p38 丝裂原活化蛋白激酶在炎症微环境作用下对牙周膜干细胞成骨分化的影响

目的 探讨p38丝裂原活化蛋白激酶(MAPK)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 2012年10月至2013年5月收集中国人民解放军总医院口腔科因治疗需要拔除的无龋前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)。采用酶联免疫吸附试验及实时定量PCR检测肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β的分泌及基因表达量,免疫荧光检测p38在细胞内的表达,Western blot法检测两种细胞成骨诱导7 d后p38及磷酸化p38(p-p38)的表达。使用p38 MAPK特异性抑制剂SB-203580干扰后体外成骨诱导7 d,实时定量PCR检测细胞成骨关键基因Runx2和碱性磷酸酶(ALP)的基因表达水平,成骨诱导21 d后茜素红染色检测PDLSCs的矿化结节形成情况。结果 P-PDLSCs的TNF-α和IL-1β分泌量均显著高于 H-PDLSCs(68.80±6.70比34.10±3.07,P=0.001;57.10±4.23比26.90±2.58,P=0.000)。P-PDLSCs中TNF-α的基因表达水平较H-PDLSCs上升1.4倍(P=0.011),IL-1β的基因表达水平也上升1.7倍(P=0.009)。P-PDLSCs的p38表达量高于H-PDLSCs;成骨诱导后两种PDLSCs 中的p-p38表达均升高,但p38表达水平有所降低。SB-203580干扰后PDLSCs 的Runx2和ALP表达水平显著降低(H-PDLSCs:P=0.044、0.036;P-PDLSCs:P=0.017、0.004),矿化结节形成数量减少。结论 在慢性炎症微环境中,p38 MAPK参与细胞的炎性反应,抑制p38后炎症微环境作用下PDLSCs的成骨分化能力也被显著抑制。

Effects of p38 Mitogen-activated Protein Kinase on Osteogenic Differentiation of Human Periodontal Ligament Stem Cells in Inflammatory Microenvironment

Objective To investigate the expression of mitogen-activated protein kinase(MAPK)in the chronic periodontitis tissue-derived in the periodontal ligament stem cells(PDLSCs)and explore its effect on the osteogenic differentiation of human PDLSCs in inflammatory microenvironment. Methods PDLSCs were obtained from human healthy individuals(H-PDLSCs)and patients with periodontitis(P-PDLSCs). The tumor necrosis factor(TNF)-α and interleukin(IL)-1β secretion and mRNA expression levels of H-PDLSCs and P-PDLSCs were detected using enzyme-linked immunosorbent assay and real-time quantitative PCR. Immunofluorescence staining was used for determining the protein levels of p38 in PDLSCs. The levels of p38 and p-p38 following culture in osteogenic medium for 7 d of H-PDLSCs and P-PDLSCs were detected using Western blotting.After the PDLSCs were stimulated with SB-203580,the p38 MAPK specific inhibitor,for 30 min and then in osteogenic induction process for 7 days,the expression levels of the osteogenic gene Runx2 and alkaline phosphatase(ALP)were determined by real-time quantitative PCR,and bone formation ability of PDLSCs was tested by alizarin red(AR)staining. Results The secretions of TNF-α and IL-1β were significantly higher in P-PDLSCs compared with H-PDLSCs(68.80±6.70 vs. 34.10±3.07,P=0.001;57.10±4.23 vs. 26.90±2.58,P=0.000). The same trend was seen in the gene expression levels of both TNF-α and IL-1β in PDLSCs(P_TNF-α=0.011,P _IL-1β=0.009). p38 was more strongly induced in P-PDLSCs cells than in H-PDLSCs.The basal level of p38 in H-PDLSCs was lower than that in P-PDLSCs cells cultured in the basic medium. However,the level of p-p38 was increased in H-PDLSCs than in P-PDLSCs under osteogenic condition. Treatment of PDLSCs with SB-203580 and then cultures under osteogenic differentiation lead to significantly decreased expressions of Runx2 and ALP in both H-PDLSCs and P-PDLSCs(H-PDLSCs:P_Runx2=0.044,P_ALP=0.036;P-PDLSCs:P_Runx2=0.017,P_ALP=0.004). Conclusions p38 MAPK is involved in the inflammatory response of PDLSCs in the chronic inflammatory microenvironment. The inhibition of p38 by SB-203580 also remarkably suppresses the osteogenic differentiation of PDLSCs in a chronic inflammatory microenvironment.