脾脏单核细胞对纤维化肝脏免疫细胞组成及免疫相关因子表达的影响

Effects of spleen monocytes on immune cell composition and immune-related factors expression in fibrotic liver

目的观察脾脏单核细胞对纤维化肝脏免疫细胞组成及免疫相关因子表达的影响。方法24只雄性C57BL/6小鼠采用随机数字法分为注射玉米油的对照组(6只)和注射CCl4的肝纤维化造模组(18只);取造模成功的小鼠12只行脾切除术,再随机分为两组分别接受脾脏单核细胞或磷酸盐缓冲液(PBS)回输,每组6只。4组小鼠继续接受玉米油或CCl4注射两周后,流式分析各组小鼠肝脏中免疫细胞差异;Luminex检测肝脏匀浆中细胞因子差异。两组间比较采用Student’s t检验。结果纤维化组小鼠肝脏中单核-巨噬细胞MoMF)比例高于对照组[(4.20±2.00)%比(1.00±0.28)%,t=-3.879,P<0.01],CD4+与CD8+T细胞比例低于对照组[(0.72±0.07)%比(0.90±0.10)%,t=3.470,P<0.01;(0.51±0.12)%比(0.98±0.38)%,t=2.917,P<0.05],但CD4+T细胞中调节性T细胞(Treg)增多[(19.75±6.43)%比(5.39±2.21)%,t’=-5.171,P<0.01];纤维化组小鼠肝脏匀浆中白细胞介素(IL)-1β、IL-13、转化生长因子-β(TGF-β)、趋化因子2(CCL2)浓度高于对照组[(24.05±10.05)pg/ml比(14.16±3.31)pg/ml,t’=-2.430,P<0.05;(45.21±9.73)pg/ml比(19.40±7.36)pg/ml,t=-5.182,P<0.01;(381.71±118.56)pg/ml比(230.19±61.58)pg/ml,t=-2.737,P<0.05;(46.58±17.92)pg/ml比(23.87±6.65)pg/ml,t=-2.911,P<0.01]。(2)肝纤维化脾切+回输组小鼠肝脏中自然杀伤细胞(NK)、MoMF及树突状细胞(DC)比例高于脾切组[(2.49±0.43)%比(1.12±0.43)%,t=-4.503,P<0.01;(6.64±1.50)%比(2.93±0.66)%,t=-4.546,P<0.01;(5.45±1.54)%比(2.59±0.66)%,t=-2.732,P<0.05],CD8+T细胞比例低于对照组[(2.28±0.67)%比(3.87±0.38)%,t=2.950,P<0.05];脾切+回输组小鼠肝脏匀浆中CCL2浓度高于脾切组[(50.09±20.55)pg/ml比(20.08±5.99)pg/ml,t=-2.804,P<0.05]。结论脾脏单核细胞参与了纤维化肝脏免疫细胞组成及相关因子表达的调控,对肝脏免疫微环境形成影响。

Objective To observe the changes of immune cell composition and immune-related factors in livers of mice with liver fibrosis,and study the effects of splenic monocytes on the change these cells and factors.Methods Twenty-four male C57BL/6 mice were randomly divided into two groups.CCl4 was injected intraperitoneally to establish liver fibrosis mouse model(18 mice),and corn oil injection group was used as control(6 mice).Splenectomy was performed on 12 of the mice with liver fibrosis.After then,these splenectomized mice were divided into two groups and received splenic CD11b+monocyte or PBS transfusion i.v.respectively.All of the mice received another 2-week injection of corn oil or CCl4.Hepatic single cell suspension of each group was prepared and the frequencies of immune cells were analyzed by flow cytometry.Liver homogenates were prepared,and expressions of cytokines and chemokines in the liver homogenate of each groups were detected by Luminex multifactor analysis.Student’s t test was used for comparison between the two groups.Results(1)The frequency of monocyte-derived macrophage(MoMF)in the liver of mice in the liver fibrosis group is higher than the control group[(4.20±2.00)%vs.(1.00±0.28)%,t=-3.879,P<0.01].The frequencies of CD4+and CD8+T cells in the liver of mice in the liver fibrosis group is lower than the control group[(0.72±0.07)%vs.(0.90±0.10)%,t=3.470,P<0.01;(0.51±0.12)%vs.(0.98±0.38)%,t=2.917,P<0.05),but the frequency of regulatory T cells(Treg)in the CD4+T cells was higher[(19.75±6.43)%vs.(5.39±2.21)%,t’=-5.171,P<0.01).The concentrations of interleukin(IL)-1β,IL-13,transforming growth factor-β(TGF-β)and chemokine chemokine 2(CCL2)in the liver homogenate of mice with liver fibrosis were higher than those of control group[(24.05±10.05)pg/ml vs.(14.16±3.31)pg/ml,t’=-2.430,P<0.05;(45.21±9.73)pg/ml vs.(19.40±7.36)pg/ml,t=-5.182,P<0.01;(381.71±118.56)pg/ml vs.(230.19±61.58)pg/ml,t=-2.737,P<0.05;(46.58±17.92)pg/ml vs.(23.87±6.65)pg/ml,t=-2.911,P<0.01].(2)The frequencies of natural killer cells(NK),MoMF and dendritic cells(DC)in the livers of mice receiving splenic monocyte transfusion were higher than the splenectomy group[(2.49±0.43)%vs.(1.12±0.43)%,t=-4.503,P<0.01;(6.64±1.50)%vs.(2.93±0.66)%,t=-4.546,P<0.01;(5.45±1.54)%vs.(2.59±0.66)%,t=-2.732,P<0.05].The frequency of CD8+T cells in the livers of mice receiving splenic monocyte transfusion were lower than the splenectomy group[(2.28±0.67)%vs.(3.87±0.38)%,t=2.950,P<0.05).The concentration of CCL2 in liver homogenate of mice receiving splenic monocyte transfusion was higher than that of control group[(50.09±20.55)pg/ml vs.(20.08±5.99)pg/ml,t=-2.804,P<0.05].Conclusion Spleen-derived monocytes are involved in the regulation of immune cell composition and factor expression in the fibrotic livers,which affects the microenvironment of fibrotic liver.