流式细胞术分析小鼠肝纤维化时机体免疫细胞的变化

Analysis of immune cell changes by flow cytometry in mice liver fibrosis

目的探讨小鼠肝纤维化时外周血和肝组织浸润的不同免疫细胞的变化及其意义。方法将C57BL/6小鼠采用数字表法随机分成肝纤维化组和对照组,每组5只,肝纤维化组腹腔注射四氯化碳(carbon tetrachloride,CCl4),建立肝纤维化模型,对照组注射等量0.9%氯化钠注射液。采用生化法测定小鼠血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)水平;HE染色、Masson染色观察肝组织病理变化;流式细胞术分析外周血和肝组织中浸润的调节性T细胞(regulatory T cells,Tregs)、NK1.1+、CD4+及CD8+细胞的比例变化,比较组间差异。结果流式分析显示纤维化小鼠肝组织中Tregs比例CD25+Foxp3+/CD4+为(10.63±1.50)%,明显高于对照组(1.80±0.66)%,差异有统计学意义(P<0.01),外周血的Tregs上升不明显,纤维化组与对照组分别为(7.40±0.85)%、(5.27±1.30)%;纤维化小鼠肝组织浸润的NK1.1+细胞和血液中的NK1.1+细胞占淋巴细胞的比例分别低于对照组〔(9.53±2.25)%vs(19.80±5.97)%,P<0.05;(0.38±0.13)%vs(1.06±0.63)%,P<0.05〕;纤维化小鼠肝组织和血中的CD4+细胞及CD4+/CD8+比值也有所下降,但差异无统计学意义。结论小鼠肝纤维化时,肝组织中浸润的CD4+CD25+Foxp3+调节性T细胞升高,NK1.1+细胞下降,CD4+细胞下降。

Objective To investigate the changes and significances of different immune cells in the peripheral blood and liver- infiltrating lymphocytes（LILs） in mice liver fibrosis. Methods C57BL/6 mice were randomly divided into the model group and the control group. The mice of liver fibrosis were induced by intraperitoneal injection of carbon tetrachloride （ CC14 ） , at the same time, the control group received the equal volume physiological saline. The serums were collected for detecting ALT of mice. Histopathological changes of liver were observed by HE and Masson staining. The frequencies of peripheral and Tregs, NK1.1 ＋ cells, CD4 ^＋ cells and CD8 ^＋ cells were analyzed by flow cytometry. The data were evaluated by t-test. Results In liver, it showed that Tregs frequency, CD25^＋ Foxp3^＋/CD4＋ in the model group was（10.63±1.50） %, which was significantly higher as compared with that in the control group（ P〈 0.01 ）. However, peripheral Tregs in the model group only increased slightly[ （7.40±0.85） % vs （5.27±1.30） % ^＋. Strikingly, there were less NK1. 1 cells in both liver and blood in the model group as compared with those of the control group [ （ 9.53 ± 2.25 ） % vs （19.80±5.97） %, P〈0.05 ; （0.38＋0.13） % vs （ 1.06±0.63 ） %, P〈0.05 ] respectively. Though CD4^＋ cells and the ratio of CD4＋/ CD8＋ decreased in liver and blood, no statistically significance was observed. Conclusion During liver fibrosis, intrahepatie CD4^＋ CD25^＋FoxD3＋ regulatory T cells are increased, while NK1.1＋ cells decreased; CD4 cells are also decreased.