摘要：

目的: 探究糖酵解与舍格伦综合征（Sjögren′s syndrome, SS）疾病发展的关系。方法: 利用磁激活细胞分选（magnetic-activated cell sorting，MACS）法提取CD4⁺T细胞，实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）法检测糖酵解节点因子的转录情况。选用雌性NOD/Ltj小鼠作为SS模型鼠，XF96代谢分析仪检测CD4⁺T细胞的糖酵解速率。采用饮水法给予NOD/Ltj小鼠糖酵解抑制2-脱氧葡萄糖（2-deoxy-D-glucose, 2-DG）。戊巴比妥钠腹腔注射麻醉小鼠进行刺激性唾液流率检测；提取小鼠下颌下腺并进行HE染色和病理性评分；多重免疫荧光组织化学（multiplex immunohistochemistry，mIHC）法对小鼠的免疫细胞进行标记； RT-qPCR检测NOD/Ltj小鼠中Th1与Th17代表基因干扰素-γ（interferon-γ, IFN-γ）和白细胞介素-17（interleukin-17, IL-17）水平变化。结果: SS患者的CD4⁺T细胞中，HK2、PKM和MYC等糖酵解因子表达上调。同时，SS疾病小鼠CD4⁺T细胞糖酵解潜能也高于对照组小鼠。抑制糖酵解可有效恢复小鼠唾液流率、减少淋巴细胞尤其是CD4⁺T细胞的浸润，抑制Th1和Th17相关因子在病变唾液腺的表达。结论: SS发病时，CD4⁺T细胞存在高水平的糖酵解，并部分参与了疾病进程。

关键词: 糖酵解, CD4⁺T细胞, 舍格伦综合征, 自身免疫疾病

Abstract:

Objects: This study intends to explore the relationship between glycolysis and the development of Sjögren′s syndrome(SS）. Methods: CD4⁺T cells were extracted by magnetic-activated cell sorting (MACS), and the expression of glycolytic genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Female NOD/Ltj mice were utilized as the SS mice model in this study. XF96 metabolic analyzer was used to detect the glycolytic rate in CD4⁺T cells of NOD/Ltj mice. The glycolytic inhibitor, 2-deoxy-D-glucose(2-DG）, was dissolved in drinking water to the treatment of NOD/Ltj mice. Mice were anesthetized by intraperitoneal injection with pentobarbital sodium for salivary flow administration. The submandibular glands were extracted and underwent HE staining and pathological evaluation. Multiplex immunohistochemistry (mIHC) was used to label the immune cells of mice. RT-qPCR was utilized to determine the level change of Th1 and Th17 representative genes(interferon-γ, IFN-γ and interleukin-17, IL-17) in NOD/Ltj mice. Results: In CD4⁺T cells of SS patients, the expression of glycolysis-related genes including HK2, PKM and MYC was up-regulated. Compared with control mice, the glycolytic capacity of CD4⁺T cells in NOD/Ltj mice was also elevated. In vivo inhibition of glycolysis could effectively restore the salivary flow rate, reduce the infiltration of lymphocytes, especially CD4⁺T cells, and inhibit the expression of Th1 and Th17 related factors in diseased salivary glands. Conclusion: In this study, we determined that the glycolytic level was abnormally up-regulated in CD4⁺T cells, which might partly participate in the development of SS.

Key words: glycolysis, CD4⁺T cell, Sjögren′s syndrome, autoimmune disease