趋化因子CXCL13对TNF-α诱导的人骨关节滑膜细胞RANKL表达的影响

Influence of CXCL13 on expression of RANKL in human synovial cells induced by TNF-α

目的探讨趋化因子CXCL13对TNF-α诱导的人骨关节滑膜细胞调控细胞核因子-κB受体活化因子配体(RANKL)表达的影响。方法 (1)体外培养人骨关节滑膜细胞,并给予10μg/m L TNF-α处理,分别在培养0、6、12、24 h时采用免疫荧光法检测CXCL13表达。(2)人滑膜细胞给予10μg/m L TNF-α处理24 h,将培养液更换为含0、5、10、25 ng/m L CXCL13的培养液继续培养24 h,或将培养液更换为含25 ng/m L CXCL13的培养液分别作用0、1、3、6、24 h;以不加TNF-α及CXCL13处理的常规培养细胞作为对照细胞。收集各浓度、各时间点细胞,采用Western blotting法检测RANKL蛋白相对表达量。结果 (1)TNF-α作用0、6、12、24 h时细胞CXCL13相对表达量(荧光强度)分别为0.907±0.350、0.823±0.730、0.710±0.660、0.653±0.850,组间比较P均<0.05。(2)对照细胞RANKL蛋白相对表达量为0.956±0.014,25 ng/m L CXCL13处理0、1、3、6、24 h时RANKL蛋白相对表达量分别为1.543±0.047、1.366±0.026、0.883±0.026、0.367±0.034、0.246±0.015;0、5、10、25 ng/m L CXCL13作用24 h时RANKL蛋白相对表达量分别为0.287±0.007、0.189±0.008、0.069±0.004、0.022±0.002;上述各组、各时间及各浓度细胞比较P均<0.05。结论趋化因子CXCL13能够在一定浓度和时间内抑制TNF-α诱导的人骨关节滑膜细胞RANKL蛋白表达。

Objective To investigate the effect of CXCL13 on the expression of receptor activator of NF-κB ligand（ RANKL） in human synovial cells induced by tumor necrosis factor-α（ TNF-α）. Methods（1） The human synovial cells were cultured in vitro and were treated with 10 g/mL TNF-α. At 0,6,12,and 24 h,we used immunofluorescence to detect the CXCL13 expression.（2） The human synovial cells cultured in vitro were treated with 10 g/mL TNF-α for 24 h,after that,we replaced the culture fluid with medium containing 0,5,10,and 25 ng/mL CXCL13 and continued to culture for 24 h,or replaced the culture fluid with medium containing 25 ng/mL CXCL13 and continued to culture for 0,1,3,6,and 24 h. The relative expression of RANKL was detected by Western blotting. The normal cultured cells without treatment of TNF-α and CXCL13 were used as the control group. Results（1）The expression levels of CXCL13（ fluorescence intensity） were 0. 907 ± 351,0. 823 ± 735,0. 710 ± 664,and 0. 653 ± 853 in cells treated with TNF-α at 0,6,12,and 24 h（ all P〈0. 05）.（2）The expression of RANKL protein was 0. 956 ± 0. 014 in the control group,and the expression levels of RANKL protein in cells treated with 25 ng/mL CXCL13 for 0,1,3,6,and 24 h were 1. 543 ± 0. 047,1. 366 ± 0. 026,0. 883 ± 0. 026,0. 367 ± 0. 034,and 0. 246 ± 0. 015; the expression levels of RANKL protein in cells treated with different contentrations（ 0,5,10,25 ng/mL） of CXCL13 were 0. 287 ± 0. 007,0. 189 ± 0. 008,0. 069 ± 0. 004,and 0. 022 ±0. 002; significant difference was found between them（ all P〈0. 05）. Conclusion CXCL13 can inhibit the expression of RANKL protein of osteoarticular synovial cells induced by TNF-α in a certain concentration and time.