调节性B细胞在骨质疏松动物中促进种植体骨结合的研究

Postmenopausal osteoporosis (PMOP) is a metabolic bone disorder which can be　induced by Th17, and it is considered contraindicated implant placement. The　imbalance between Treg and Th17 always indicate the severity of the disease. The　mechanism of Bregs which can maintain the balance of Treg/Th17 in PMOP remains　unclear. Previous data shows that Bregs can significantly reduce the inflammation　in periodontitis and the bone absorption. The experiment is set up to further understand the mechanism how Breg promote　the function of Treg as well as it depress Th17 to suppress the inflammation.In current study, an osteoporosis rat model will be set firstly. The secrection of Breg/Th17/Treg will be measured. Breg will be transferred into osteoporosis rats after the dental implants are put into maxillary. The interaction between Breg and Treg/Th17; the antigen specific and Breg’s inflammatory suppression will be　investigated. Flowcytometry,　Immunohistological chemistry will be used to find out the mechanism of Bregs/Th17/Treg in inflammation and bone absorption. The signal of　Breg to active osteoblast will be explored to provide valuable insights into the potential B cell-directed therapeutics in humans as well.

绝经后骨质疏松（PMOP）患者由于持续发生病理性骨吸收，种植成功率低。炎症细胞Th17的功能亢进与骨吸收程度显著相关，因此抑制Th17功能成为治疗PMOP的热点。调节性B细胞（Breg）是近年新发现的重要亚群，不但可直接抑制Th17细胞功能，还能促进Treg对Th17的抑制作用。目前Breg在PMOP中少见报道。本课题组前期研究已显示：炎症细胞诱导的破骨细胞分化可被Breg所抑制，炎性骨吸收减少。为进一步探讨Breg在PMOP中的调节机制，本项目拟首先构建PMOP模型，观察疾病中Breg、Th17、Treg等的表达水平；其次，通过回输体外增殖的Breg，研究Th17/Treg数量及相关基因的变化，阐明调控的作用和机制；最后评估炎症控制及骨结合水平，进一步分析和确证Breg/Th17/Treg调控通路对种植体周围骨再生相关基因的影响，为控制种植区骨的炎症吸收,促进早期骨结合提供新的治疗靶点。

绝经后骨质疏松（PMOP）患者由于持续发生病理性骨吸收，种植成功率低。炎症细胞Th17的功能亢进与骨吸收程度显著相关，因此抑制Th17功能成为治疗PMOP的热点。调节性B细胞（Breg）是近年新发现的重要亚群，不但可直接抑制Th17细胞功能，还能促进Treg对Th17的抑制作用。目前Breg在PMOP中少见报道。本课题组以Breg的功能为核心研究内容，通过体外实验确认了以P. gingivalis LPS (10 μg/ml) 和CpG (10μ M)联合刺激来获得Breg的增殖活化，并将其回输入炎症性骨吸收的小鼠动物模型检验其功能及作用机理。实验结果显示P. g LPS和CpG的联合刺激能显著提高CD19+IL-10+细胞的数量，并验证了CD1dhi CD5+ B是产生IL-10的主要细胞。通过卵巢摘除术建立PMOP小鼠模型后，可以发现小鼠外周血中的CD19+CD1dhi CD5+ B细胞、CD4+CD25+FoxP3+细胞降低时间点与IL-17+T细胞增加时间点一致，趋势相反，呈现负相关联系。通过体内实验，验证体外回输增殖的Breg能够抑制P. g细菌相关的结扎模型的牙槽骨吸收。Breg抑制RANKL/OPG, TNF-α, and IL-1β 但是促进牙龈 IL-10mRNA的表达。采用Vivo track荧光标记技术，验证了Pan B细胞胞在小鼠中会趋化至炎症区域，通过浸润于牙齿周围组织发挥作用。回输活化的Breg至拔除一侧第一磨牙的小鼠体内，12周后进行骨组织计量学测定回输Breg的OVX小鼠显著优于无回输的OVX小鼠。骨组织形态学观察显示Breg回输能有效改善PMOP小鼠的成骨。回输Breg后的小鼠外周血中CD19+CD1dhighCD5+的细胞以及IL-10+Breg细胞的数量显著高于未回输的OVX小鼠。而未回输Breg的OVX鼠外周血中IL-17+T细胞的数量显著高于回输Breg的鼠，具统计学差异。