中文摘要：

目的 探讨细胞外调节蛋白激酶（extracellular signal-regulated kinase,ERK）通路在牙周膜干细胞（periodontal ligament stem cells,PDLSC）内皮向分化中的作用,为PDLSC分化调控提供实验基础.方法 使用血管内皮生长因子（vascular endothelial growth factor,VEGF）和碱性成纤维细胞生长因子（basic fibroblast growth factor,b-FGF）联合诱导PDLSC向内皮细胞分化（诱导组）,对诱导分化的细胞使用ERK1/2磷酸化阻断剂U0126进行处理（诱导＋U0126组）,同时用二甲基亚砜（dimethylsulfoxide,DMSO）作为对照（诱导＋DMSO组）,空白对照组为未经诱导的PDLSC;蛋白质印迹法检测诱导组0、1、3、6及12h的胞内磷酸化ERK1/2 （p-ERK1/2）表达水平;各组诱导7d后提取细胞RNA,实时荧光定量PCR法检测细胞内CD31、血管内皮钙黏素（vascular endothelial-cadherin,VE-cadherin）和VEGF mRNA的表达情况;各组诱导14d,流式细胞计数法检测CD31＋和VE-cadherin＋细胞比例,基质胶管腔形成实验检测细胞的管腔形成能力.计量资料采用均值±标准差表示,两组间比较采用独立样本t检验;多组间比较采用单因素方差分析.结果 诱导1、3h后p-ERK1/2与ERK1/2比值分别升高至1.24±0.12、1.03±0.24,均显著高于诱导前（0.58±0.17）（P＜0.01）;实时荧光定量PCR结果显示,诱导＋U0126组CD31、VEGF mRNA相对表达水平分别降至0.09±0.18、0.49±0.17,均显著低于诱导组细胞（P＜0.05）;流式细胞检测结果显示,诱导＋U0126组CD31＋、VE-cadherin＋细胞比值分别降至5.22±0.85、3.56±0.87,均显著低于诱导组细胞（P＜0.05）;基质胶管腔形成实验显示诱导组的分支节点数、管腔数目、管腔长度分别升高至62.3±10.0、145.0±14.8及（32 129.7±4 413.9）像素,而诱导＋U0126组分别下降至7.0±2.7、33.5±6.4及（15 951.0±758.1）像素,均显著低于诱导组（P＜0.05）.结论 PDLSC内皮分化过程受ERK通路正向调控,阻断ERK1/2磷酸化可以抑?

英文摘要：

Objective To investigate the effect of extracellular signal-regulated kinase（ERK） signaling pathway on the endothelial differentiation of periodontal ligament stem cells（PDLSC）.Methods Human PDLSC was cultured in the medium with vascular endothelial growth factor（VEGF） and basic fibroblast growth factor（b-FGF） to induce endothelial differentiation.Endothelial inducing cells was incubated with U0126,a specific p-ERK1/2 inhibitor.PDLSC from one person were randomly divided into four groups：control group,endothelial induced group,endothelial induced ＋ DMSO group and endothelial induced＋U0126 group.The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0,1,3,6 and 12 hours during endonthelial induction.The mRNA expressions of CD31,VE-cadherin,and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction（qRT-PCR） after a 7-day induction.The proportion of CD3F to VE-cadherin＋cells was identified by flow cytometry,and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction.The measurement data were statistically analyzed.Results Phosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively,during the endothelial induction（P〈0.01）.The mRNA expressions of CD31 and VEGF in induced＋U0126 group were decreased to 0.09±0.18 and 0.49±0.17,which were both significantly different with those in induced group（P〈0.05）.The proportion of CD31 ＋ to VE-cadherin＋ cells of induced＋U0126 group were decreased to 5.22±0.85 and 3.56±0.87,which were both significantly different with those in induced group（P〈0.05）.In Matrigel assay,the branching points,tube number and tube length were decreased to 7.0±2.7,33.5±6.4,and （15 951.0±758.1） pixels,which were all significantly different with those in induced group（P〈0.05）.Conclusions The endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway.Inhibit