Smad通路参与应力诱导颞下颌关节滑膜细胞蛋白多糖4表达的研究

Involvement of Smad pathway in proteoglycan 4 expression induced by hydrostatic pressure in temporomandibular 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.

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 temporomandibnlar 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 immunoflnorescence 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 SIM31542 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 PRG4 protein induced by IHP. Conclusions Smad signal acts as an essential signal pathway to regulate PRG-4 expression induced by IHP.