体外氧糖剥夺培养条件促进人牙髓细胞内质网应激的研究  

Study of endoplasmic reticulum stress in human dental pulp cells induced by oxygen⁃glucose deprivation cul⁃ture in vitro

作　　者：李莉芬[1] 朱亚琴[1] 江龙[1] LI Lifen;ZHU Yaqin;JIANG Long(Department of General Dentistry,Shanghai Ninth People's Hos-pital,College of Stomatology,Shanghai Jiao Tong University School of Medicine,National Center for Stomatology,Na-tional Clinical Research Center for Oral Disease,Shanghai key Laboratory of Stomatology,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院口腔综合科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床研究中心,上海市口腔医学重点实验室

出　　处：《口腔疾病防治》2022年第4期245-250,共6页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金项目(81700949)。

摘　　要：目的通过氧糖剥夺(oxygen⁃glucose deprivation,OGD)体外模拟细胞缺血缺氧,观察人牙髓细胞(hu⁃man dental pulp cells,hDPCs)内质网应激变化,为缺血缺氧条件调控hDPCs的机制研究提供依据。方法应用无糖DMEM培养液联合低氧培养(体积分数2%O2)构建hDPCs OGD模型,体外模拟hDPCs缺血缺氧,设置对照组和实验组。对照组:常规培养;实验组:OGD处理0、2、4、8 h。MTT检测hDPCs OGD处理0、2、4、8 h后细胞存活率;qRT⁃PCR检测hDPCs内质网应激关键分子:剪切X盒结合蛋白1(splicing x⁃box binding protein 1,sXBP1)、活化转录因子4(activating transcription factor 4,ATF4)、C/EBP同源蛋白(C/EBP homologous protein,chop)mRNA水平,Western blot检测内质网应激关键蛋白:磷酸化蛋白激酶样内质网激酶(phosphorylated RNA⁃activated protein kinase⁃like ER⁃resident kinase,p⁃perk)、磷酸化真核起始因子⁃2α(phosphorylated eukaryotic initia⁃tion factor⁃2α,p⁃eIF2α)表达水平。结果相较于OGD处理0 h,OGD培养hDPCs 2、4、8 h后,死亡细胞增多,细胞存活率显著下降(P<0.05)。与对照组相比,OGD处理4 h后,hDPCs内质网应激关键信号分子sXBP1、ATF4、CHOP mRNA表达水平升高;内质网应激关键蛋白p⁃perk、p⁃eIF2α表达增加,差异均有统计学意义(P<0.05)。结论hDPCs OGD培养条件下内质网应激水平明显升高。Objective Oxygen⁃glucose deprivation(OGD)is used to mimic ischemia in vitro to observe whether en⁃doplasmic reticulum(ER)stress is involved in human dental pulp cells(hDPCs)after OGD and to better understand the regulatory mechanism of hDPCs in ischemia.Methods hDPCs were cultured in glucose⁃free DMEM and hypoxia(vol⁃ume fraction 2%O2)to establish an hDPCs OGD model in vitro,which mimics hDPCs ischemia in vitro.hDPCs were di⁃vided into a control group(normal culture)and an experimental group(OGD 0 h,2 h,4 h and 8 h groups).After pre⁃treatment with OGD for 0,2,4 and 8 h,hDPC viability was measured by methylthiazol tetrazolium(MTT)assay.qRT⁃PCR was used to detect the mRNA expression of ER stress markers[splicing x⁃box binding protein1(sXBP1),activating transcription Factor 4(ATF4)and C/EBP homologous protein(chop)].Western blot was used to detect the protein ex⁃pression of ER stress markers[phosphorylated RNA⁃activated protein kinase⁃like ER⁃resident kinase(p⁃perk)and phos⁃phorylated eukaryotic initiation factor⁃2α(p⁃eIF2α)].Results Compared with OGD 0 h group,cell viability of hDPCs decreased when exposed to OGD treatment for 2 h,4 h and 8 h.Compared with the control group,mRNA expressions of ER stress makers(sXBP1,ATF4 and chop)and the protein expressions of ER stress protein markers(p⁃perk andp⁃eIF2α)increased in OGD treatment cells after 4 h were higher in OGD cells.The differences were statistically signifi⁃cant(P<0.05).Conclusion The results indicate that ER stress response is involved in hDPCs in OGD treatment.

关 键 词：人牙髓细胞 氧糖剥夺 内质网应激 剪切X盒结合蛋白1 活化转录因子4 C/EBP同源蛋白 蛋白激酶样内质网激酶 真核起始因子⁃2α 

分 类 号：R78[医药卫生—口腔医学]