组蛋白去乙酰化酶3对恒定型自然杀伤性T细胞发育和功能的调控作用

doi:10.13481/j.1671-587x.20200201

吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (02): 205-213.doi: 10.13481/j.1671-587x.20200201

• 基础研究 • 下一篇

组蛋白去乙酰化酶3对恒定型自然杀伤性T细胞发育和功能的调控作用

郑全辉¹, 雷冰¹, 袁鸿儒¹, 马剑楠¹, 刘兆基¹, 张郡¹, 刘倚博¹, 张冰², 张爱红³, 郑爱华³, 张颖⁴, 李会婷⁴, 田枫⁴

1. 河北省慢性疾病重点实验室河北省唐山市慢性病基础研究重点实验室华北理工大学基础医学院免疫学系, 河北唐山 063210;
2. 河北医科大学第三医院骨外科, 河北石家庄 050051;
3. 河北省唐山市工人医院ICU, 河北唐山 063000;
4. 北京大学医学部实验动物科学部, 北京 100191

收稿日期:2019-06-20 发布日期:2020-04-07
通讯作者: 郑全辉,副教授,硕士研究生导师(Tel:0315-8805516,E-mail:1078209929@qq.com) E-mail:1078209929@qq.com
作者简介:郑全辉(1973-),男,河北省唐山市人,副教授,理学博士,主要从事T细胞发育与分化方面的研究。
基金资助:
国家自然科学基金面上项目资助课题（81373111）；河北省卫健委科研基金资助课题（20190105）；华北理工大学大学生创新创业训练计划资助课题（X2018193）；华北理工大学教育教学改革研究与实践项目资助课题（Y1750-21）

Regulatoy effects of HDAC3 on development and function of iNKT cells

ZHENG Quanhui¹, LEI Bing¹, YUAN Hongru¹, MA Jiannan¹, LIU Zhaoji¹, ZHANG Jun¹, LIU Yibo¹, ZHANG Bing², ZHANG Aihong³, ZHENG Aihua³, ZHANG Ying⁴, LI Huiting⁴, TIAN Feng⁴

1. Hebei Key Laboratory for Chronic Diseases, Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan 063210, China;
2. Department of Immounology, Department of Orthopedics, Third Hospital, Hebei Medical University, Shijiazhuang 050051, China;
3. Tangshan Worker's Hospital, Hebei Province, Tangshan 063000, China;
4. Department of Laboratory Animal Science, Health Science Center, Peking University, Beijing 100191, China

Received:2019-06-20 Published:2020-04-07

摘要/Abstract

摘要： 目的：探讨组蛋白去乙酰化酶3（HDAC3）缺失对恒定型自然杀伤性T细胞（iNKT）数量、发育表型和细胞因子产生功能的影响，确定HDAC3在iNKT细胞发育和功能发挥中的调控作用。方法：采用T细胞特异的hdac3基因敲除（HDAC3KO）及其野生型正常对照（WT）小鼠，每种小鼠各取3~5只，分离小鼠胸腺、脾脏、淋巴结和肝脏淋巴细胞，采用细胞表面抗体染色和流式细胞术检测HDAC3对iNKT细胞数量和发育表型的变化，实验至少重复3次。采用HDAC3KO小鼠及其同系B6.SJL小鼠，每种小鼠各取4~6只；提取以上2种小鼠骨髓细胞，混合后经尾静脉注入经γ射线照射的B6.SJL小鼠，以制备骨髓混合嵌合体模型，8周后流式细胞术检测不同骨髓来源iNKT细胞在胸腺的产生和发育，实验重复3次。选取HDAC3KO和WT小鼠，每种各取小鼠3~5只；采用iNKT细胞特异激活剂半乳糖神经酰胺腹腔注射4 h后，收集各组小鼠脾脏淋巴细胞和血清，分别采用细胞内染色、流式细胞术和酶联免疫吸附实验（ELISA）法检测HDAC3作用下iNKT细胞因子水平，实验至少重复3次。结果：与WT小鼠比较，HDAC3KO小鼠胸腺和外周免疫器官中iNKT细胞比例和数量均明显降低（P<0.05），且HDAC3KO小鼠胸腺iNKT细胞中CD44-NK1.1-和CD44+NK1.1-细胞比例明显升高（P<0.05），而CD44+NK1.1+、CD122+、CD69+和DX5+细胞比例明显降低（P<0.05）。骨髓混合嵌合体实验，与正常B6.SJL小鼠来源的骨髓细胞比较，来源于HDAC3KO小鼠的骨髓细胞产生的iNKT细胞数量明显减少（P<0.05），同时iNKT细胞中CD44+NK1.1+细胞比例也明显降低（P<0.05）。细胞因子胞内染色和ELISA法检测，与WT小鼠比较，HDAC3KO小鼠iNKT细胞和血清中IFN-γ和IL-4水平均明显降低（P<0.05）。结论：HDAC3缺失通过内源性机制导致iNKT细胞数量减少，细胞发育成熟受阻；同时HDAC3缺失导致iNKT细胞因子产生功能明显降低。提示HDAC3在iNKT细胞的发育和功能发挥中具有重要调控作用。

关键词: 组蛋白去乙酰化酶3, 恒定型自然杀伤性T细胞, 细胞发育, 细胞功能

Abstract: Objective: To investigate the effects of histone deacetylation enzyme 3 (HDAC3) on the quantity, developmental phenotypes and cytokine production function of the invariant nature killer T cells (iNKT), and to determine the regulatory effects of HDAC3 on the development and function of the iNKT cells. Methods: The T cell-specific hdac3 gene knockout (HDAC3KO) mice and their wild type normal control (WT) mice were used, and 3-5 mice were selected from every kind of mice in experiment. The lymphocytes from the thymus, spleen, lymph nodes and liver of the HDAC3KO and WT mice were separated, and the effects of HDAC3 on the number and developmental phenotypes of the iNKT cells were detected by cell surface antibody staining and flow cytometry methods. The experiment was repeated at least three times. The HDAC3KO mice and their homologous B6.SJL mice were used in the mixed hematopoietic chimerism experiment, and 4-6 mice were selected from every kind of mice. The bone marrow cells from HDAC3KO and B6.SJL mice were extracted, and after mixed together they were injected into the γ-ray irradiated B6.SJL mice via tail vein to prepare the bone marrow mixed chimeric models. Eight weeks later,the production and development of iNKT cells in the thymus from different bone marrows were detected by flow cytometry. The experiment was repeated for three times. The HDAC3KO and WT mice (3-5 mice from every kind of mice) were selected and introperitoneally injected by iNKT cell specific stimulator α-Galcer; 4 h later, the spleen lymphocytes and serum were collected,and the cytokine levels in the iNKT cells after treated with HDAC3 were detected by intracellular staining, flow cytometry and ELISA methods. The experiment was repeated at least 3 times. Results: Compared with the WT mice, the ratios and the number of iNKT cells in the thymus and peripheral immune organs of the HDAC3KO mice were significantly decreased(P<0.05);the ratios of CD44-NK1.1- and CD44+NK1.1-cells in the iNKT cells in thymus of the HDAC3KO mice were significantly increased(P<0.05); and the ratios of CD44+NK1.1+, CD122+, CD69+ and DX5+ cells were significantly decreased(P<0.05).The experiment of mixed bone marrow chimerism showed that the number of iNKT cells from the bone marrow cells of the HDAC3KO mice was significantly decreased compared with the bone marrow cells of the B6.SJL mice(P<0.05),and the ratio of CD44+NK1.1+ cells in the iNKT cells from the bone marrow cells of the HDAC3KO mice was significantly lower than that of the iNKT cells from the B6.SJL mice (P<0.05).The results of intracellular staining and ELISA showed that the levels of IFN-γ and IL-4 in the iNKT cells and serum of the HDAC3KO mice were significantly decreased compared with the WT mice(P<0.05). Conclusion: HDAC3 deficiency results in the decrease of the number of iNKT cells and the blocking of development and maturation of iNKT cells by an endogenous pathway.In addition, HDAC3 deficiency results in the significant reduction of cytokine production in the iNKT cells. All the results indicate that HDAC3 plays an important role in the regulation of iNKT cell development and function.

Key words: histone deacetylase 3, invariant nature killer T cells, cell development, cell function

中图分类号:

R392.11

郑全辉, 雷冰, 袁鸿儒, 马剑楠, 刘兆基, 张郡, 刘倚博, 张冰, 张爱红, 郑爱华, 张颖, 李会婷, 田枫. 组蛋白去乙酰化酶3对恒定型自然杀伤性T细胞发育和功能的调控作用[J]. 吉林大学学报(医学版), 2020, 46(02): 205-213.

ZHENG Quanhui, LEI Bing, YUAN Hongru, MA Jiannan, LIU Zhaoji, ZHANG Jun, LIU Yibo, ZHANG Bing, ZHANG Aihong, ZHENG Aihua, ZHANG Ying, LI Huiting, TIAN Feng. Regulatoy effects of HDAC3 on development and function of iNKT cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(02): 205-213.

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组蛋白去乙酰化酶3对恒定型自然杀伤性T细胞发育和功能的调控作用

Regulatoy effects of HDAC3 on development and function of iNKT cells

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