炎症微环境作用下经典及非经典Wnt通路平衡对牙周膜干细胞成骨分化的调控作用

The crosstalk between canonical and noncanonical Wnt signaling pathway in osteoblast differentiation of periodontal ligament stem cells in inflammatory microenvironments

目的 探讨炎症微环境作用下经典Wnt/β-联蛋白（β-catenin）与非经典Wnt/Ca^2＋通路的平衡对牙周膜干细胞（periodontal ligament stem cells,PDLSC）成骨分化的调控作用.方法 收集解放军总医院口腔科2015年3至6月因治疗需要拔除的前磨牙和第三磨牙8颗及慢性牙周炎牙齿8颗,培养慢性炎症组织来源的PDLSC （PDLSC from patients diagnosed as periodontitis,P-PDLSC）与正常组织来源的PDLSC （PDLSC obtained from a healthy microenvironment,H-PDLSC）.RNA干扰转染β-联蛋白至H/P-PDLSC,观察细胞状态并用蛋白质印迹法检测转染效果.实时定量PCR技术检测转染后Runt相关转录因子2 （Runt-related transcription factor 2,Runx2）、β-联蛋白及Nemo样激酶（nemo like kinase,NLK） mRNA表达.成骨诱导3d后蛋白质印迹法检测Ca^2＋/钙调素依赖型蛋白激酶Ⅱ（Ca^2＋/calmodulin-dependent protein kinaseⅡ,CaMKⅡ）和NLK的表达,免疫荧光检测CaMKⅡ的表达.结果 蛋白质印迹法检测RNA干扰24 h抑制H/P-PDLSC siRNA β-联蛋白转染组中β-联蛋白的表达.成骨诱导3d后实时定量PCR结果显示,P-PDLSC siRNA β-联蛋白组Runx2（4.553±0.659）显著高于P-PDLSC空质粒转染组（1.918±0.315）（P=0.000）,NLK mRNA表达（7.341±1.331）亦显著高于空质粒转染组（5.664±0.792）（P=0.030）;与之相应的是蛋白质印迹法检测P-PDLSC siRNA β-联蛋白组与P-PDLSC空质粒转染组相比CaMKⅡ、NLK蛋白表达水平上升.免疫荧光检测结果显示,成骨诱导后H-PDLSC及P-PDLSC siRNA β-联蛋白组CaMKⅡ表达增强且高于H/P-PDLSC空质粒转染组.结论 炎症微环境作用下经典/非经典Wnt通路在PDLSC成骨分化过程中均发挥重要作用,抑制β-联蛋白可增强非经典Wnt/Ca^2＋通路,促进干细胞的成骨分化.

Objective To investigate the crosstalk between canonical Wnt/β-catenin and noncanonical Wnt/Ca^2＋ pathway in osteoblast differentiation process of periodontal ligament stem cell （PDLSC） in inflammatory microenvironments.Methods PDLSCs were obtained from human healthy individuals（H-PDLSC） and patients with periodontitis（P-PDLSC）.The H/P-PDLSCs were transfected with β-catenin siRNA.Cell morphology was observed under fluorescent microscope and transfection efficiency was easured by Western blotting after transfection of PDLSC.The mRNA expressions of Runt-related transcription factor 2（Runx2）,β-catenin and nemo like kinase（NLK） were detected by real time PCR,the protein expressions of calciun/calmodulin-dependent protein kinase Ⅱ （CaMK Ⅱ） and NLK were examined by Western blotting and the CaMK Ⅱ was observed by immunofluorescence staining,respectively.Results The β-catenin expressions in H/P-PDLSCs were inhibited specifically and efficiently by treatment of β-catenin-siRNA for 24 h.After a 3-day-osteogenic process,results of real-time quantitative PCR showed that the Runx2 mRNA expression in P-PDLSC siRNA β-catenin transfected group（4.553 ± 0.659） was significantly higher than that in P-PDLSC empty plasmid control group（l.918 ±0.315）（P=0.000）.A similar trend was observed in the NLK mRNA expression tests（7.341 ± 1.331 vs.5.664 ± 0.792）（P=0.030）.Accordingly,the protein expression levels of CaMK Ⅱ,NLK were higher in P-PDLSC siRNA β-catenin transfected group than that in P-PDLSC empty plasmid control group in osteogenic differentiation condition for 3 days.CaMK Ⅱ was more strongly induced in P-PDLSC siRNA β-catenin group than that in P-PDLSC empty plasmid control group after PDLSC cultured in osteogenic medium for 3 days.Conclusions Both canonical Wnt/β-catenin and noncanonical Wnt/Ca2＋ pathway could regulate the osteogenic differentiation potential of P-PDLSC.Suppression of β-catenin by siRNA promoted osteogenic differentiation via increasing noncanonical Wnt signaling pathway of PDLSC in inflammatory microenvironments.