CAJ | 학술논문

目的：初步探讨Sonic Hedgehog（Shh）信号通路分子Smoothened（Smo）对类风湿关节炎（RA）成纤维滑膜细胞（FLS）RhoA/ROCK通路相关分子的影响。方法收集病情活动（DAS28≥3.2）RA 患者关节镜手术或关节置换术切除的滑膜组织，组织块培养法培养 RA-FLS 作为细胞模型，流式细胞术检测CD55阳性率鉴定细胞，然后分别予Smo分子激动剂Purmorphamine或抑制剂KAAD-Cyclopamine处理，应用GST-pull down法检测RhoA活性，Western blot检测ROCK活性与Smo蛋白表达。结果与对照组相比，RA-FLS经Purmorphamine刺激后，活性RhoA蛋白、磷酸化MYPT1蛋白及Smo蛋白均上调（P＜0.05）；经KAAD-Cyclopamine处理后，活性RhoA蛋白、磷酸化MYPT1蛋白及Smo蛋白均下调（P＜0.05）。结论 RA-FLS Smo表达可影响RhoA/ROCK信号的传导。Smo可能参与了RA-FLS中Shh信号通路非经典途径的调控。
목적：초보탐토Sonic Hedgehog（Shh）신호통로분자Smoothened（Smo）대류풍습관절염（RA）성섬유활막세포（FLS）RhoA/ROCK통로상관분자적영향。방법수집병정활동（DAS28≥3.2）RA 환자관절경수술혹관절치환술절제적활막조직，조직괴배양법배양 RA-FLS 작위세포모형，류식세포술검측CD55양성솔감정세포，연후분별여Smo분자격동제Purmorphamine혹억제제KAAD-Cyclopamine처리，응용GST-pull down법검측RhoA활성，Western blot검측ROCK활성여Smo단백표체。결과여대조조상비，RA-FLS경Purmorphamine자격후，활성RhoA단백、린산화MYPT1단백급Smo단백균상조（P＜0.05）；경KAAD-Cyclopamine처리후，활성RhoA단백、린산화MYPT1단백급Smo단백균하조（P＜0.05）。결론 RA-FLS Smo표체가영향RhoA/ROCK신호적전도。Smo가능삼여료RA-FLS중Shh신호통로비경전도경적조공。
Objective By investigating the expression of Sonic Hedgehog signaling pathway-associated factor Smoothened (Smo) of fibroblast-like synoviocytes from active rheumatoid arthritis (RA-FLS) and its effect on RhoA/ROCK signaling pathway, to study the regulation of Smo in non-canonical Hh pathway. Methods The synovial tissue which was taken from surgery of RA patients was mechanically separated and used to establish the cell model of primary RA-FLS by using explants-culture. Furthermore, fibroblasts'cell purity was identified by testing positive expression of CD55 with flow cytometry. The RA-FLS was treated with Purmorphamine (Smo agonist), KAAD-Cyclopamine (Smo inhibitor) or DMSO (as blank control). The RhoA activity was measured by a pull-down assay;The ROCK activity (expression of MYPT1 protein) and the expression of Smo protein were assessed by Western blot. Results Compared with the control group, the protein expression of Smo, the activities of RhoA and ROCK (expression of MYPT1 protein) were increased in Purmorphamine group while decreased in KAAD-Cyclopamine groups (P<0.05). Conclusion Smo may contribute to the modulation of the RhoA/ROCK pathway and also be the keypoint which links the Shh signaling pathway and the Rho pathway together in RA-FLS.