6例非酒精性脂肪性肝病小鼠肠道B细胞的变化

doi:10.6040/j.issn.1671-7554.0.2022.0686

摘要/Abstract

摘要： 目的探索非酒精性脂肪性肝病(NAFLD)小鼠肠道B细胞的变化。方法将12只雄性C57BL/6J小鼠随机分为实验组和对照组,每组6只。实验组喂以45%高脂肪饮食诱导发生NAFLD,对照组喂以正常饮食。16周后处死小鼠,留取两组肝脏、小肠黏膜、结肠黏膜、肠系膜淋巴结(MLN)、派氏结(PP)及小肠内容物。采用苏木精-伊红染色法确认实验组发生NAFLD,采用免疫组织化学染色法及实时定量PCR法检测小肠及结肠黏膜中B细胞的分布和含量。采用流式细胞术分析MLN和PP中B细胞比例。采用酶联免疫法检测小肠内容物sIgA的水平。采用免疫磁珠法分选MLN中的B细胞进行体外培养,加入脂多糖(LPS)或抗CD40和抗IgM(BCR)刺激,采用细胞因子流式技术法检测培养液上清白介素-6、白介素-10及肿瘤坏死因子-α的水平。结果实验组和对照组小肠及结肠的B细胞散在分布于固有层中。与对照组相比,实验组MLN和PP中B细胞比例增多(P_MLN-B细胞=0.025,P_PP-B细胞=0.004)。实验组小肠内容物中sIgA的含量较对照组明显升高(P=0.042),并且其PP中IgA+B细胞比例升高(P=0.023)。体外实验显示,实验组MLN中的B细胞接受刺激后分泌IL-6增多(P_LPS<0.001,P_BCR=0.003)。结论 NAFLD小鼠PP及MLN中B细胞比例增多,不仅小肠内sIgA含量增多,并且其MLN中B细胞接受刺激后分泌促炎性IL-6增多。

关键词: 非酒精性脂肪性肝病, B细胞, 肠道, 肠肝轴, 炎症因子

Abstract: Objective To investigate the changes of intestinal B lymphocytes in mice with non-alcoholic fatty liver disease(NAFLD)induced by high fat diet. Methods A total of 12 male C57BL/6J mice were randomly divided into the experimental group and control group, with 6 in either group. The experimental group was fed with 45% high fat diet, while the control group with normal diet. After 16 weeks, the mice were killed to collect liver, small intestine mucosa, colon mucosa, mesenteric lymph nodes(MLN), Peyers patches(PP)and small intestine contents. Hepatic steatosis in experimental group was confirmed with hematoxylin and eosin(HE)staining. The distribution and expression of B cells in small intestine and colon mucosa were detected with immunohistochemical staining and real-time quantitative PCR, respectively. Proportion of B and T lymphocytes in the MLN and PP were analyzed with flow cytometry. The sIgA level in the small intestinal contents was detected with enzyme linked immunosorbent assay(ELISA). B lymphocytes were enriched with a magnetic-activated cell sorting method and cultured in vitro with or without the stimulation of lipopolysaccharide(LPS)or anti-CD40/IgM(BCR). The levels of IL-6, IL-10 and TNF-α in the cultural supernatants were monitored with cytometric bead arrays(CBA). Results B lymphocytes scattered throughout the lamina in both groups. The proportion of B lymphocytes was increased significantly in MLN and PP in experimental group than in control group(P_MLN-B=0.025, P_PP-B=0.004). The experimental group had higher level of sIgA in the small intestinal contents(P=0.042), which was in line with the higher proportion of IgA+ B lymphocytes in the PP(P=0.023). B lymphocytes in MLN in the experimental group secreted more IL-6 upon the stimulation of LPS or anti-CD40/IgM(BCR)(P_LPS<0.001, P_BCR=0.003)than in the control group. Conclusion Proportion of B lymphocytes in PP and MLN increased significantly in NAFLD mice. Small intestine secreted higher level of sIgA, and B lymphocytes in MLN produced more IL-6 upon stimulation in NAFLD mice.

Key words: Non-alcoholic fatty liver disease, B lymphocyte, Gut, Gut-liver axis, Inflammatory cytokine

中图分类号:

R575

张凤,吴哲,徐俊,刘玉兰. 6例非酒精性脂肪性肝病小鼠肠道B细胞的变化[J]. 山东大学学报 (医学版), 2022, 60(9): 67-73.

ZHANG Feng, WU Zhe, XU Jun, LIU Yulan. Changes of intestinal B lymphocytes in 6 mice with non-alcoholic fatty liver disease[J]. Journal of Shandong University (Health Sciences), 2022, 60(9): 67-73.

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