miR-23a 和 miR-30a对牙周炎微环境下成骨细胞作用机制研究

Chronic inflammatory disease,such as periodontitis,are the common causes of bone tissue destruction. Osteoblast differentiation is a key step in proper skeletal development, miRNAs regulate mechanism of osteoblast differentiation, the physiological role of non-coding small RNAs, especially miRNAs in osteoblast differentiation in inflammation microenvironment remain elusive. Here,through comprehesive analysis of miRNA expression during periodontitis, we show that miR-23a and miR-30a, previously viewed as osteoblast-specific miRNAs, are key regulative miRNAs in periodontitis. The objective of this study was to explore the properties of miR-23a and miR-30a involving osteoblast differentation in inflammation microenvionment. After RNA extraction of gingival tissues, miRNA profiles were analyzed by microarray, and expression level of selective miR-23a and miR30a were confirmed by real-time quantiative reverse transcription polymerase chain reaction (RT-PCR). Analyses using three computational methods, Targetscan, miRNADA and pictar were combined to identify common targets of these miRNAs. Finally,targets of miR-23a and miR-30a were confirmed by immunohistochemistry in periodontitis and exploring the role of miR-23a, miR-30a and its targets by interferring test in periodontitis. These studies help to establish knowledgeable elaborate networks of functionally tested miRNAs and it targets in bone tissue destruction of periodontitis, and provide us new means in inflammatory bone disease therapy through miR-23a, miR-30a and its targets.

牙周炎性骨质缺失是最常见的严重危害口腔健康的疾病之一。研究表明，炎性微环境下成骨细胞功能障碍是导致牙周持续性骨缺失的主要原因。miRNA参与了成骨细胞分化的调控，但在炎症微环境下miRNA如何影响成骨细胞的功能尚无报道。本课题组在筛选并鉴定牙周炎症敏感的miRNA分子群的基础上，发现miR-23a和miR-30a在调节成骨细胞分化过程中扮演重要角色。本研究拟利用体内外实验阐明炎症微环境下miR-23a和miR-30a对成骨细胞功能的调控作用；通过稳定同位素标记蛋白质谱、RISC免疫共沉淀和生物信息学分析研究miR-23a和miR-30a参与炎性骨缺失的调节途径；利用免疫组化、干预实验技术探索miR-23a和miR-30a所调节的特定靶分子在炎性牙周组织中的表达和功能。以期为深入理解牙周炎性骨缺失发生的分子机制和探讨治疗牙周炎骨质丢失提供新的策略。

牙周炎骨质缺损是最常见的严重危害口腔健康的疾病之一。研究表明，炎性微环境下骨生成障碍是导致牙周持续性骨缺失的主要原因。从与牙周炎和成骨细胞分化相关的miRNA分子群筛选出miR-23a和30a，发现miR-23a和30a具有调节成骨细胞分化和炎性因子活性双重功能，推测miR-23a和30a在炎性微环境下牙周骨缺损中可能发挥有调控炎症和骨代谢平衡双重作用。PCR检测体内外炎性牙周组织和牙周膜干细胞在炎性微环境下，miR-23a和30a表达明显增高；并表达量与炎性因子刺激的时间正相关性；合成miR-23a和30a模拟物mimics和抑制物inhibitor，转染牙周膜干细胞，上调/下调牙周膜干细胞内miR-23a和30a，观察牙周膜干细胞骨代谢和炎症进程特定因子BMP、OC、OPN、Runx2、ALP、TNF、COL-1、IL-1、IL-6表达改变，茜素红和碱磷酶染色观察上调/下调牙周膜干细胞内miR-23a和30a对矿化进程的改变。初步验证miR-23a和30a参与调控牙周炎骨缺损，具有调节成骨细胞分化和炎性因子活性双重功能。建立小鼠牙周炎模型。合成miR-23a和30a的模拟物和抑制物tagonir和antagonir，体内转染7天小鼠牙周炎性模型，采用微量注射方法体内转染3、7、11天，microCT检测观察活体小鼠炎性缺损区组织的变化，观察miR-23a和30a抑制物能有利于骨修复。通过计算机生物信息学软件miRanda，TargetScan和PicTar预测miR-23a和miR-30a可能的靶基因，继而体外干预牙周膜细胞内miR-23a和miR-30a的表达水平，检测已经预测到的可能靶基因的表达情况，STAT5B为miR-23a的靶基因、Runx2为miR-30a的靶基因：升高miR-23a和miR-30a的表达可抑制牙周膜细胞成骨作用和炎症反应。