增强PTPN2降低糖尿病性牙周炎炎症程度的分子机制研究

Periodontal disease, a worldwide complication of diabetes mellitus, is characterized by significantly increased inflammatory cytokines (TNF-α、INF-γ、IL-6) in serum and gingival cervical fluid. Current therapeutic approaches for diabetic periodontal disease like scaling and root planning only lead to variable and unpredictable clinical results. There is a lack of effective way that can prevent or reduce the prevalence of this disease. Recent studies shown that T-cell protein tyrosine phosphatase non-receptor type 2 (PTPN2)is a important factor that can be used to aid in prevention for diabetic periodontitis and regenerate periodontium, because 1)the expression of T-cell protein tyrosine phosphatase is strongly associated with diabetes; 2) JAK tyrosine kinases and STAT can serve as direct substrates for PTPN2; 3) inflammatory cytokine-instigated JAK/STAT-signaling pathways inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. Our preliminary data demonstrate a reduced expression of PTPN2 in gingival epithelial tissue of diabetic periodontitis and a decreased secretion of inflammatory factors in diabetic peridontitis after ehancement of PTPN2. As such, the central hypothesis in this application is that PTPN2 may help enhance the defensive ability of periodontal tissue in diabetes via diphosphate protein substrates in JAK/STAT signaling pathway. To address our hypothesis, we propose to: 1) Identify a novel approach to establishing a cell and mouse model that mimics clinical diabetic periodontitis in an efficient way; 2) Quantify the expression of PTPN2 in primary cultures of gingival epithelial cells and in mice with diabetic peridontitis.3) Define the mechanism of inhibition of inflammatory cytokine in periodontal tissue by the examination of regulation of JAK/STAT signal transduction pathways by tranfecting PTPN2-lentiviral vector both in vitro and in vivo model. Identification of the molecular mechanisim of PTPN2 that could increase defensive ability of the periodontal tissue through transcriptional mechanisms may have long-reaching implications in the study of periodontal disease and diabetes in general. It is our hope that the results from this pilot study will be a foundation to develop an in-depth study of mechanism of diabetic peridontitis and to augment current therapies for this disease.

糖尿病性牙周炎是一种易造成牙周组织显著破坏的难治性疾病，是目前牙周疾病治疗的难点和热点之一。最新研究表明蛋白酪氨酸磷酸酶非受体型2(PTPN2)在糖尿病及其并发症发展过程中起重要作用。本课题组前期研究发现糖尿病性牙周炎小鼠牙龈上皮组织PTPN2表达明显降低，增加其表达能显著降低该小鼠模型血清炎症因子水平，但其机制尚不清楚。JAK/STAT信号通路能被多种炎症因子激活，PTPN2对其关键因子的去磷酸化作用可能是PTPN2降低炎症反应的原因。本课题组拟用已构建成功的糖尿病性牙周炎细胞和小鼠模型，采用分子生物学、免疫荧光、Micro-CT、基因/蛋白芯片等技术，通过慢病毒载体增强PTPN2表达，研究PTPN2抑制炎症因子介导的JAK/STAT通路在细胞内信号转导过程中磷酸化作用，探讨其在糖尿病牙周炎炎症发生发展过程中的关键作用点及负性调控机制，为寻找治疗糖尿病性牙周炎的新途径提供理论依据。

糖尿病性牙周炎是一种易造成牙周组织显著破坏的难治性疾病，蛋白酪氨酸磷酸酶非受体型2(PTPN2)为25VD3/VDR通路上的关键调节蛋白，在糖尿病及其并发症发展过程中起重要作用。本课题假设PTPN2负性调控炎症，增强PTPN2能改善糖尿病状态牙龈上皮的防御功能，并进行了系列研究。首先，成功构建稳定的糖尿病牙周炎体内、外模型，应用免疫荧光、分子生物学等技术在高糖炎症状态的牙龈上皮细胞中，首次发现25VD3通过VDR增加PTPN2的表达，抑制JAK1、STAT3表达及其磷酸化，减少高糖状态下的牙龈上皮细胞TNF-α的分泌，从而降低炎症水平。CRISPR/Cas9技术特异性的敲除牙龈上皮细胞PTPN2基因后，上皮细胞中JAK1、STAT3表达及其磷酸化显著增加，炎症反应加重；PTPN2敲出后，25VD3不能降低其表达，表明25VD3在牙龈上皮的防御反应中起重要作用是通过调控PTPN2的表达及其下游JAK/STAT通路蛋白的磷酸化来实现的，PTPN2为VD3作用中的关键点。我们进一步应用课题构建的1型和2型糖尿病牙周炎小鼠模型，应用免疫组化、分子生物学等技术验证了PTPN2在25VD3/VDR介导的伴糖尿病牙龈上皮防御调节机制中的作用，发现增强PTPN2的表达后，糖尿病牙周炎小鼠的牙龈上皮炎症状态显著减轻，小鼠血清中TNF-α、IL-6等炎症因子的分泌下降，上皮组织中炎症相关蛋白NF-κB、TLR4、JAK1、STAT3表达及其磷酸化表达显著降低，牙槽骨吸收明显下降，表明增强PTPN2后小鼠牙龈上皮防御能力增强，牙周炎症状改善明确。除此之外，本课题还进行了一部分延伸研究，针对糖尿病患者的全身和牙周局部高炎症水平的状态，设计了具有良好的控释效果和引导组织细胞再生特性的生物材料，发明了可用于牙周治疗的血糖感应控释抗炎药物的支架材料及其制备方法，并经动物实验证明能有效恢复糖尿病动物牙周组织缺损效，有良好的推广应用的前景，为糖尿病牙周炎的治疗提供了理论依据和实验基础，研究成果发表论文14篇，其中SCI文章9篇，并多次在国际国内学术会议上报告交流。与国外合作导师联合培养博士研究生2名，硕士研究生5名，研究成果申报国家发明专利获得授权1项，获得市级科技进步奖励1项。