白细胞介素10抑制枯否细胞诱导的肝星状细胞激活的研究

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 2d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 2ng/ml和 2 0ng/ml的IL 10处理。 2d后 ,分别采用3 H 胸腺嘧啶脱氧核苷 (3 H 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 .0 5 )和α SMA表达 (P >0 .0 5 )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 ;2ng/ml和 2 0ng/ml的IL 10分别使共培养组HSC的增殖降低了 2 3%和 33% ,α SMA表达降低了 35 %和 4 9% ,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 .0 5或P <0 .0 0 1)。HSC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 .0 1) ;2ng/ml和 2 0ng/ml的IL 10分别使共培养组的TNF α量降低了 2 7% (P <0 0 1)和 36 % (P<0 0 1) ,使KC单独培养组的TNF α量降低了 2 9% (P <0 0 5 )和 4

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.