脂多糖诱导的内质网应激在牙周膜干细胞中的表达及其对成骨分化的影响  

Effect of endoplasmic reticulum stress on the expression and osteogenic differentiation of periodontal ligament stem cells

作　　者：薛芃[1] 李蓓[2] 谈珺[1] 安莹[1] 金岩[2] 王勤涛[1] 

机构地区：[1]第四军医大学口腔医学院牙周科军事口腔医学国家重点实验室,西安710032 [2]第四军医大学口腔医学院组织工程中心,西安710032

出　　处：《中华口腔医学杂志》2015年第9期548-553,共6页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81271137、81470742）

摘　　要：目的 探讨使用脂多糖模拟炎症微环境下牙周膜干细胞（periodontal ligament stem cells,PDLSC）中内质网应激（endoplasmic reticulum stress,ERS）的表达差异及对成骨分化的影响,初步证明ERS可以调控炎症微环境下的PDLSC,使其与正常来源的PDLSC相比成骨分化能力产生差异.方法 原代和有限稀释法克隆化培养正常来源的PDLSC,使用ERS激动剂毒胡萝卜素（thapsigargin,TG）观察是否可以诱导PDLSC发生ERS;实时定量PCR检测使用炎性因子脂多糖刺激是否可以诱导PDLSC发生ERS;使用含有成骨诱导培养基的TG和ERS抑制剂4-苯基丁酸（4-phenylbutyrate,4-PBA）刺激PDLSC,实验组分为PDLSC＋ TG组、PDLSC＋脂多糖组及PDLSC＋脂多糖＋4-PBA组,对照组为单纯成骨诱导培养基诱导的PDLSC组,实时定量PCR、茜素红染色及氯化十六烷基吡啶检测其成骨分化能力的差异.结果 炎性因子体外模拟炎症环境可观察到ERS被激活,PDLSC＋TG组6h时PERK、GRP78、ATF4及CHOP mRNA表达量均显著高于PDLSC组（分别为1.49±0.24、2.77±0.60、1.75±0.16、2.16±0.32）,P＜0.05;PDLSC＋脂多糖组PERK、CHOP mRNA表达量均在6h时达峰值（分别为1.76±0.08、2.31±0.17）,且与PDLSC组相比差异均有统计学意义（P＜0.05）.模拟牙周炎症微环境下的ERS状态可抑制PDLSC的成骨分化,PCR结果显示PDLSC＋ TG组RUNX2、ALP、OCN mRNA表达量为0.73±0.06、0.01 ±0.00、0.20±0.06,均显著低于PDLSC组（P＜0.05）;PDLSC＋脂多糖组RUNX2、ALP、OCN mRNA表达量分别为0.80±0.06、0.48±0.05、0.29±0.04,均显著低于PDLSC组（P＜0.05）;PDLSC＋脂多糖＋4-PBA组RUNX2、ALP、OCN mRNA表达量分别为1.10±0.09、0.74±0.05、0.67±0.13,均显著高于PDLSC＋脂多糖组（P＜0.05）.茜素红染色及氯化十六烷基吡啶定量结果和PCR结果相符.结论 体外使用脂多糖模拟炎症微环境,可具有同ERS激活剂TG相同的作用,刺激PDLSC,使其ERS被激活,进而成骨分化受到抑制;加入EObjective To determine the activity of endoplasmic reticulum stress（ERS） and its effect on osteogenic differentiation of periodontal ligament stem cells（PDLSC） in inflammatory microenvironment.Methods PDLSC were obtained from the primary culture of the human tooth and cloned with limited diluted method.Real-time reverse transcription（RT）-PCR was used to examine the different expression of thapsigargin（TG） treated PDLSC and lipopolysaccharide（LPS） treated PDLSC.Real-time RT-PCR,alizarin red staining and cetyl pyridine chloride quantitative analyze were used to examine the osteogenic differentiation of PDLSC,TG ＋ PDLSC,LPS ＋ PDLSC and LPS ＋ PDLSC ＋ 4-PBA.Results Protein kinase receptorlike endoplasmic reticulum kinase（PERK）,glucose regulated protein 78 （GRP78）,transcription activation factor 4（ATF4）,CCAAT/enhancer-binding protein-homologous protein （CHOP） mRNA expression in group PDLSC＋TG in 6 h were respectively 1.49±0.24,2.77±0.60,1.75±0.16,2.16±0.32,which were all greater than that in group PDLSC（P〈0.05）.PERK,CHOP mRNA expression reached the peak at 6 h（1.76±0.08,2.31 ±0.17） and were greater than group PDLSC（P〈0.05）.ERS could suppress osteogenic differentiation of TG＋PDLSC and LPS＋PDLSC.The runt-related transcription factor-2 （RUNX2）,alkaline phosphatase（ALP）,osteocalcin（OCN） mRNA expression of group TG ＋ PDLSC was respectively 0.73±0.06,0.01±0.00,0.20±0.06（P〈0.05）.The RUNX2,ALP,OCN mRNA expression of group LPS＋PDLSC was respectively 0.80±0.06,0.48±0.05,0.29±0.04（P〈0.05）.The RUNX2,ALP,OCN mRNA expression of group PDLSC ＋TG ＋4-PBA was respectively 1.10±0.09,0.74±0.05,0.67±0.13,which were greater higher than that of group LPS＋PDLSC（P〈O.05）.Conclusions ERS was activated in PDLSC and suppressed osteogenic differentiation of PDLSC,which can simulate inflammatory microenvironment in vitro.This effect can be recovered by using ERS inhibitor 4-PBA.

关 键 词：牙周膜 干细胞 细胞分化 内质网应激 炎症微环境 

分 类 号：R78[医药卫生—口腔医学]