炎症微环境中WDR72对牙周膜干细胞成骨分化的调节作用

Role of WDR72in regulating osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment

目的探讨肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的炎症微环境中WDR72对牙周膜干细胞(periodontal ligament stem cells,PDLSCs)成骨分化的调节作用及机制。方法收集前磨牙样本,分离培养PDLSCs并采用流式细胞术鉴定其干细胞特性。取对数生长期PDLSCs分为对照组(成骨诱导培养液培养)、TNF-α组(含10μg/L TNF-α的成骨诱导培养液培养),培养第7天,采用实时荧光定量PCR法检测碱性磷酸酶(alkaline phosphatase,ALP)、骨钙蛋白(osteocalcin,OCN)、RUNX2mRNA相对表达量,采用Western blot法检测WDR5、WDR7、WDR72蛋白相对表达量,筛选出2组表达有差异的蛋白WDR72进行质粒转染;培养第21天采用茜素红染色法观察钙结节并检测540nm波长处吸光度值(OD_(540))。对数生长期PDLSCs分为对照质粒组(成骨诱导培养液培养并转染对照质粒)、对照质粒+TNF-α组(含10μg/L TNF-α的成骨诱导培养液培养并转染对照质粒)、WDR72质粒+TNF-α组(用含10μg/L TNF-α的成骨诱导培养液培养并转染WDR72质粒),培养第7天采用实时荧光定量PCR法检测ALP、OCN、RUNX2mRNA相对表达量,采用Western blot法检测WDR72蛋白相对表达量;培养第21天采用茜素红染色法观察钙结节并检测OD_(540)值。结果PDLSCs表面干细胞标志物CD44、CD90阳性表达率均>99%,CD45、CD34阳性表达率均<1%,分离培养的PDLSCs符合间充质干细胞特性。培养第7天TNF-α组ALP、OCN、RUNX2mRNA(0.56±0.11、0.56±0.10、0.58±0.08)及WDR72蛋白(0.40±0.02)相对表达量均低于对照组(1.00±0.15、1.00±0.13、1.00±0.16、0.72±0.02)(P<0.05),WDR5、WDR7蛋白相对表达量与对照组比较差异均无统计学意义(P>0.05)。培养第21天TNF-α组较对照组钙结节数量少、染色浅,TNF-α组OD_(540)值(0.70±0.04)低于对照组(1.90±0.12)(t=9.479,P<0.001)。培养第7天对照质粒+TNF-α组ALP、OCN、RUNX2mRNA(0.57±0.12、0.59±0.11、0.53±0.13)及WDR72蛋白(0.35±0.02)相对表达量均低于对照质粒组(1.00±0.18、1.00±0.14、1.00±0.12、0.78±0.03)和WDR72质粒+TNF-α组(0.95±0.13、0.93±0.15、0.97±0.13、1.10±0.08)(P<0.05)。培养第21天对照质粒+TNF-α组较对照质粒组和WDR72质粒+TNF-α组钙结节数量少、染色浅;对照质粒+TNF-α组OD_(540)值(0.72±0.06)低于对照质粒组(1.87±0.14)和WDR72质粒+TNF-α组(1.69±0.16)(t=7.437,P<0.001;t=5.717,P<0.001)。结论TNF-α诱导的炎症微环境抑制PDLSCs成骨分化与其抑制WDR72表达有关,WDR72参与PDLSCs成骨分化的调控。

Objective To investigate the regulatory role and mechanism of WDR72in osteogenic differentiation of periodontal ligament stem cells(PDLSCs)under tumor necrosis factor-α(TNF-α)induced inflammatory microenvironment.Methods The premolars samples were collected,PDLSCs were isolated and cultured,and their stem cells were identified by flow cytometry.The PDLSCs in logarithmic growth phase were divided into control group(cultured with osteogenic induction medium)and TNF-αgroup(cultured with osteogenic induction medium containing 10μg/L TNF-α).On the 7th day of culture,the relative expressions of alkaline phosphatase(ALP),osteocalcin(OCN)and RUNX2mRNAs were detected by real-time fluorescence quantitative PCR,and the relative expressions of WDR5,WDR7and WDR72proteins were detected by Western blot.The differentially expressed WDR72protein was screened in two groups and were grouped for plasmid transfection.On the 21st day of culture,the calcium nodules were observed by alizarin red staining and the optical density at 540nm(OD_(540))value was detected.The PDLSCs in logarithmic growth phase were divided into control plasmid group(cultured with osteogenic induction medium and transfected with control plasmid),control plasmid+TNF-αgroup(cultured with osteogenic induction medium containing 10μg/L TNF-αand transfected with control plasmid),and WDR72plasmid+TNF-αgroup(cultured with osteogenic induction medium containing 10μg/L TNF-αand transfected with WDR72plasmid).On the 7th day of culture,the relative expressions of ALP,OCN and RUNX2mRNAs were detected by real-time fluorescence quantitative PCR,and the relative expression of WDR72protein was detected by Western blot.On the 21st day of culture,the calcium nodules were observed by alizarin red staining and the OD_(540)value was detected.Results The positive rates of CD44and CD90on the surface of PDLSCs were higher than 99%,and the positive rates of CD45and CD34were lower than 1%,suggesting that PDLSCs isolated and cultured were consistent with the characteristics of mesenchymal stem cells.On the 7th day of culture,the relative expressions of ALP,OCN and RUNX2mRNAs as well as WDR72protein were lower in TNF-αgroup(0.56±0.11,0.56±0.10,0.58±0.08,0.40±0.02)than those in control group(1.00±0.15,1.00±0.13,1.00±0.16,0.72±0.02)(P<0.05),and the relative expressions of WDR5and WDR7proteins showed no significant differences between two groups(P>0.05).On the 21st day of culture,the number of calcium nodules was less,and the staining was lighter in TNF-αgroup compared with control group;the OD_(540)value was lower in TNF-αgroup(0.70±0.04)than that in control group(1.90±0.12)(t=9.479,P<0.001).On the 7th day of culture,the relative expressions of ALP,OCN and RUNX2mRNAs as well as WDR72protein were lower in control plasmid+TNF-αgroup(0.57±0.12,0.59±0.11,0.53±0.13,0.35±0.02)than those in control plasmid group(1.00±0.18,1.00±0.14,1.00±0.12,0.78±0.03)and WDR72plasmid+TNF-αgroup(0.95±0.13,0.93±0.15,0.97±0.13,1.10±0.08)(P<0.05).On the 21st day of culture,the number of calcium nodules was less,and the staining was lighter in control plasmid+TNF-αgroup compared with control plasmid group and WDR72plasmid+TNF-αgroup;the OD_(540)value was lower in control plasmid+TNF-αgroup(0.72±0.06)than that in control plasmid group(1.87±0.14)and WDR72plasmid+TNF-αgroup(1.69±0.16)(t=7.437,P<0.001;t=5.717,P<0.001).Conclusion The TNF-αinduced inflammatory microenvironment inhibits PDLSCs osteogenic differentiation,which is probably correlated with the inhibition of WDR72,and WDR72participates the regulation of PDLSCs osteogenic differentiation.