中文摘要：

目的：探讨非经典Wnt钙离子信号通路在慢性牙周炎组织来源的牙周膜干细胞中的表达及其对炎症微环境作用下牙周膜干细胞成骨分化能力的影响。方法：收集解放军总医院口腔科因治疗需要拔除的无龋前磨牙、第三磨牙及慢性牙周炎牙齿,培养正常及炎症微环境下的牙周膜干细胞（H-PDLSCs,P-PDLSCs）。成骨诱导3d后采用免疫荧光检测CaMKII在细胞内的表达。H-PDLSCs,P-PDLSCs成骨诱导7d后实时定量PCR检测CaMKII、NLK和Runx2 mRNA表达水平,同时采用Western blot检测两种细胞成骨诱导7d后Runx2、ALP、CaMKII、和NLK的表达。结果：免疫荧光结果显示在H-PDLSCs及P-PDLSCs的对照组中CaMKII均有弱阳性表达,成骨诱导3d后两组细胞中CaMKII的表达增强。实时定量PCR结果显示常规培养条件下CaMKII、Runx 2在H-PDLSCs与P-PDLSCs中的表达不存在显著性差异,当成骨诱导后其在两组中的表达量显著增高,但H-PDLSCs组显著高于P-PDLSCs组。P-PDLSCs中NLK的表达水平显著高于H-PDLSCs,成骨诱导后两种组织来源细胞中NLK mRNA的表达水平均有显著性升高但H-PDLSCs组显著高于P-PDLSCs组。非经典信号通路的关键蛋白CaMKII、NLK在成骨诱导7d后其表达水平升高,成骨关键蛋白Runx2及ALP的表达趋势与之一致,但P-PDLSCs低于H-PDLSCs。结论：在慢性炎症微环境中非经典Wnt信号通路参与牙周膜干细胞的成骨分化,Wnt/Ca2＋通路中CaMKII、NLK蛋白与干细胞成骨分化正相关。

英文摘要：

Objective： To investigate the osteogenic differentiation potential and the role of non-canonical Wnt/Ca2＋ signaling pathway of human periodontal ligament stem cells in inflammatory microenvironment. Methods： Periodontal ligament stem cells were obtained from healthy human individuals（H-PDLSCs） and patients with periodontitis（P-PDLSCs）. Immunofluorescence staining was used for determining the protein expression of CaMKII in PDLSCs after 3days cultured in osteogenic medium. After P-PDLSCs and H-PDLSCs were cultured in osteogenic medium for 7 days, the m RNA expressions of CaMKII, NLK and Runx2 were tested by Real time PCR, and the protein expression of Runx2,ALP, CaMKII and NLK were examined by Western Blot. Results： CaMKII was more strongly induced in H-PDLSCs cells than in P-PDLSCs cells after PDLSCs cultured in osteogenic medium for 3 days. Real-time PCR results showed no significant difference in the mRNA expression of CaMKII and Runx2 between H-PDLSCs and P-PDLSCs under normal culture conditions, while both CaMKII and Runx2 expression in H-PDLSCs group significantly higher than the P-PDLSCs group when PDLSCs were cultured in osteogenic medium for 7 days. NLK expression levels were significantly increased in H-PDLSCs and P-PDLSCs after 7days ＇ induction. When stimulated with osteogenic medium for 7d, CaMKII and NLK activation were observed in H-PDLSCs and P-PDLSCs, while the levels of CaMKII and NLK in H-PDLSCs were found higher than those in H-PDLSCs cultured in the presence or absence of osteogenic medium. Osteogenic medium enhanced the levels of Runx2 and ALP of PDLSCs especially in H-PDLSCs. Conclusion： Wnt/Ca2＋ signaling pathway takes part in regulating osteogenic differentiation of PDLSCs in inflammatory microenvironments. CaMKII, NLK positively correlated with stem cells osteogenic differentiation.