盐酸小檗碱通过EGFR介导的ERK通路促进牙周膜细胞成骨转化及牙周骨组织再生机制的研究

Because of the open environment of the mouth, it is difficult to achieve a satisfied therapeutic effect to promote bone repair for treating periodontitis by simple growth factors. Therefore, the treatment should both include controlling inflammation and promoting periodontal bone tissue regeneration. Traditional Chinese medicine has been known as the effect on anti-inflammatory, immune regulation, balance the bone metabolism, so it is very suitable for the treatment of periodontitis. Applicant had found in the early research that anti-inflammatory active ingredient berberine (BER) extracted from Coptis chinensis could promote the proliferation and osteogenesis of periodontal ligament cells (PDLC), and had a competitive effect with EGFR inhibitor. Based on that, this project will explore the effect of BER in anti-inflammatory and promoting periodontitis bone tissue repair by the periodontitis animal models on SD rats and miniature pigs，and to determine the dose-effect relationship on the overall level. The high and low expression EGFR cells will be built，and the interaction between BER and EGFR will be examined by the combination of cell membrane chromatography and radio ligand binding assay. The signal pathway of target action of BER through EGFR will be studied by molecular biology technique such as PCR Array. The relationship between BER and EGFR, ERK, the regeneration mechanism of periodontal bone tissue will be elucidated. The completion result of this project can provide experimental basis for the development of new methods and therapeutic drug research for treatment of periodontal bone destruction caused by periodontitis.

由于口腔的开放环境，对慢性牙周炎单纯使用生长因子促骨质修复，难以达到满意的治疗效果，需同时控制炎症和促进牙周骨组织再生。中药同时具有消炎杀菌、调节免疫、平衡骨代谢作用，非常适合牙周炎的治疗。申请人前期研究发现，中药黄连的抗炎活性成分小檗碱（BER）可促进牙周膜细胞（PDLC）增殖和成骨转化，且与EGFR抑制剂有竞争结合作用。本项目拟在此基础上，采用SD大鼠和小型猪牙周炎模型，考察BER抗炎及促牙周骨质修复作用，在整体水平确定BER促成骨作用的量效关系；建立EGFR过表达和沉默细胞系，通过细胞膜色谱模型、放射性配体结合分析法等，在细胞水平考察BER与EGFR相互作用关系；采用PCR-Array芯片等分子生物学技术，研究BER通过EGFR靶点作用的信号通路，阐明BER与EGFR、ERK的关系及促牙周骨组织再生机制，为开发治疗牙周炎引起牙周骨组织破坏的中药及其活性成分的新方法和药物提供实验基础。

研究背景：牙周炎是目前成年人牙齿丧失的主要病因，研发出有效的药物用于临床牙周炎的治疗有重大意义。中药同时具有的消炎杀菌、调节免疫和平衡骨代谢的作用，非常适合达到牙周炎控制炎症和促进牙周骨再生的效果。中药黄连的活性成分盐酸小檗碱(BER)和人牙周膜干细胞（hPDLSCs）具有靶向亲和效应，但其效应机制尚不明确。本研究明确了hPDLSCs具备多向分化能力；BER可促进hPDLSCs增殖和成骨转化，进而筛选出BER和hPDLSCs表面亲和的特异性靶标受体(表皮生长因子受体，EGFR)，通过研究BER和EGFR相互作用关系，明确BER通过膜表面特异性靶点作用的信号通路EGFR-ERK-FOS，初步阐明BER促进牙周组织再生的机制；hPDLSCs联合CPC支架材料能够有效促进牙周组织再生。.研究内容：1、hPDLSCs多向分化能力的研究；2、BER促进hPDLSCs的增殖和成骨效应的膜表面靶标位点的初步研究；3、hPDLSCs联合磷酸钙（CPC）支架材料促进牙周组织再生的初步研究.研究结果：1、证实hPDLSCs具有成骨、成纤维和成牙骨质多向分化的能力；2、筛选出BER亲和hPDLSCs的特异性靶标受体EGFR，可能通过EGFR-ERK-FOS通路促进牙周组织再生；3、hPDLSCs联合CPC支架材料能够有效促进牙周组织再生。.意义：1、hPDLSCs具备多向分化能力，适用于牙周局部需要达到成骨、成纤维和成牙骨质多种组织再生的要求；2、BER可能通过EGFR-ERK-FOS信号通路促进牙周组织再生，对深入研究BER的药物作用机制及药物的优化改性提供思路；3、寻找适合牙周组织再生的支架材料，对后期将BER联合hPDLSCs用于牙周组织再生的组织工程技术提供支架材料选择的研究基础。