长非编码RNA对牙髓成纤维细胞NLRP3/caspase-1炎症体的调控机制研究

Innate immunity has been viewed as the first line for host against invading pathogens. The inflammasome, a large multiprotein complex assembled by the pattern-recognition receptors (PRRs) in the cytoplasm, plays an important role in innate immune system. Human dental pulp fibroblasts (HDPF) are very important in development of dental pulp inflammation. By sensing bacterial stimuli, nucleotide-binding oligomerization domain-like receptors P3(NLRP3) in HDPF can initiate the reaction leakage of inflammation of dental pulp. While the other intracellular/extracelluar signals could effect the function of NLRP3/caspase-1 pathway. In this project,the regulative effects of heat shock RNA-1(HSR-1),a lncRNA, on NLRP3/caspase-1 inflammasome in HDPF are examined with lentiviral expression vector and high throughput screening techniques. The effects of the heat shock protein (HSP) on NLRP3/caspase-1 in HDPF are also studied by establish of overexpress and silence of HSP gene and NLRP3 gene of HDPF. More lncRNAs which can regulate NLRP3 in the gene's upstream and downstream will be screened by high-throughput sequencing technology. The aim of this study is to clarify the pathogenesis of dental pulp inflammation, explore the function of lncRNA on the resolution and outcome of dental pulpitis, and provide scientific basis on searching the therapeutic target on dental pulp inflammation.

牙髓成纤维细胞(human dental pulp fibroblast, HDPF)在牙髓炎症的发展进程中作用十分重要。它可通过胞质Nod样受体P3(NLRP3)感知刺激，启动炎症反应，而其信号通路又受到其他网络信号的影响。本研究通过慢病毒表达载体的方法，构建heat shock RNA-1(HSR-1)这一目前已知的长非编码RNA(long non-coding RNA, lncRNA)基因的过表达和沉默模型，研究HSR-1对HDPF细胞内NLRP3/Caspase-1炎症体通路的调控作用；构建NLRP3基因过表达和沉默模型并通过高通量分析来进一步筛选可以调控NLRP3/caspase-1上下游lncRNA及其功能特点。探索在牙髓炎症进程中lncRNA的调节机制，为阐明牙髓炎的发病机理，进而为探索lncRNA在牙髓组织炎症转归和消退机理，为临床寻找治疗牙髓炎新的靶点提供理论依据。

龋病是一种慢性感染性疾病。龋源相关性细菌的入侵是造成龋病的主要原因，它能够引起牙釉质、牙本质脱矿，进而造成牙髓组织损伤，引起牙髓炎症反应。研究证明NLRP3/Caspase-1炎症体通路在牙髓组织抵御细菌入侵的过程中发挥了重要作用。然而该炎症体通路是如何参与调控牙髓炎发生发展的分子机制，目前尚不清楚。长链非编码RNA（long noncoding RNA, lncRNA）在表观遗传、转录及转录后基因表达中起着重要的调控作用，其是否参与调控牙髓炎的发生发展，尚未见报道。为了深入研究lncRNA对牙髓炎的发生发展的分子调控机制，我们进行了如下实验：1、体外分离并鉴定了牙髓成纤维细胞（HDPF）。2、证明了TLR4信号通路与NLRP3的crosstalk在牙髓炎症进程中具有重要的调控作用。3、 lncRNA（HSR-1）通过热休克反应对牙髓炎症发展进程起了重要调控作用。4、 lncRNA（ANCR）对于牙源性干细胞生物学性能具有重要调控作用。下调ANCR能够有效提高牙周膜干细胞增殖能力；下调ANCR能够有效促进牙髓干细胞、牙周膜干细胞、根尖牙乳头干细胞成骨、成脂、成神经分化潜能。5、非编码小RNA（micro RNA, miR）（miR-223和miR-22）对HDPF中NLRP3炎症体的转录后调控起着重要作用。我们对非编码RNA在牙髓组织及HDPFs中NLRP3/Caspase-1炎症复合体通路的调节机制进行了初步的探索和研究，这些结果有助于我们进一步研究该炎症体通路，揭开炎症的发生发展机制，从而进一步为临床转化医学研究创造条件。

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