CAJ | 학술논문

目的探讨RhoA/Rho激酶(ROCK)信号通路对类风湿关节炎(RA)患者成纤维样滑膜细胞(FLS)迁移、侵袭和增殖的调控作用.方法 RA患者FLS来源于11例活动性RA患者滑膜组织.另设骨关节炎(OA)组和外伤(正常对照组)为对照.用不同浓度的RhoA抑制剂C3转化酶或ROCK抑制剂Y27632处理RA患者FLS,在迁移和侵袭实验中用10％胎牛血清(FBS)作为诱导物,在增殖实验中以IL-1β为刺激剂.RhoA活性检测采用pull down方法；ROCK活性用磷酸化肌球蛋白结合亚单位1(MYPT1)蛋白表达来表示,磷酸化MYPT1蛋白检测采用免疫印迹法；FLS迁移和侵袭能力采用Transwell小室测定；细胞增殖检测采用MTT法.结果新鲜分离的RA患者FLS中RhoA和ROCK活性显著高于OA患者和正常对照组,单独FBS诱导组、C3转化酶处理组和Y27632处理组迁移细胞数分别为85±14、51±15、42 ±11,侵袭Matrigel基质细胞数分别为64±13、39±12、26±9,单独FBS诱导组与其他两组比较差异均有统计学意义(均P＜0.05),C3转化酶和Y27632对RA FLS的细胞骨架的聚合亦有显著的抑制作用.C3转化酶和Y27632呈剂量依赖性地抑制RA FLS增殖.结论 RhoA/ROCK信号通路对RA FLS的迁移、侵袭和增殖具有调控作用,通过抑制过度活化的ROCK可能有利于抑制RA关节破坏.
목적 탐토RhoA/Rho격매(ROCK)신호통로대류풍습관절염(RA)환자성섬유양활막세포(FLS)천이、침습화증식적조공작용.방법 RA환자FLS래원우11례활동성RA환자활막조직.령설골관절염(OA)조화외상(정상대조조)위대조.용불동농도적RhoA억제제C3전화매혹ROCK억제제Y27632처리RA환자FLS,재천이화침습실험중용10％태우혈청(FBS)작위유도물,재증식실험중이IL-1β위자격제.RhoA활성검측채용pull down방법；ROCK활성용린산화기구단백결합아단위1(MYPT1)단백표체래표시,린산화MYPT1단백검측채용면역인적법；FLS천이화침습능력채용Transwell소실측정；세포증식검측채용MTT법.결과 신선분리적RA환자FLS중RhoA화ROCK활성현저고우OA환자화정상대조조,단독FBS유도조、C3전화매처리조화Y27632처리조천이세포수분별위85±14、51±15、42 ±11,침습Matrigel기질세포수분별위64±13、39±12、26±9,단독FBS유도조여기타량조비교차이균유통계학의의(균P＜0.05),C3전화매화Y27632대RA FLS적세포골가적취합역유현저적억제작용.C3전화매화Y27632정제량의뢰성지억제RA FLS증식.결론 RhoA/ROCK신호통로대RA FLS적천이、침습화증식구유조공작용,통과억제과도활화적ROCK가능유리우억제RA관절파배.
Objective To evaluate the modulation of RhoA/Rho kinase (ROCK),a small Rho GTPase,on migration,invasion and proliferation of fibroblast like synoviocytes (FLS) from rheumatoid arthritis (RA) patients.Methods RA FLS were collected from active RA patients.And 10％ fetal bovine serum (FBS) and intedeukin-1β (IL-1β) were used as stimuli in migration and proliferation experiments respectively.RhoA activity was measured by pull down assay while ROCK activity by Western blot.FLS migration and invasion in vitro were measured by the Transwell chamber method.And thiazolyl blue tetrazolium bromide (MTT) test was used to detect cell proliferation.Results There were increased activities of RhoA and ROCK in ex vivo FLS from RA versus OA patients and healthy control.The migrated cell number of FBS-induced,C3-treated and Y27632-treated groups was 85 ± 14,51 ± 15 and 42 ± 11 respectively.The Matrigel invading cell number of 3 groups was 64 ± 13,39 ± 12 and 26 ± 9 respectivcly.Statistical differences existed in cell number between FBS-induced,C3-treated or Y27632-treated group(P ＜ 0.05)in above migration and invasion experiments.Inhibition of RhoA and ROCK activity also suppressed the cytoskeletal reorganization and proliferation of RA FLS.Conclusion Increased RhoA/ROCK activity may contribute to abnormal migration,invasion and proliferation of RA FLS.Thus inhibition of ROCK activity may be a new therapeutic target for RA.