中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2014年
2期
101-105
,共5页
徐婷%吴慧玲%冯剑颖%谷志远
徐婷%吳慧玲%馮劍穎%穀誌遠
서정%오혜령%풍검영%곡지원
颞下颌关节%蛋白多糖4%滑膜细胞
顳下頜關節%蛋白多糖4%滑膜細胞
섭하합관절%단백다당4%활막세포
Temporomandibular joint%Proteoglycan-4%Synovial fibroblasts
目的 观察应力刺激对大鼠颞下颌关节滑膜细胞蛋白多糖4(proteoglycan 4,PRG-4)表达的影响,并探讨其可能的作用机制.方法 将体外培养的20只SD大鼠颞下颌关节滑膜细胞分为两组,加力组施加间歇静压力,压力值100 kPa,加载频率为4 h/d;对照组为未加载应力的滑膜细胞.采用蛋白质印迹法和细胞免疫荧光技术分别在实验各时间点检测Smad通路和p38丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)通路相关蛋白的表达变化;应用转化生长因子(transforming growth factor,TGF)β1受体抑制剂SB431542预处理后,蛋白质印迹法和实时荧光定量PCR技术观察72 h后对照组和加力组PRG-4表达的变化.结果 蛋白质印迹法检测显示,间歇静压力作用1h后,Smad-2、-3蛋白磷酸化水平显著升高,分别在2、4h达到峰值.而在实验1、2及4h观察p38蛋白磷酸化水平与对照组相比无明显变化.细胞免疫荧光结果显示,间歇静压力作用使Smad-2、-3蛋白发生核转位,且胞内荧光信号比对照组明显增强(24.11 ±4.70) (P ＜0.05).实时荧光定量PCR检测显示,间歇静压力促进滑膜细胞PRG-4 mRNA的表达量(1.48 ±0.08)显著高于对照组(P＜0.05),加入SB431542预处理后再加力,PRG-4 mRNA表达量较加力组显著降低(0.47 ±0.05) (P ＜0.05).蛋白质印迹法检测显示,各组PRG-4蛋白表达量受抑制趋势与实时荧光定量PCR结果相一致.结论 Smad信号通路是参与调节间歇静压力诱导滑膜细胞PRG-4表达的一条重要途径.
目的 觀察應力刺激對大鼠顳下頜關節滑膜細胞蛋白多糖4(proteoglycan 4,PRG-4)錶達的影響,併探討其可能的作用機製.方法 將體外培養的20隻SD大鼠顳下頜關節滑膜細胞分為兩組,加力組施加間歇靜壓力,壓力值100 kPa,加載頻率為4 h/d;對照組為未加載應力的滑膜細胞.採用蛋白質印跡法和細胞免疫熒光技術分彆在實驗各時間點檢測Smad通路和p38絲裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)通路相關蛋白的錶達變化;應用轉化生長因子(transforming growth factor,TGF)β1受體抑製劑SB431542預處理後,蛋白質印跡法和實時熒光定量PCR技術觀察72 h後對照組和加力組PRG-4錶達的變化.結果 蛋白質印跡法檢測顯示,間歇靜壓力作用1h後,Smad-2、-3蛋白燐痠化水平顯著升高,分彆在2、4h達到峰值.而在實驗1、2及4h觀察p38蛋白燐痠化水平與對照組相比無明顯變化.細胞免疫熒光結果顯示,間歇靜壓力作用使Smad-2、-3蛋白髮生覈轉位,且胞內熒光信號比對照組明顯增彊(24.11 ±4.70) (P ＜0.05).實時熒光定量PCR檢測顯示,間歇靜壓力促進滑膜細胞PRG-4 mRNA的錶達量(1.48 ±0.08)顯著高于對照組(P＜0.05),加入SB431542預處理後再加力,PRG-4 mRNA錶達量較加力組顯著降低(0.47 ±0.05) (P ＜0.05).蛋白質印跡法檢測顯示,各組PRG-4蛋白錶達量受抑製趨勢與實時熒光定量PCR結果相一緻.結論 Smad信號通路是參與調節間歇靜壓力誘導滑膜細胞PRG-4錶達的一條重要途徑.
목적 관찰응력자격대대서섭하합관절활막세포단백다당4(proteoglycan 4,PRG-4)표체적영향,병탐토기가능적작용궤제.방법 장체외배양적20지SD대서섭하합관절활막세포분위량조,가력조시가간헐정압력,압력치100 kPa,가재빈솔위4 h/d;대조조위미가재응력적활막세포.채용단백질인적법화세포면역형광기술분별재실험각시간점검측Smad통로화p38사렬원격활단백격매(mitogen activated protein kinase,MAPK)통로상관단백적표체변화;응용전화생장인자(transforming growth factor,TGF)β1수체억제제SB431542예처리후,단백질인적법화실시형광정량PCR기술관찰72 h후대조조화가력조PRG-4표체적변화.결과 단백질인적법검측현시,간헐정압력작용1h후,Smad-2、-3단백린산화수평현저승고,분별재2、4h체도봉치.이재실험1、2급4h관찰p38단백린산화수평여대조조상비무명현변화.세포면역형광결과현시,간헐정압력작용사Smad-2、-3단백발생핵전위,차포내형광신호비대조조명현증강(24.11 ±4.70) (P ＜0.05).실시형광정량PCR검측현시,간헐정압력촉진활막세포PRG-4 mRNA적표체량(1.48 ±0.08)현저고우대조조(P＜0.05),가입SB431542예처리후재가력,PRG-4 mRNA표체량교가력조현저강저(0.47 ±0.05) (P ＜0.05).단백질인적법검측현시,각조PRG-4단백표체량수억제추세여실시형광정량PCR결과상일치.결론 Smad신호통로시삼여조절간헐정압력유도활막세포PRG-4표체적일조중요도경.
Objective To examine the expression of proteoglycan 4 (PRG-4) induced by hydrostatic pressure in rat temporomandibular synovial fibroblasts and investigate the possible mechanism.Methods The cultured rat temporomandibular synovial fibroblasts were subjected to 100 kPa magnitude intermittent hydrostatic pressure (IHP) at frequency of 4 h/day,and the static group served as control.The expressions of Smad pathway proteins and p38MAPK pathway proteins were analyzed by Western blot and immunofluorescence staining.Then the cells were incubated with SB431542,the inhibitor of transforming growth factor (TGF)-[β receptor.Western blot and reverse transcription PCR were used to detect the PRG-4 expression after 72 h.Results The expression of phosphorylated Smad-2 and phosphorylated Smad-3 were increased after 1 h of IHP,reaching a maximum after 2 h and 4 h of IHP,respectively.However,the protein content of phosphorylated p38 did not vary significantly.In addition,IHP induced nuclear translocation of Smad-2/-3,and the immunofluorescence staining signal intensity markedly increased (24.11 ± 4.70) (P ＜ 0.05).The levels of PRG-4 mRNA were significantly increased by IHP (1.48 ± 0.08)(P ＜ 0.05).Treatment of cells with SB431542 could decrease the expression of PRG-4 mRNA significantly after IHP (0.47 ± 0.05) (P ＜ 0.05).In addition,SB431542 inhibited the expression of PRG-4 protein induced by IHP.Conclusions Smad signal acts as an essential signal pathway to regulate PRG-4 expression induced by IHP.
