Aspirin联合牙周膜干细胞再生全脱位牙牙周组织机制研究

We found there was low tooth retention rate after routine dental replantation treatment caused by unavoidable external root resorption postoperative. This result directly impact on children's facial appearance and psychological health.The study found, PDLSCs have the ability to regenerate PL and cementum meanwhile.Therefore, we try to transplant the auto-PDLSCs into non-immunosuppressive mini pigs CAT model, and found that cells transplantation group was getting better periodontal regeneration, but it still can not avoid the external root resorption occurrencing, it suggested that the immune cells may impacted the transplanted PDLSCs tissue repair ability. Aspirin, representatives of non-steroidal anti-inflammatory drug, had been shown to have a role in stimulate stem cells regulate body immune function. However, the mechanism of Aspirin to regulate local immune of CAT, and promote PDLSCs regenerate ideal functional periodontal tissueion remains to be seen. Therefore, in present study we will around the scientific hypothesis "Aspirin can potentially promote Treg to regulate PDLSCs system, and via anti-inflammatory cytokines release to delay PDLSCs apoptosis, and promote the body into the wound healing circulation, to get the ideal periodontal regeneration", we proposed from in vitro cells detect and in vivo transplantation experiments in RvD1-LxA4-PGE2/FGF2 pathway , and using a variety of molecular biology and biochemistry experiments to explore the mechanism of Aspirin promote PDLSCs regeneration founctional periodontal tissue of CAT l, and provide new ideas and theoretical basis for the clinical treatment for CAT.

降低牙根吸收，获得功能性牙周组织再生是提高牙全脱位延迟再植术成功率的关键。牙周膜干细胞是牙周再生的理想细胞，以往研究发现单独应用牙周膜干细胞无法有效阻止牙根吸收，需控制局部微环境的炎症免疫反应以提高其组织修复能力。Aspirin被证实具有炎症免疫调节功能，但能否促进牙周膜干细胞在牙全脱位延迟再植术中的功能及机制尚不清楚。本课题在前期基础上，拟开展Aspirin对局部Treg的调节作用及牙周膜干细胞功能影响的研究，探讨在牙全脱位延迟再植动物模型局部应用Aspirin对微环境改善、牙周膜干细胞功能的影响及在再生修复中的作用。明确Aspirin可否通过Treg，进而调控牙周膜干细胞RvD1-LxA4-PGE2/FGF2信号通路并改变其功能。通过上述研究，我们可以发现促进牙全脱位延迟再植术及功能性牙周组织再生的候选药物、应用方法及作用机制，为提高牙全脱位延迟再植效果提供理论依据及新的治疗途径。

牙齿全脱位（Completely avulsed teeth,CAT）是儿童牙外伤常见且严重的一类，目前牙回植术（Tooth replantation,TR）因难掌控回植时间，组织活力及患者免疫水平等因素，疗效不佳。查阅文献后，我们将焦点放在组织活力的延续、免疫环境调节及创口初期愈合调控机制上来，以期对上述因素的负面影响。因此，主要研究内容为：1.体外分离犬 T 细胞和牙周膜干细胞（Periodontal ligament stem cells,PDLSCs ），对其进行纯化和鉴定；2.探索阿司匹林（Aspirin） 和 Treg 对犬PDLSCs 生物学功能改变的影响及作用机制 ；3构建 Beagle 犬CAT延迟再植动物模型，在体内进一步观察 Aspirin 对PDLSCs 再生牙周组织的效果和相关基因的表达，通过以上三部分内容的探究，回答Aspirin是否可以上调Treg细胞水平，干预PDLSCs表达创伤初期愈合关键因子：脂养素A4（(lipoxin A4，LxA4)和消散素D1(Resolvins D1,RvD1)，从而促进PDLSCs再生牙周组织过程，且降低干扰因素的影响。结果初步验证了250mg/mlAspirin是最佳诱导Treg增殖比率的给药浓度。在对犬PDLSCs LxA4受体FPR2-shRNA慢病毒转染来下调LxA4水平后，发现不管是否是单独使用Aspirin还是Treg诱导，PDLSCs的增殖能力，迁移能力以及分化能力都出现了下调，从而证实了LxA4在犬PDLSCs的细胞修复能力上起着关键调节作用。实验中还验证了250mg/mlAspirin联合Treg细胞共培养后，PDLSCs的各项修复指标极分化能力都是最佳的。此外，研究也验证了Treg细胞比率的增加和PDLSCs生物学活力是相互调节的。体内研究发现，在使用Aspirin诱导的实验组里，造模后1d,7d,14d龈沟液的渗出是最少的，术后微量蛋白固相芯片检测也是实验组炎性介质水平最低，与对照组之间差异有统计学意义。体内造模前和4w后拍摄X光和CT，影像学显示回植了PDLSCs的实验组，不管是否使用Aspirin均较无PDLSCs回植组更接近正常牙周影像。而组织切片再次印证了这一点。因此，我们认为Aspirin可以尝试成为CATTR功能性牙周组织再生的候选药物。