肝脏CD4+CD25+Foxp3+调节性T细胞在小鼠急、慢性肝损伤中的表达差异及意义

doi:10.3969/j.issn.1006-7795.2014.04.020

摘要/Abstract

摘要：

目的探究肝内CD4+CD25+ Foxp3+调节性T细胞（regulatory T cell，Tregs）在急、慢性肝损伤中的表达差异及意义。方法使用四氯化碳（carbon tetrachloride，CCL₄）一次或多次给予C57BL/6J小鼠腹腔注射，分别制备急性（24 h）和慢性肝损伤（6周）模型，对照组小鼠腹腔注射等体积0.9%氯化钠注射液。病理切片Masson染色观察不同模型小鼠肝内病理变化情况，运用流式细胞分析法和免疫荧光检测小鼠肝内Tregs的表达。Real-time PCR检测小鼠肝内Ⅰ型胶原、Ⅲ型胶原、白细胞介素-1β（interloukin-1β，IL-1β）、IL-6、IL-10、转化生长因子-β（transforming growth factor，TGF-β）的表达。结果 Masson染色显示急性肝损伤小鼠的肝细胞大片坏死伴出血，慢性肝损伤小鼠肝脏假小叶形成，同时Ⅰ型胶原、Ⅲ型胶原表达增加。流式细胞分析显示Tregs在急性和慢性组小鼠肝脏表达都有升高，但在慢性组升高的幅度比急性组大（P<0.01），与免疫荧光结果一致。肝内抑制性细胞因子IL-10、TGF-β在慢性肝损伤组升高显著（P<0.05），而促炎因子IL-1β、IL-6在急性肝损伤组升高显著（P<0.01）。结论肝内Tregs在急、慢性肝损伤中表达不同。在急性肝损伤中，尤其是急性肝损伤恢复期，Tregs部分增加，抑制炎性反应扩散，促进机体恢复；在慢性肝损伤中，Tregs表达显著增加，形成免疫耐受，抑制免疫清除，肝纤维化形成。

关键词: 调节性T细胞, 急性肝损伤, 慢性肝损伤, 免疫抑制

Abstract:

Objective The present study focuses on the differences and significance of intrahepatic CD4+CD25+Foxp3+ regulatory T cells(Tregs) between acute and chronic liver injury. Methods C57BL/6J mice were treated with carbon tetrachloride(CCL₄) by intraperitoneal injection, to prepare acute(24 h) and chronic liver injury(6 weeks) model; control mice were injected with saline. The pathological changes of livers in different mouse models were observed by Masson staining; the expression of intrahepatic Tregs was analyzed by flow cytometry and immunofluorescence staining; the mRNA levels of collagen-Ⅰ, collagen-Ⅲ, IL-1β、IL-6、IL-10、TGF-β were detected by real-time PCR. Results Masson staining showed a large liver cell necrosis with bleeding in acute liver injury mice; however, pseudolobule formed in the liver of chronic liver injury mice, while type-Ⅰ, type-Ⅲ collagen expression was increased. Tregs expression has elevated in both acute and chronic liver injury mice, but chronic injury group increased in magnitude larger than acute injury group(P<0.01). Intrahepatic inhibitory cytokine IL-10, TGF-β in chronic liver injury group were increased larger than that in acute injury liver group(P<0.05); while the pro-inflammatory cytokines IL-1β, IL-6 in mice with acute liver injury were enhanced more significantly comparing to mice in chronic liver injury(P<0.01). Conclusion The expression of intrahepatic Tregs was different between acute and chronic liver injury. In acute liver injury, especially in the recovery of acute liver injury, Tregs increased in part to inhibit the proliferation of inflammation, and promote the body's recovery; in chronic liver injury, Tregs were significantly increased to induce formation of immune tolerance, suppress immune clearance, finally led to hepatic fibrosis.

Key words: regulatory T cells, acute liver injury, chronic liver injury, immune suppression

中图分类号:

R575

张晓慧, 刘新, 白丽, 陈煜, 段钟平. 肝脏CD4+CD25+Foxp3+调节性T细胞在小鼠急、慢性肝损伤中的表达差异及意义[J]. 首都医科大学学报, 2014, 35(4): 483-487.

Zhang Xiaohui, Liu Xin, Bai Li, Chen Yu, Duan Zhongping. Differences of liver CD4+CD25+Foxp3+ regulatory T cells in mice with acute and chronic liver injury[J]. Journal of Capital Medical University, 2014, 35(4): 483-487.

参考文献

[1] Vignali D A A, Collison L W, and Workman Creg J. How regulatory T cells work[J]. Nat Rev Immunol, 2008,8(7):523-532.
[2] Yang X H, Yamagiwa S, Ichida T, et al. Increase of CD4+CD25+ regulatory T-cells in the liver of patients with hepatocellular carcinoma[J]. J. Hepatol, 2006,45:254-262.
[3] El-Badawy O H, Sayed D, Badary M S, et al. Relations of regulatory T cells with hepatitis markers in chronic hepatitis B virus infection[J]. Hum Immunol, 2012,73(4):335-341.
[4] Yoshizawa K, Abe H, Kubo Y, et al. Expansion of CD4(+)CD25(+)FoxP3(+) regulatory T cells in hepatitis C virus-related chronic hepatitis, cirrhosis and hepatocellular carcinoma[J]. Hepatol Res, 2010,40(2):179-187.
[5] Liang X S, Li C Z, Zhou Y, et al. Changes of Treg and Th17 cells balance in the development of acute and chronic hepatitis B virus infection[J]. BMC Gastroenterol, 2012,12:43.
[6] 刘新,娄金丽,白丽,等.流式细胞术分析小鼠肝纤维化时机体免疫细胞的变化[J].首都医科大学学报,2013,34(4).
[7] Breous E, Somanathan S, Vandenberghe L H, et al. Hepatic regulatory T cells and Kupffer cells are crucial mediators of systemic T cell tolerance to antigens targeting murine liver[J]. Hepatology, 2009,50(2):612-621.
[8] Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+regulatory T cells in immunological tolerance to self and non-self[J]. Nat Immunol, 2005,6:345-352.
[9] Lu L, Feng M, Gu J, et al. Restoration of intrahepatic regulatory T cells through MMP-9/13-dependent activation of TGF-β is critical for immune homeostasis following acute liver injury[J]. J Mol Cell Biol, 2013,5(6):369-379.
[10] StooP J N, vanderMolen R G, Baan C C, et al. Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infeetion[J]. Hepatology, 2005,41,771-778.
[11] 张静.蒙族慢性乙型肝炎患者NK细胞的特征与肝损伤相关性分析[J].中国煤炭工业医学杂志,2013,16(7):1073-1077.
[12] 程仕彤,冯鹭,刘丽娜,等.α-干扰素对肝纤维化小鼠肝组织中Foxp3~+调节性T细胞的影响[J].中国医科大学学报,2013,42(5):402-405.
[13] Katz S C, Ryan K, Ahmed N, et al. Obstructive jaundice expands intrahepatic regulatory T cells, which impair liver T lymphocyte function but modulate liver cholestasis and fibrosis[J]. J Immunol, 2011,187(3):1150-1156.