Interleukin-10 inhibits the activation of cult ured rat hepatic stellate cells induced by Kupffer cells

目的研究白细胞介素10(IL-10)对与枯否氏细胞(KC)共培养的肝星状细胞(HSC)激活的影响,并初步探讨其作用机制.方法采用肝脏离体胶原酶灌注消化,及密度梯度离心的方法来分离培养HSC和KC; 将原代培养2 d的HSC和KC分为HSC单独培养、KC单独培养和HSC与KC共培养3大组,分别加2 n g/ml和20 ng/ml的IL-10 处理.2 d后,分别采用3H-胸腺嘧啶脱氧核苷(3H-TdR) 掺入法检测HSC的增殖,Western印染法检测HSC中α-平滑肌肌动蛋白(α-SMA)的表达和酶联免疫吸附分析(ELISA)法检测培养上清中肿瘤坏死因子-α(TNF-α)的含量. 结果 IL-10对单独培养的HSC的增殖(P>0.05)和α-SMA 表达(P>0.05) 无明显影响.共培养组HSC的增殖和α-SMA 表达分别增加了2.4倍和6倍;2 ng/ml和20 ng/ ml的IL-10分别使共培养组HSC的增殖降低了23%和33%,α-SMA 表达降低了35%和49%,均呈浓度依赖性,和HSC单独培养组相比,差异有显著意义(P<0.05或P<0.001).HS C单独培养组未检测出TNF-α;共培养组的TNF-α量比KC单独培养组增加了74%(P<0. 01);2 ng/ml和20 ng/ml的IL-10分别使共培养组的TNF-α量降低了27%(P<0.01) 和36%(P<0.01),使KC单独培养组的TNF-α量降低了29%(P<0.05)和42%( P<0.01),均呈浓度依赖性.结论 IL-10通过抑制KC分泌细胞因子而抑制KC诱导的HSC激活,在减轻肝纤维化中发挥作用.To investigate the effect of interleukin-10 (IL-10) on the activation of cultured rat hepatic stellate cells (HSC) induced by Kupffer cells (KC) and relevant mechanisms.HSC and KC were isolated and purified from rat liver by collagenase IV perfusion and density gradient centrifugation with Nycodenz. After primary culture for 2 days, HSC and KC were divided randomly into three groups: HSC group, KC group and HSC + KC group (coculture group), then were stimulated by IL-10 of concentrations of 2 ng/ml or 20 ng/ml respectively. After 2 days, the proliferation of HSC were determined with (3)H-TdR incorporating test, the expression of alpha-smooth muscle actin (alpha-SMA) in HSC was detected by Western blotting, and tumor necrosis factor-alpha (TNF-alpha) protein concentration in the supernatant was determined by ELISA.IL-10 showed no significant effect on HSC proliferation (P > 0.05) and alpha-SMA expression (P > 0.05) in HSC group. The levels of proliferation and alpha-SMA expression of HSC in coculture group were 2.4 times (P < 0.001) and 6 times (P < 0.001) higher respectively than that in HSC groups. In coculture group, HSC proliferation and alpha-SMA expression were decreased by 23% (P < 0.001) and 35% (P < 0.05) respectively after stimulation of 2 ng/ml IL-10, and were decreased by 33% (P < 0.001) and 49% (P < 0.05) respectively after stimulation of 20 ng/ml IL-10 in a dose dependent way. TNF-alpha was not detected in HSC group. The level of TNF-alpha in HSC + KC coculture group was 74% higher than that in KC group (P < 0.01). 2 ng/ml and 20 ng/ml IL-10 reduced the TNF-alpha level by 27% (P < 0.01) and 36% (P < 0.01) respectively in coculture group, and reduced the TNF-alpha level by 29% (P < 0.05) and 42% (P < 0.01) respectively in KC group in a dose dependent way.IL-10 reduces the level of TNF-alpha secreted by KC dose-dependently. Through reducing cytokine production by KC, IL-10 inhibits the activation of cultured rat HSC induced by KC, which may play a protective role against liver fibrosis.国家自然科学基金PubMed中文核心期刊要目总览(PKU)中国科学引文数据库(CSCD)02104-1078