细胞因子对人视网膜色素上皮细胞syndecan-1表达的影响

Effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial cells

目的检测细胞因子对人视网膜色素上皮(RPE)细胞syndecan-1表达的影响,并探讨其作用的信号转导途径。方法体外培养人RPE细胞,采用逆转录聚合酶链反应和免疫荧光法检测正常细胞syndecan-1mRNA、蛋白的表达;免疫荧光法定性、定量观察不同刺激因子作用下syndecan-1的表达变化,包括:7、35ng/ml肿瘤坏死因子(TNF)-α刺激24h,1、6μg/ml脂多糖(LPS)刺激11h,7ng/mlTNF-α刺激不同时间(0～24h,每2h为1个时间点,共13个时间点),30%单核/巨噬细胞株(THP-1细胞)上清液刺激3、14、43h;细胞外信号调节激酶(ERK)1/2通路特异性阻断剂PD098059预处理2h后对30%THP-1细胞上清液作用的调节;30%THP-1细胞上清液刺激细胞3h后0.25%胰酶作用5min,噻唑蓝法检测细胞贴壁情况。结果在正常RPE细胞中可检测到syndecan-1mRNA和蛋白的表达;未受刺激的RPE细胞细胞膜、细胞浆及核仁呈强的黄绿色荧光,细胞核呈浅绿色荧光;随TNF-α、LPS浓度及时间增加,荧光强度呈减弱趋势(P<0.01);30%THP-1细胞上清液刺激后细胞荧光强度明显减弱(P<0.001)。50μmol/LPD098059预处理2h后可部分阻断30%THP-1细胞上清液的下调作用。与对照组相比,THP-1细胞上清液作用3h后贴壁细胞数下降(P<0.05)。结论TNF-α和LPS可下调体外培养的人RPE细胞syndecan-1的表达;30%THP-1细胞上清液可下调RPE细胞syndecan-1的表达、促使细胞脱壁,且该作用至少通过了ERK1/2信号转导通路。

Objective To investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial （RPE） cells and the signal transduction pathway. Methods Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor （TNF）-α for 24 hours, 1 and 6 μg/ml lipopolysaccharide （LPS） for 11 hours, 7 ng/ml TNF-α for 0 to 24 hours （once per 2 hours, and 13 times in total）, and 30% supernatant of monocyte/macrophage strain （THP-1 cells） for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059 [the specific inhibitor of extracellular signal regulated kinase（ERK） 1/21 for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay. Results In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and strong syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-α or LPS increased, the fluorescence intensity decreased （P〈0. 01）, and after exposed to 30% supernatant of THP-1 cells, weaker fluorescence intensity was detected （P〈0. 001）. Pretreatment with 50 μmol/L PD098059 for 2 hours partly inhibited the effect of THP-1 cells supernatant. After exposed to 30% supernatant of THP-1 cells for 3 hours, the number of attached cells decreased compared with the controls （P 〈 0. 05）. LPS down-regulate the expression of syndecan-1 in cultured human RPE cells Conclusions TNF-α and The supernatant of THP-1 cells down-regulates the expression of syndecan-1 and lessens the cells attaching, which is at least mediated by ERK 1/2 pathway.