肝纤维化小鼠调节性T淋巴细胞的变化及其意义

Changes of regulatory T cells related to CClcinduced liver fibrosis in mice

目的探讨小鼠肝纤维化模型外周血和肝组织浸润的CD4＋CD25＋Foxp3＋调节性T淋巴细胞（Tregs）的变化及其意义。方法将10只C57BL／6小鼠随机均分成模型组和对照组，模型组腹腔注射cch建立肝纤维化模型，对照组注射等量等渗盐水。用生物化学法测定血清ALT水平；HE、Masson染色后在光学显微镜下观察肝组织病理学变化；用流式细胞仪检测外周血和肝组织中浸润的Tregs、NK1．1＋、CD4＋及CD8＋细胞的比例变化；免疫荧光检测肝内Foxp3细胞的表达；RT-PCR检测白细胞介素6、白细胞介素10、转化生长因子D和Foxp3的mRNA表达水平。用t检验分析组间差异。符合方差齐陛的两样本均数比较采用独立样本t检验。结果模型组小鼠肝组织中Tregs比例CD25＋Foxp3＋／CD4＋为10．63％±1．50％，高于对照组（1．80％±0．66%）（P〈0．01），外周血的Tregs上升并不明显，模型组与对照组分别为6．00%±0．62％、5．33％±2．86％；模型组肝组织Foxp3表达增多，Foxp3mRNA表达升高（P〈0．05）。模型组肝组织浸润的和血中的NK1．1＋细胞占淋巴细胞的比例分别低于对照组（9．53％±2．25％比19．80％土5．97％，t=3．219，P〈0．05；0．38％±0．13％比1．06％±0．63％，t=2．375，）P〈0．05）；模型组小鼠肝组织和外周血中的CD4＋细胞及CD4＋／CD8＋比值也有所下降，但差异无统计学意义（P〉0．05）。模型组肝组织中Th2型细胞因子转化生长因子DmRNA表达升高（P〈0．01）。结论肝纤维化时，肝组织中浸润的CD4＋CD25＋Foxp3＋Tregs升高，NK1．1＋细胞下降，Tregs可能通过转化生长因子β抑制了NK1．1＋细胞的作用。

Objective To investigate liver fibrosis-related changes of CD4＋CD25＋Foxp3＋ regulatory T cells （Tregs） in peripheral blood and in liver-infiltrating lymphocytes （LILs） using a mouse model. Methods C57BL/6 mice were randomly divided into a model group and a control group. The model group received intraperitoneal injection of carbon tetrachloride （CC14） to induce liver fibrosis, and the control group received an equal volume of physiological saline. Serum was collected for detection of alanine aminotransferase level. Histopathological changes in liver were assessed by microscopic observation of tissues stained by hematoxylin- eosin and Masson. Frequencies of peripheral and intrahepatic Tregs, NK1.1＋ cells, CD4＋ and CD8＋ cells were analyzed by flow cytometry. Intrahepatic Foxp3＋ cells were detected by immunofluorescence. Liver expression of IL-6, IL-10, TGFI3 and Foxp3 was measured by RT-PCR detection of mRNA. Inter-group differences were evaluated by t-test. Results The model group showed a significantly higher frequency of intrahepatic CD25＋Foxp3＋/CD4＋ Tregs （10.63% ＋ 1.50% vs. control group： 1.80% ＋ 0.66%; P 〈 0.01） but only slightly higher frequency of peripheral Tregs （6.00% ± 0.62% vs. 5.33% ± 2.86%）. The model group also showed significantly higher levels of intrahepatic Foxp3＋ cells and of Foxp3 mRNA （both P 〈 0.05）, but significantly lower frequencies ofNK1.1 cells in LILs （9.53% ± 2.25% vs. 19.80% ＋ 5.97%; P 〈 0.05） and in peripheral blood （0.38%± 0.13% vs. 1.06% q- 0.63%;P 〈 0.05）. The CD4＋ cell frequency and the CD4＋/CD8＋ ratio were lower in L1Ls and peripheral blood of the model group, but none differed significantly from the control group. The intrahepatic expression of TGFI3 mRNA was significantly higher in the model group （P 〈 0.01）. Conclusion In liver fibrosis, intrahepatic CD4＋CD25＋Foxp3＋ Tregs are increased while NKI.1＋ cells are decreased. Tregs may suppress NK1.1 ＋ cells through a mechanism involving TGFβ.