目的:初步观察Sonic Hedgehog信号通路分子Smoothened(Smo)在类风湿关节炎(rheumatoid arthritis,RA)滑膜血管内皮细胞的表达及其生物学意义.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,免疫组化检测滑膜组织Smo蛋白表达情况.采用人脐静脉内皮细胞系EA.hy926作为滑膜血管内皮细胞的模型,予不同浓度肿瘤坏死因子α(TNF-α)处理,Western blotting检测Smo蛋白表达;采用RNAi技术转染体外合成的特异性Smo-siRNA,应用Western blotting检测沉默效果;转染siRNA 24 h后,经TNF-α/放线菌素D(actinomycin D,ActD)诱导细胞凋亡,CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡情况.结果:RA患者滑膜组织Smo表达高于对照组,以血管内皮细胞表达尤为明显.EA.hy926细胞经TNF-α刺激后,Smo蛋白表达上调(P<0.05).RNA干扰EA.hy926细胞Smo表达后,细胞存活率为(24.30±0.45)%,低于阴性对照组的(36.86±0.62)%(P<0.05),细胞凋亡率为(48.00±1.96)%,高于阴性对照组的(31.70±0.82)%(P<0.05).结论:Smo可能参与了RA患者滑膜组织血管内皮细胞凋亡的调控.%AIM: To investigate the expression of Sonic Hedgehog signaling pathway-associated factor Smoothened ( Smo) and its role in endothelial cells in synovial tissue of active rheumatoid arthritis ( RA) . METHODS: Smo expression in synovial tissue from 4 RA patients and 4 patients with trauma or meniscal injury ( without arthritis, used for control) was detected by the method of immunohistochemistry. Human umbilical vein endothelial cell line EA. hy926 was used as the model of synovial vascular endothelial cells. The expression of Smo was detected by Western blotting after TNF-α treatment. The small interfering RNA (siRNA) specifically targeting Smo gene was synthesized and transfected into EA. hy926 cells. The interference efficiency of the siRNA on the production of Smo protein was determined by Western blotting. The cells were treated with TNF-a and actinomycin D (ActD) 24 h after siRNA transfection. The cell survival rate was determined by CCK-8 assay and the apoptotic rate was examined by flow cytometry. RESULTS; Smo was highly expressed in synovial tissue from active RA patients, especially in endothelial cells as compared with control group. TNF-a significantly increased the protein expression of Smo in EA. hy926 cells. EA. hy926 cells transfected with Smo-siRNA showed a significant decrease in the cell viability with the cell survival rate of (24.30 ±0.45)% and the apoptotic rate of (48. 00 ±1.96)% , as compared with those in negative control group [(36.86 ±0.62)% and (31.70 ±0.82)% , respectively] . CONCLUSION: Smo may play a role in the regulation of apoptosis in endothelial cells in RA synovium.
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