中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2015年
9期
548-553
,共6页
薛芃%李蓓%谈珺%安莹%金岩%王勤涛
薛芃%李蓓%談珺%安瑩%金巖%王勤濤
설봉%리배%담군%안형%금암%왕근도
牙周膜%干细胞%细胞分化%内质网应激%炎症微环境
牙週膜%榦細胞%細胞分化%內質網應激%炎癥微環境
아주막%간세포%세포분화%내질망응격%염증미배경
Periodontal ligament%Stem cells%Cell differentiation%Endoplasmic reticulum stress%Inflammatory microenvironment
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ERS抑制剂4-PBA后可使脂多糖抑制成骨分化的作用得到恢复.
目的 探討使用脂多糖模擬炎癥微環境下牙週膜榦細胞(periodontal ligament stem cells,PDLSC)中內質網應激(endoplasmic reticulum stress,ERS)的錶達差異及對成骨分化的影響,初步證明ERS可以調控炎癥微環境下的PDLSC,使其與正常來源的PDLSC相比成骨分化能力產生差異.方法 原代和有限稀釋法剋隆化培養正常來源的PDLSC,使用ERS激動劑毒鬍蘿蔔素(thapsigargin,TG)觀察是否可以誘導PDLSC髮生ERS;實時定量PCR檢測使用炎性因子脂多糖刺激是否可以誘導PDLSC髮生ERS;使用含有成骨誘導培養基的TG和ERS抑製劑4-苯基丁痠(4-phenylbutyrate,4-PBA)刺激PDLSC,實驗組分為PDLSC+ TG組、PDLSC+脂多糖組及PDLSC+脂多糖+4-PBA組,對照組為單純成骨誘導培養基誘導的PDLSC組,實時定量PCR、茜素紅染色及氯化十六烷基吡啶檢測其成骨分化能力的差異.結果 炎性因子體外模擬炎癥環境可觀察到ERS被激活,PDLSC+TG組6h時PERK、GRP78、ATF4及CHOP mRNA錶達量均顯著高于PDLSC組(分彆為1.49±0.24、2.77±0.60、1.75±0.16、2.16±0.32),P＜0.05;PDLSC+脂多糖組PERK、CHOP mRNA錶達量均在6h時達峰值(分彆為1.76±0.08、2.31±0.17),且與PDLSC組相比差異均有統計學意義(P＜0.05).模擬牙週炎癥微環境下的ERS狀態可抑製PDLSC的成骨分化,PCR結果顯示PDLSC+ TG組RUNX2、ALP、OCN mRNA錶達量為0.73±0.06、0.01 ±0.00、0.20±0.06,均顯著低于PDLSC組(P＜0.05);PDLSC+脂多糖組RUNX2、ALP、OCN mRNA錶達量分彆為0.80±0.06、0.48±0.05、0.29±0.04,均顯著低于PDLSC組(P＜0.05);PDLSC+脂多糖+4-PBA組RUNX2、ALP、OCN mRNA錶達量分彆為1.10±0.09、0.74±0.05、0.67±0.13,均顯著高于PDLSC+脂多糖組(P＜0.05).茜素紅染色及氯化十六烷基吡啶定量結果和PCR結果相符.結論 體外使用脂多糖模擬炎癥微環境,可具有同ERS激活劑TG相同的作用,刺激PDLSC,使其ERS被激活,進而成骨分化受到抑製;加入ERS抑製劑4-PBA後可使脂多糖抑製成骨分化的作用得到恢複.
목적 탐토사용지다당모의염증미배경하아주막간세포(periodontal ligament stem cells,PDLSC)중내질망응격(endoplasmic reticulum stress,ERS)적표체차이급대성골분화적영향,초보증명ERS가이조공염증미배경하적PDLSC,사기여정상래원적PDLSC상비성골분화능력산생차이.방법 원대화유한희석법극륭화배양정상래원적PDLSC,사용ERS격동제독호라복소(thapsigargin,TG)관찰시부가이유도PDLSC발생ERS;실시정량PCR검측사용염성인자지다당자격시부가이유도PDLSC발생ERS;사용함유성골유도배양기적TG화ERS억제제4-분기정산(4-phenylbutyrate,4-PBA)자격PDLSC,실험조분위PDLSC+ TG조、PDLSC+지다당조급PDLSC+지다당+4-PBA조,대조조위단순성골유도배양기유도적PDLSC조,실시정량PCR、천소홍염색급록화십륙완기필정검측기성골분화능력적차이.결과 염성인자체외모의염증배경가관찰도ERS피격활,PDLSC+TG조6h시PERK、GRP78、ATF4급CHOP mRNA표체량균현저고우PDLSC조(분별위1.49±0.24、2.77±0.60、1.75±0.16、2.16±0.32),P＜0.05;PDLSC+지다당조PERK、CHOP mRNA표체량균재6h시체봉치(분별위1.76±0.08、2.31±0.17),차여PDLSC조상비차이균유통계학의의(P＜0.05).모의아주염증미배경하적ERS상태가억제PDLSC적성골분화,PCR결과현시PDLSC+ TG조RUNX2、ALP、OCN mRNA표체량위0.73±0.06、0.01 ±0.00、0.20±0.06,균현저저우PDLSC조(P＜0.05);PDLSC+지다당조RUNX2、ALP、OCN mRNA표체량분별위0.80±0.06、0.48±0.05、0.29±0.04,균현저저우PDLSC조(P＜0.05);PDLSC+지다당+4-PBA조RUNX2、ALP、OCN mRNA표체량분별위1.10±0.09、0.74±0.05、0.67±0.13,균현저고우PDLSC+지다당조(P＜0.05).천소홍염색급록화십륙완기필정정량결과화PCR결과상부.결론 체외사용지다당모의염증미배경,가구유동ERS격활제TG상동적작용,자격PDLSC,사기ERS피격활,진이성골분화수도억제;가입ERS억제제4-PBA후가사지다당억제성골분화적작용득도회복.
Objective 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.
