesis is devided into three parts: .Part one: 1.The TLR4/TRAF6/NF-κB signal axis is examined in COS tissues obtained during submandibular gland surgey and in experimental rabbit COS models by PCR and IHC examination. 2.The inflammatory level is subjected to histomorphometric examinations, then the relationship between TLR4/TRAF6/NF-κB expression and the degree of inflammation is analysed. .Part two: 1.HSG and rat submandibular gland cells are cultrued, P.g.-LPS is used to stimulate the cells. The contribution of LPS to cytokines production is deternined by PCR and ELISA. TLR4/NF-κB signal activity is determined by western immublotting. 2.After siRNAs(containing a mixture of four siRNAs) targeting human TLR4 or TRAF6 sequences, and overexpression those gene in the two kinds of cells, P.g.-LPS is used to stimulate the cells. Compare the results between step 1 and step 2. Part three: 1. Explore the microRNA (miR) differential expression profile between the HSG cell with and without inflammation which is stimulated by P.g.-LPS by the method of microRNA array and Real-time PCR. 2. The significance of the differentiated expressed miR is analyzed by bio-informational methods. 3. The miR with differential expression is overexpressed and inhibited in HSG. P.g.-LPS is used to stimulate the cells. Then compare the results between the two kinds of HSG cell that is with or without miR intervention.

NF-κ B 是炎症反应中重要的转录因子，它的反常激活可导致多种炎性疾病的发生。慢性阻塞性涎腺炎为口腔颌面部常见疾病，在其病理过程中，细菌感染所引发的免疫炎症发挥了重要作用。本课题旨在通过对人体病变组织检测和建立动物模型，以及体外细胞学实验，深入研究NF-κ B 炎性通路活化在慢性阻塞性涎腺炎中的作用，并探索其分子机制。在此基础上，选择目前研究较多miRNA 作为切入点，应用miRNA 片分析和Real time-PCR 技术，筛选出炎症感染颌下腺细胞中差异表达的miRNA，并研究上述miRNA 改变对涎腺细胞生物学作用以及对NF-κ B 介导炎性效应的影响。实验结果对于进一步认识阻塞性涎腺炎的感染和免疫学机制，从而为其诊治提供新的理论依据和方法具有重要意义。

背景：慢性阻塞性涎腺炎（chronic obstructive sialadenitis，COS）是一种临床常见疾病，病因较为复杂，其确切致病机理尚不明确。核转录因子kappaB（Nuclear factor-kappaB，NF-κ B）是天然免疫和获得性免疫以及炎症反应中一种重要的核转录因子，目前被认为是治疗控制多种炎性疾病的靶点，但其在COS中的作用尚未见研究。.主要研究内容：本研究建立了兔COS的动物模型，探索了颌下腺在慢性炎症过程中的变化；收集了患者的颌下腺组织，检测了模式识别受体和自噬相关蛋白的表达，分析了其相互之间的关系；体外进行了HSG细胞炎症模型的建立，并在此基础上探究了模式识别受体TLR4和NOD2在炎症过程中对激活自噬的协同作用；检测了HSG细胞IL-17 mRNA和蛋白表达水平，探究了IL-17刺激对HSG细胞分泌IL-6的影响，及NF-κB在其中的作用。.重要结果、关键数据及其科学意义：建立了部分颌下腺导管结扎诱导的兔慢性颌下腺炎模型，组织学检测显示颌下腺有明显的炎症改变。通过对人颌下腺组织的检测，发现炎症组织内模式识别受体和自噬相关蛋白的表达水平显著升高，并在导管细胞中存在共定位。体外实验，在HSG细胞炎症模型上发现TLR4和NOD2可激活自噬，并具有协同作用；发现IL-17以剂量和时间依赖的方式提高了HSG细胞内IL-6的表达水平，其机制与ERK, p38 MAPK, Akt以及NF-κB等信号通路相关。