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目的:探讨氟西汀对体外培养的人结膜上皮细胞细胞炎性因子IL-1和TNF-α的表达及丝裂原活化蛋白激酶(MAPK)信号通路的影响。方法:混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入不同浓度的氟西汀(0,1,2.5,5,10,20,40μmol/L),CCK-8法检测细胞增殖率;免疫荧光检测ERK1/2、P-ERK1/2、IL-1、TNF-α在对照组及氟西汀组(5μmol/L)HConEpiC细胞中的定位;Western Blot检测HConEpiC细胞中ERK、P-ERK、IL-1、TNF-α蛋白表达水平表达。结果:随着氟西汀浓度增加HConEpiC细胞增殖率呈缓慢降低状态,以氟西汀浓度为5μmol/L进一步探讨其对蛋白量表达的影响。对细胞免疫荧光照片进行光密度分析发现,氟西汀组ERK1/2的累积光密度(IOD)值较对照组差异无统计学意义,提示氟西汀干预对HConEpiC中ERK1/2蛋白的表达无明显影响;氟西汀组P-ERK1/2、IL-1、TNF-α的IOD值较对照组均明显增高(P<0.05),提示氟西汀干预后HConEpiC中P-ERK1/2、IL-1、TNF-α蛋白表达增多;Western Blot实验检测各组蛋白量结果显示,氟西汀组与对照相比,ERK1/2蛋白量表达无明显差异;氟西汀组P-ERK1/2、IL-1、TNF-α蛋白表达水平较对照组升高(P<0.05)。结论:氟西汀可激活培养的正常人结膜上皮细胞内MAPK-ERK信号通路并导致炎性相关因子的产生。
Objective: To investigate the effect of fluoxetine on the expression of IL-1 and TNF-α and mitogen-activated protein kinase (MAPK) in cultured human conjunctival epithelial cells. Methods: Human conjunctival epithelial cells were cultured by mixed digestive fluid culture method and identified by immunohistochemistry. The cells were cultured for 3 or 4 passages, fluoxetine (0, 1, 2.5, 5, 10, 20 and 40μmol / L) was added to the cell culture medium, and cell proliferation rate was measured by CCK- The localization of ERK1 / 2, P-ERK1 / 2, IL-1 and TNF-αin control group and fluoxetine group (5μmol / L) HConEpiC cells were detected by immunofluorescence. The expressions of ERK and P-ERK , IL-1, TNF-α protein expression levels. Results: The proliferation rate of HConEpiC cells gradually decreased with the increase of fluoxetine concentration. The effect of fluoxetine concentration was 5μmol / L on the protein expression. The results of optical density analysis of immunofluorescence showed that the cumulative optical density (IOD) value of ERK1 / 2 in fluoxetine group was not significantly different from that in control group, suggesting that the expression of ERK1 / 2 protein in HConEpiC (P <0.05), suggesting that the IOD values of P-ERK1 / 2, IL-1 and TNF-α in fluoxetine group were significantly higher than those in control group The expression of ERK1 / 2 protein in fluoxetine group was not significantly different from that in control group. The levels of P-ERK1 / 2, The levels of IL-1 and TNF-α were higher than those in control group (P <0.05). Conclusion: Fluoxetine can activate the MAPK-ERK signal pathway in cultured human normal conjunctival epithelial cells and cause the production of inflammatory related factors.