摘要
目的明确肿瘤坏死因子(TNF-α)增强血管紧张素Ⅱ(AngⅡ)对人成纤维样滑膜细胞(FLS)增殖、迁移与侵袭的作用及其部分机制。方法体外培养正常人FLS,用AngⅡ(10-9、10-8、10-7、10-6、10-5mol/L)、TNF-α(20 ng/ml)单用或联合使用,刺激48 h后,采用CCK8试剂盒检测FLS增殖功能;Transwell小室法检测迁移与侵袭功能;激光共聚焦、免疫荧光或蛋白免疫印迹法检测AngⅡ受体及G蛋白偶联受体激酶2(GRK2)的表达。结果 AngⅡ(10-7、10-6、10-5mol/L)能促进人FLS的增殖(P<0.05),最适浓度为10-7mol/L;TNF-α(20 ng/ml)能显著增强FLS的增殖(P<0.05);AngⅡ(10-7mol/L)与TNF-α(20 ng/ml)联合使用可进一步促进FLS增殖(P<0.05);单用AngⅡ(10-7mol/L)、TNF-α(20 ng/ml)或者联合使用都能显著促进FLS的迁移(P<0.01)与侵袭能力(P<0.05);单用AngⅡ可显著升高FLS血管紧张素Ⅱ1型受体(AT1R)的表达水平(P<0.05),GRK2水平有上升趋势(P>0.05);单用TNF-α可显著升高FLS的AT1R和GRK2的蛋白表达水平(P<0.05);AngⅡ与TNF-α联合使用,AT1R与GRK2的表达水平显著升高(P<0.05);GRK2抑制剂可以下调AngⅡ与TNF-α联合诱导的FLS迁移、侵袭功能(P<0.01)。结论 AngⅡ促进FLS增殖、迁移和侵袭,TNF-α可以促进AngⅡ介导的FLS增殖、侵袭和转移,其机制可能与上调FLS的AT1R和GRK2表达有关。
Objective To identify the effects and mechanisms of tumor necrosis factor α（ TNF-α） enhances human fibroblast-like synoviocyte（ FLS） proliferation,migration,and invasion induced by angiotensin Ⅱ（ Ang Ⅱ）.Methods FLS was stimulated by different concentrations of Ang Ⅱ（ 10-9,10-8,10-7,10-6,10-5mol / L） combined with or without TNF-α（ 20 ng / ml） for 48 h,and then CCK8 assay and Transwell chamber were used to test FLS proliferation,migration and invasion. The expressions of Ang Ⅱ receptors（ AT1 R and AT2R） and GRK2 were measured by immunofluorescence,laser confocal or Western blot. Results Ang Ⅱ（ 10-7,10-6,10-5mol /L） could promote the proliferation of FLS（ P〈 0. 05）,10-7mol / L was the optimum concentration. TNF-α（ 20 ng / ml） significantly enhanced the proliferation of FLS（ P 〈0. 05）. Ang Ⅱ（ 10-7mol / L） combined with TNF-α（ 20 ng /ml） had a further promotion on FLS proliferation（ P〈 0. 05）. The migration and invasion of FLS were significantly increased by Ang Ⅱ,TNF-α or their combination（ migration： P〈 0. 01,and invasion： P 〈0. 05）. The expression of AT1 R was increased（ P 〈0. 05）,but the expression of GRK2 only had a rising trend induced by AngⅡ（ P 〉0. 05）. The expressions of AT1 R and GRK2 on FLS were increased significantly induced by TNF-α（ P〈 0. 05） and their combination group（ P 〈0. 05）. GRK2 inhibitor could down-regulate the combination group effects on FLS such as migration and invasion（ P〈 0. 01）. Conclusion Ang Ⅱ promotes FLS proliferation,migration and invasion. TNF-α could promote FLS proliferation,migration and invasion induced by Ang Ⅱ through up-regulating the expressions of AT1 R and GRK2 on FLS.
出处
《安徽医科大学学报》
CAS
北大核心
2016年第9期1238-1243,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81503084
81330081
81302784)
