摘要
目的探讨低氧环境对人牙周膜成纤维细胞(periodontal ligament cells,PDLCs)成骨分化的影响,以及低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)在该过程中的作用。方法组织块法原代分离牙周膜标本中PDLCs,采用激光共聚焦检测波形蛋白与细胞角蛋白表达。PDLCs分别在常氧(20%体积分数O_2)培养以及低氧(1%体积分数O_2)培养12~72 h,检测常氧组与低氧组PDLCs碱性磷酸酶(alkaline phosphatase,ALP)活性,比较成骨标志物ALP、I型胶原(Collogen-1,COL1)、成骨特异性转录因子(runt related transcription factor 2,RUNX2)的m RNA表达量变化,Western免疫印迹检测HIF-1α表达水平。进一步转染小干扰RNA(HIF-1α-si RNA 1,2),检测低氧环境中PDLCs的HIF-1α水平、ALP活性与成骨标志物m RNA表达变化。采用SPSS 13.0软件包对数据进行统计学分析。结果组织块法成功获取PDLCs,PDLCs中波形蛋白阳性表达、细胞角蛋白阴性表达。低氧环境中培养48 h后PDLCs中ALP活性下降,ALP、COL1、RUNX2的mRNA表达量显著降低,但HIF-1α蛋白表达升高。HIF-1α-si RNA成功敲低PDLCs中HIF-1α表达,敲低HIF-1α的PDLCs在低氧环境中的ALP活性升高,ALP、COL1、RUNX2的mRNA表达量增加。结论长时间低氧处理能抑制PDLCs成骨分化,敲低HIF-1α表达可逆转低氧对PDLCs成骨分化的抑制作用。
Objective To investigate the effects of hypoxia on osteogenic differentiation of periodontal ligament cells(PDLCs)and the role of hypoxia inducible factor1α(HIF1α)in this process.Methods Human PDLCs were isolated and identified by checking the expression of vimentin and cytokeratin.PDLCs were cultured in normoxia(20%O2)or hypoxia(1%O2)for12?72h.Changes of alkaline phosphatase(ALP)activity and mRNA expressions of osteogenic markers ALP,collagenI(COL1)and runt related transcription factor2(RUNX2)were detected.Western blot was used to detect the expression of HIF?1α.After transfected with HIF1α?siRNA,the expressions of HIF1αand osteogenic differentiation markers were furthered detected.The statistics were analyzed with SPSS13.0.Results Positive vimentin but negative cytokeratin were observed in primary cultured PDLCs.ALP activity and mRNA expressions of ALP,COL1and RUNX2were decreased in PDLCs in hypoxia for48h,while HIF?1αexpression was increased.After knockingdown of HIF1αwith siRNA,HIF?1αwas significantly reduced in PDLCs under hypoxia,while ALP activity and mRNA expressions of osteogenic markers were significantly increased.Conclusion Hypoxia may inhibit osteogenic differentiation of PDLCs via upregulated HIF1α.
作者
庞静雯
吴亚霖
徐婷
庄秀妹
PANG Jingwen;WU Yalin;XU Ting;ZHUANG Xiumei(Guangzhou Haizhu District Hospital of Stomatology, Guangzhou 510220, China;Department of Stomatology, Sun Yat.sen Memorial Hospital Affiliated to Sun Yat-sen University, Guangzhou 510120, China)
出处
《口腔疾病防治》
2017年第8期488-493,共6页
Journal of Prevention and Treatment for Stomatological Diseases
基金
国家自然科学基金青年基金(81600899)
关键词
牙周炎
人牙周膜成纤维细胞
低氧
成骨分化
低氧诱导因子-1Α
Periodontitis
Periodontal ligament cells
Hypoxia
Osteogenic differentiation
Hypoxia inducible factor-1α
