炎症微环境作用下牙周膜干细胞免疫调节及再生功能变化及其调控机制研究

Microenvironment is critical for regulation the function of stem cell. Osteogenic differentiation and immune function of stem cells are affected after the invasion of inflammation. There is little know about stem cell immune function in chronic inflammatory microenvironment, and there is great controversy adjustment mechanism. Periodontitis is a chronic inflammatory disease associated with immune. Periodontal ligament tissue-derived mesenchymal stem cells (PDLSCs) are capable of being used in stem-cell-mediated therapies and tissue engineering. PDLSCs have dual function of both the immune regulation and tissue regeneration ability, which make them has broad application prospects in the treatment of immune-related tissue defects. Our previous studies have shown that inflammatory microenvironments reduces differentiation potentials of PDLSCs, inhibition of the Wnt pathway can partially restore the regenerative capacity of inflammatory PDLSCs. Recent studies have found that the regenerative capacity of PDLSCs was decreased while its immunomodulatory capacity was upregulated in the state of periodontitis, however the mechanism still unknown. We will first study the influence and the role of key cytokines in PDLSCs get from/ under inflammatory microenvironment. We will then investigate the interactions between PDLSCs and immune cells. We will further determine the mechanism of PDLSCs immunomodulatory capacity and regeneration functional under inflammatory microenvironment. Finally, we will explore novel strategies via immunoregulation and regeneration to restore the function of PDLSCs in periodontal disease microenvironment. We believe that the completion of these studies will provide critical information about how to regulate immune status and osteogenic differentiation potential of stem cells in inflammatory microenvironment, then provide new strategies for functional periodontal tissue regeneration .

微环境对调控干细胞功能至关重要，慢性炎症组织中干细胞再生能力下降，机理尚不明确。慢性牙周炎是免疫相关的炎性疾病，牙周膜干细胞（PDLSCs）具有组织再生和免疫调节的双重功能，直接参与牙周组织修复再生。我们前期研究表明炎症抑制PDLSCs成骨分化，Wnt通路调控可部分恢复炎症微环境下PDLSCs 的再生能力，不能达到正常水平；近期研究发现牙周炎条件下PDLSCs 再生能力下降而免疫调节功能上调，其原因与调控机理有待阐明。本项目拟在前期工作基础上全面比较正常和炎症组织来源PDLSCs的再生潜能和免疫调节能力，确定关键炎症因子对 PDLSCs的影响；明确PDLSCs与免疫细胞相互作用；阐明调控PDLSCs免疫调节和组织再生双重功能的分子机制，在此基础上建立恢复炎症微环境下 PDLSCs再生能力的方法。此研究将为基于干细胞技术的牙周炎治疗提供新的方法和理论依据。

慢性牙周炎是免疫相关的炎性疾病，牙周膜干细胞(PDLSCs)具有组织再生和免疫调节的双重功能，直接参与牙周组织修复再生。我们前期研究表明炎症抑制PDLSCs成骨分化，Wnt通路调控可部分恢复炎症微环境下PDLSCs 的再生能力，不能达到正常水平；近期研究发现牙周炎条件下PDLSCs 再生能力下降而免疫调节功能上调，其原因与调控机理有待阐明。因此本研究系统研究其作用机制。首先我们经过体外及体内实验证实炎症条件下PDLSCs具备干细胞的特性但是成骨分化能力显著降低。我们通过免疫磁珠法从人外周血白膜层获取了CD3+T细胞，并进行了HPDLSCs、IPDLSCs与CD3+T细胞的不同比例（1：10、1：20、1：50、1：100）共培养，发现在炎症条件下PDLSCs对CD3+T细胞的增殖抑制能力也降低了。随后我们的研究证实了与非经典Wnt / Ca2+通路密切相关的蛋白P38MAPK参与细胞的炎性反应，抑制p38后炎症条件下PDLSCs 的成骨分化能力也被显著抑制，同时，Wnt关键蛋白GSK3β的磷酸化可影响Wnt通路抑制PDLSCs 成骨分化；炎症微环境作用下经典/非经典Wnt通路在牙周膜干细胞成骨分化过程中均发挥重要作用，SiRNA抑制β-catenin 可增强非经典Wnt/Ca2+ 通路从而促进干细胞的成骨分化；过表达β-catenin对H-PDLSCs成骨分化能力的影响远高于对P-PDLSCs的影响。过表达及SiRAN β-catenin调控Wnt信号通路后 HPDLSCs及IPDLSCs与CD3+T细胞共培养后成骨能力降低，增殖能力降低。上述结果表明通过靶向抑制炎症微环境作用下干细胞中β-catenin蛋白，在成骨条件下可有效的上调非经典Wnt信号通路的活化水平，恢复Wnt经典与非经典信号通路的平衡，进而恢复炎症组织来源干细胞的成骨分化能力。通过调控经典Wnt信号通路关键分子β-catenin 可提高炎症作用下PDLSCs 的免疫调节能力。此研究结合Wnt信号通路调控PDLSCs免疫调节和组织再生双重功能的分子机制，在此基础上建立恢复炎症微环境下 PDLSCs再生能力的方法。 将为基于干细胞技术的牙周炎治疗提供新的方法和理论依据。