内质网应激在牙周膜细胞成骨分化中的表达

Study of endoplasmic reticulum stress response in osteogenic differentiation of human periodontal ligament cells

目的 :观察人牙周膜细胞(periodontal ligament cells,PDLCs)成骨分化过程中内质网应激水平的变化及其影响,以便更好地了解PDLCs成骨分化的机制。方法:原代培养人PDLCs,并体外诱导成骨,通过碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色鉴定其成骨特性;PCR和Western印迹检测体外成骨诱导时PDLCs内质网应激关键分子的表达。采用SPSS17.0软件包对数据进行统计学分析。结果:体外诱导成骨培养的PDLCs ALP染色及茜素红染色增强,矿盐沉积增加。同时,体外成骨诱导的PDLCs内质网应激关键分子剪切x盒结合蛋白1(splicing x-box binding protein-1,s XBP1)、活化转录因子4(activating transcription factor 4,ATF4)、葡萄糖调节蛋白质78(glucoseregulated protein 78,GRP78)m RNA表达水平升高。Western印迹检测显示,成骨诱导的PDLCs内质网应激关键分子磷酸化蛋白激酶样内质网激酶(phosphorylated the RNA-activated protein kinase-like ER-resident kinase,p-PERK)、磷酸化真核起始因子2α(phosphorylated eukaryotic initiation factor-2α,p-e IF2α)表达增加。结论:牙周膜细胞成骨分化时内质网应激水平明显升高。

PURPOSE： The aim of this study was to establish whether endoplasmic reticulum （ER） stress is involved in osteogenic differentiation of periodontal ligament cells （PDLCs） and to better understand the mechanism of PDLCs osteogenic differentiation. METHODS： PDLCs were isolated from extracted teeth and cultured in presence or absence of osteogenic medium, which can induce osteogenic differentiation of PDLCs. Alkaline phosphatase and alizarin red S staining were performed to characterize the osteogenic differentiation of PDLCs. The mRNA and protein levels of ER stress markers were examined by RT-PCR and Western blot analysis. The data were analyzed with SPSS 17.0 software package. RESULTS： Cell treated with osteogenic medium showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation compared with untreated controls. Treatment with osteogenic induction resulted in up-regulation of genes, such as splicing x-box binding protein-1 （sXBP1）, activating transcription factor 4 （ATF4） and glucose-regulated protein 78 （GRP78）. The expressions of ER stress protein markers, phosphorylated RNA-activated protein kinase-like ER-resident kinase （p-PERK）, phosphorylated eukaryotic initiation factor-2α （p-eIF2α）, increased in osteogenic induction cells compared with controls. CONCLUSIONS： The results indicate that ER stress response is involved in the process of osteogenic differentiation of PDLCs.