IL-10调控HaCaT细胞增殖及分化的分子机制

The study of molecular mechanism of regulation of IL-10on proliferation and differentiation of HaCaT cells

目的探讨白细胞介素10(IL-10)对皮肤角质形成细胞(HaCaT)增殖影响和对氯化钙(CaCl 2)诱导的角质形成细胞分化标志物的表达影响及其可能的分子机制。方法以不同浓度IL-10(0、3、10、30 ng/ml)处理HaCaT细胞不同时间(0、24、48、72 h),MTS分析细胞增殖,流式细胞仪检测细胞周期;IL-10(终浓度为10 ng/ml)预处理HaCaT 1 h,加入或不加CaCl 2(终浓度为1.2 mmol/L)培养24、48、72 h,Western blot检测IL-10对HaCaT分化标志物表达影响;丝裂原蛋白激活激酶-胞外信号调节激酶(MAPKs-ERK1/2)特异性抑制剂PD98059及磷脂酰肌醇激酶-丝氨酸/苏氨酸激酶(PI3K-AKT)特异性抑制剂LY294002预处理HaCaT细胞,分别提取细胞总RNA和蛋白,荧光定量PCR(RT-qPCR)和Western blot检测IL-10对HaCaT细胞分化标志物(Keratin1、Keratin5、Involucrin)表达影响。结果MTS结果显示,在72 h内,IL-10(30 ng/ml和较低浓度)对HaCaT细胞增殖无影响;流式细胞分析结果提示IL-10不影响HaCaT细胞周期进程。Western blot分析显示,IL-10上调HaCaT角质形成细胞角质细胞分化标志物Involucrin表达,而对Keratin1及Keratin5没有显著的影响。机制研究分析显示,IL-10能够活化ERK1/2和AKT,增加其磷酸化水平;RT-qPCR和Western blot结果显示PD98059及LY294002部分阻断IL-10诱导的Involucrin的表达。结论IL-10在一定浓度范围内不影响HaCaT的增殖;IL-10部分通过MAPKs-ERK1/2和PI3K-AKT途径上调HaCaT分化标志物Involucrin表达。

Objective To investigate the effects of interleukin(IL)-10 on the proliferation of HaCaT cells and CaCl 2 induced expression of differentiation markers and its possible molecular mechanisms.Methods HaCaT cells were treated with various concentrations of IL-10(0,3,10,30 ng/ml)for different time(0,24,48,72 h),cell proliferation was measured using MTS,and cell cycle was determined by flow cytometry.HaCaT cells were pretreated with IL-10(final concentration 10 ng/ml)for 1 h,then incubated with or without CaCl 2(final concentration 1.2 mmol/L)for 24,48,72 h,Western blot was performed to detect the effect of IL-10 on the expression of HaCaT keratinocyte differentiation markers.After pretreatment of HaCaT cells with PD98059,an inhibitor of mitogen-activated kinase-ERK1/2,and LY294002,an inhibitor of phosphatidylinositol kinase-serine/threonine kinase(PI3K-AKT),the total RNA and proteins were extracted separately,real time quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to examine the influence of IL-10 on the expression of differentiation markers(Keratin1,Keratin5,Involucrin).Results MTS results revealed that IL-10(30 ng/ml and lower doses)did not alter the proliferation of HaCaT cells in 72 h.Flow cytometry analysis demonstrated that IL-10 had no significant influence on cell cycle progression.The results of Western blot showed that IL-10 upregulated the expression of differentiation markers Involucrin,while there was no significant effect on Keratin1 and Keratin5.Mechanism research analysis demonstrated that IL-10 could activate ERK1/2 and AKT,increase their phosphorylation levels;RT-qPCR and Western blot results showed that PD98059 and LY294002 partially blocked IL-10 induced Involucrin expression.Conclusion At a particular concentration range,IL-10 has little effect on HaCaT proliferation,but it partially upregulates the expression of differentiation marker Involucrin via the MAPKs-ERK1/2 and PI3K-AKT pathways.