CAJ | 학술논문

目的初步探讨类风湿关节炎(RA)滑膜组织血管内皮细胞Smo蛋白的表达,并观察肿瘤坏死因子(TNF)-α作用于人脐静脉内皮细胞系EA.hy926细胞后Sonic Hedgehog(Shh)信号通路相关分子的表达,及特异性抑制Smo对细胞凋亡的影响.方法收集4例病情中度活动的RA患者的滑膜组织,同时收集4例外伤或半月板损伤(无关节炎)者滑膜组织作为对照组,免疫组织化学法检测滑膜组织血管内皮细胞Smo蛋白表达情况.予不同浓度TNF-α或联合不同浓度环巴胺处理EA.hy926细胞,实时荧光定量聚合酶链反应(PCR)检测Shh、Ptch1、Smo、Gli1 mRNA表达.予不同浓度环巴胺处理EA.hy926细胞,加入TNF-α+放线菌素D(ActD)诱导细胞凋亡,采用细胞增殖与毒性试剂盒(CCK-8)检测细胞存活率,流式细胞术检测细胞凋亡情况.两样本均数比较采用t检验,多个样本均数比较采用单因素方差分析.结果 RA患者滑膜血管内皮细胞Smo阳性表达率为(81±23)％,高于对照组阳性表达率(20±17)％(P＜0.05).EA.hy926细胞经TNF-α刺激后,Shh、Smo mRNA表达上调(P＜0.05),Ptch1 mRNA表达无明显变化(P＞0.05),Gli1 mRNA表达下调(P＜0.05).TNF-α与环巴胺共同作用后,Shh、Smo和Gli1 mRNA表达下调(P＜0.05).不同浓度环巴胺(分别为2、4、8μmol/L)作用后的细胞存活率分别为(57±6)％、(44±8)％、(32±5)％,低于TNF-α/ActD组细胞存活率(64±6)％(P＜0.05),细胞凋亡率分别为(12.4±3.3)％、(14.5±2.7)％、(15.7±2.4)％,高于TNF-α/ActD组细胞凋亡率(7.1±1.3)％(P＜0.05).结论 Shh信号通路在RA患者滑膜组织血管内皮细胞中存在激活.特异性抑制Shh信号通路,可以促进内皮细胞凋亡.Shh信号通路可能参与TNF-α作用于内皮细胞后的抗凋亡调控机制.
목적 초보탐토류풍습관절염(RA)활막조직혈관내피세포Smo단백적표체,병관찰종류배사인자(TNF)-α작용우인제정맥내피세포계EA.hy926세포후Sonic Hedgehog(Shh)신호통로상관분자적표체,급특이성억제Smo대세포조망적영향.방법 수집4례병정중도활동적RA환자적활막조직,동시수집4예외상혹반월판손상(무관절염)자활막조직작위대조조,면역조직화학법검측활막조직혈관내피세포Smo단백표체정황.여불동농도TNF-α혹연합불동농도배파알처리EA.hy926세포,실시형광정량취합매련반응(PCR)검측Shh、Ptch1、Smo、Gli1 mRNA표체.여불동농도배파알처리EA.hy926세포,가입TNF-α+방선균소D(ActD)유도세포조망,채용세포증식여독성시제합(CCK-8)검측세포존활솔,류식세포술검측세포조망정황.량양본균수비교채용t검험,다개양본균수비교채용단인소방차분석.결과 RA환자활막혈관내피세포Smo양성표체솔위(81±23)％,고우대조조양성표체솔(20±17)％(P＜0.05).EA.hy926세포경TNF-α자격후,Shh、Smo mRNA표체상조(P＜0.05),Ptch1 mRNA표체무명현변화(P＞0.05),Gli1 mRNA표체하조(P＜0.05).TNF-α여배파알공동작용후,Shh、Smo화Gli1 mRNA표체하조(P＜0.05).불동농도배파알(분별위2、4、8μmol/L)작용후적세포존활솔분별위(57±6)％、(44±8)％、(32±5)％,저우TNF-α/ActD조세포존활솔(64±6)％(P＜0.05),세포조망솔분별위(12.4±3.3)％、(14.5±2.7)％、(15.7±2.4)％,고우TNF-α/ActD조세포조망솔(7.1±1.3)％(P＜0.05).결론 Shh신호통로재RA환자활막조직혈관내피세포중존재격활.특이성억제Shh신호통로,가이촉진내피세포조망.Shh신호통로가능삼여TNF-α작용우내피세포후적항조망조공궤제.
Objective To investigate Smoothened (Smo) expression in endothelial cells of synovial tissues in active rheumatoid arthritis (RA),and the expression of Sonic Hedgehog (Shh) signaling pathwayassociated factors after TNF-α treatment in EA.hy926 cells,and the effects of specific inhibitor of Smo (cyclopamine) on the apoptosis of EA.hy926 cells.Methods The Smo expression in endothelial cells in synovial tissue from 4 RA patients and 4 patients with traumatic or meniscal injury (with no arthritis,act as control group) were detected by immunohistochemistry assay.EA.hy926 cells were treated with different concentrations of TNF-α or TNF-α together with different concentrations of cyclopamine,and Shh,Ptch1,Smo,Gli1 mRNA expression levels were detected by real time-PCR.EA.hy926 cells were co-cultured with three different concentrations of cyclopamine for 24 hours before the addition of TNF-α and ActinomycinD (ActD).The cell survival rate was detected using CCK-8,and the population of apoptotic cells was detected using a flow cytometry.T-test and one-way ANOVA were used for statistical analysis.Results The positive expression rate of Smo in endothelial cells of synovial tissue in RA group was (81±23)％,which was higher than that in the control group (20±17)％ (P＜0.05).After being treated with TNF-α,the expressions of Shh and Smo mRNA in EA.hy926 cells increased,while the expression of Gli1 mRNA decreased (P＜0.05),and the expression of Ptch1 mRNA did not change significantly (P＞0.05).The expressions of Shh,Smo and Gli1 mRNA were down-regulated (P＜0.05).EA.hy926 cells treated with different concentrations of cyclopamine (2,4 and 8 μmol/L) showed a significant decrease in cell viability,in cell survival rates (57±6)％,(44±8)％ and (32±5)％ compared with that of TNF-α/ActD group (64±6)％ (P＜0.05),and cell apoptosis rates [(12.4±3.3)％,(14.5±2.7)％ (15.7±2.4)％] compared with that of TNF-α/ActD group (7.1±1.3)％ (P＜0.05).Conclusion Shh pathway is activated in endothelial cells of synovial tissue in active RA.The apoptosis in endothelial cells is promoted after cyclopamine treatment.Shh pathway may play an important role in the antiapoptotic regulatory mechanism of endothelial cells.