LED蓝光照射对人牙周膜干细胞血管向分化的影响

Effect of LED blue light irradiation on vascular differentiation of human periodontal ligament stem cells

目的探讨LED蓝光照射调控胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)信号通路对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)血管向分化的促进作用及可能机制。方法hPDLSCs随机分为0 J/cm^(2)组、0.5 J/cm^(2)组、1 J/cm^(2)组、3 J/cm^(2)组、5 J/cm^(2)组,均给予光功率密度100 mW/cm^(2),波长420~470 nm的LED蓝光照射,照射剂量分别为0、0.5、1、3、5 J/cm^(2),光照时间分别为0、5、10、30、50 s。采用体外血管形成实验检测hPDLSCs管腔形成情况,采用实时荧光定量PCR法检测hPDLSCs血管形成相关基因血管内皮生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、血管内皮钙黏蛋白(vascular endothelial-cadherin,VE-cadherin)mRNA相对表达量,筛选促进hPDLSCs血管向分化的最佳光照剂量。hPDLSCs随机分为0 J/cm^(2)组(正常培养)、0 J/cm^(2)+U0126组(正常培养+ERK1/2信号通路阻断剂U0126处理)、5 J/cm^(2)组(5 J/cm^(2)LED蓝光照射)、5 J/cm^(2)+U0126组(5 J/cm^(2)LED蓝光照射+U0126处理),处理第7天采用实时荧光定量PCR法检测各组细胞VEGF、bFGF、VE-cadherin mRNA相对表达量,采用Western blot法检测p-ERK1/2蛋白相对表达量。结果(1)光照后第7、14天0、0.5、1、3、5 J/cm^(2)组hPDLSCs管腔形成数量均依次增多(P<0.05)。(2)光照后第7天,0.5、1、3、5 J/cm^(2)组hPDLSCs VEGF mRNA相对表达量均高于0 J/cm^(2)组(P<0.05),1、3、5 J/cm^(2)组依次增高(P<0.05);1、3、5 J/cm^(2)组hPDLSCs bFGF mRNA相对表达量均高于0、0.5 J/cm^(2)组(P<0.05),5 J/cm^(2)组高于1、3 J/cm^(2)组(P<0.05);0、0.5、1、3、5 J/cm^(2)组hPDLSCs VE-cadherin mRNA相对表达量依次增高(P<0.05)。光照后第14天,0.5、1、3、5 J/cm^(2)组hPDLSCs VEGF mRNA相对表达量均高于0 J/cm^(2)组(P<0.05),3、5 J/cm^(2)组均高于0.5 J/cm^(2)组(P<0.05),5 J/cm^(2)组高于1 J/cm^(2)组(P<0.05);0、0.5、1、3、5 J/cm^(2)组hPDLSCs bFGF、VE-cadherin mRNA相对表达量均依次增高(P<0.05)。(3)0 J/cm^(2)+U0126组hPDLSCs p-ERK1/2蛋白(0.178±0.012)及VEGF(0.896±0.020)、bFGF(0.806±0.032)、VE-cadherin(0.816±0.068)mRNA相对表达量均低于0 J/cm^(2)组(0.597±0.032、1.003±0.046、1.004±0.063、1.003±0.085)(P<0.05),5 J/cm^(2)组(1.174±0.015、1.650±0.060、1.514±0.046、1.620±0.054)均高于0 J/cm^(2)组(P<0.05);5 J/cm^(2)+U0126组hPDLSCs p-ERK1/2蛋白(0.627±0.010)及VEGF(1.374±0.062)、bFGF(1.333±0.079)mRNA相对表达量均低于5 J/cm^(2)组(P<0.05),VE-cadherin mRNA相对表达量(1.607±0.016)与5 J/cm^(2)组比较差异无统计学意义(P>0.05);4组hPDLSCs ERK1/2蛋白相对表达量比较差异无统计学意义(P>0.05)。(4)5 J/cm^(2)组hPDLSCs p-ERK1/2蛋白相对表达量与VEGF、bFGF、VE-cadherin mRNA相对表达量均呈正相关(r=0.904,P<0.001;r=0.904,P<0.001;r=0.792,P=0.002)。结论LED蓝光照射导致ERK1/2信号通路中ERK1/2蛋白磷酸化增加,促进hPDLSCs血管向分化。

Objective To investigate the role of LED blue light in promoting the vascular differentiation of human periodontal ligament stem cells(hPDLSCs)by regulating extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway and its possible molecular mechanism.Methods The hPDLSCs were randomly divided into 0,0.5,1,3and5J/cm^(2)groups with a light power density of 100mW/cm^(2)and light length of 420to 470nm at doses of 0,0.5,1,3and5J/cm^(2)for 0,5,10,30and 50s,respectively.The hPDLSCs lumen formation was determined by in vitro vessel formation assay.Real-time fluorescence quantitative PCR was used to detect the relative expressions of hPDLSCs angiogenic gene vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF),and vascular endothelial-cadherin(VE-cadherin)mRNAs.The optimal light dose was chosen for promoting vascular differentiation of hPDLSCs.The hPDLSCs were randomly divided into 0J/cm^(2)group(normal culture),0J/cm~2+U0126group(normal culture+ERK1/2blocker U0126treatment),5J/cm^(2)group(5J/cm^(2)LED blue light irradiation),and 5J/cm~2+U0126group(5 J/cm^(2)LED blue light irradiation+U0126treatment).The relative expressions of VEGF,bFGF and VE-cadherin mRNAs were determined by real-time fluorescence quantitative PCR by day 7of treatment.The relative expression of p-ERK1/2protein was determined by Western blot.Results The number of hPDLSCs lumens increased successively in turn in 0,0.5,1,3and 5J/cm^(2)groups by day 7and 14of light exposure(P<0.05).By day 7of light exposure,the relative expression of VEGF mRNA was higher in 0.5,1,3and 5J/cm^(2)groups than that in 0J/cm^(2)group(P<0.05),and increased successively in turn in 1,3and 5J/cm^(2)groups(P<0.05).The relative expressions of bFGF mRNA was higher in 1,3and 5J/cm^(2)groups than that in 0and 0.5J/cm^(2)group(P<0.05),and higher in 5J/cm~2group than that in 1and 3J/cm^(2)group(P<0.05).The relative expression of VE-cadherin mRNA increased successively in turn in 0,0.5,1,3and 5J/cm^(2)groups(P<0.05).By day 14of light exposure,the relative expression of VEGF mRNA was higher in 0.5,1,3and 5J/cm^(2)groups than that in 0J/cm^(2)group(P<0.05),higher in 3and 5J/cm~2groups than that in 0.5J/cm^(2)group(P<0.05),and higher in 5J/cm^(2)group than that in 1J/cm^(2)group(P<0.05).The relative expressions of bFGF and VE-cadherin mRNAs increased successively in turn in 0,0.5,1,3and 5J/cm^(2)groups(P<0.05).The relative expressions of p-ERK1/2protein,VEGF mRNA,bFGF mRNA and VE-cadherin mRNA were lower in 0J/cm~2+U0126group(0.178±0.012,0.896±0.020,0.806±0.032,0.816±0.068)than those in 0J/cm~2group(0.597±0.032,1.003±0.046,1.004±0.063,1.003±0.085)(P<0.05),and higher in 5J/cm^(2)group(1.174±0.015,1.650±0.060,1.514±0.046,1.620±0.054)than those in 0J/cm^(2)group(P<0.05).The relative expressions of p-ERK1/2protein,VEGF mRNA and bFGF mRNA were lower in 5J/cm~2+U0126group(0.627±0.010,1.374±0.062,1.333±0.079)than those in 5J/cm^(2)group(P<0.05).The relative expression of VE-cadherin mRNA showed no significant difference between 5J/cm~2+U0126group(1.607±0.016)and 5J/cm^(2)group(P>0.05).The relative expression of p-ERK1/2protein showed no significant difference among 4groups(P>0.05).The relative expression of p-ERK1/2protein was positively correlated with the relative expressions of VEGF,bFGF and VE-cadherin mRNAs in 5J/cm^(2)group(r=0.904,P<0.001;r=0.904,P<0.001;r=0.792,P=0.002).Conclusion LED blue light irradiation leads to an increase of p-ERK1/2protein in ERK1/2signaling pathway,which promotes the vascular differentiation of hPDLSCs.