神经递质γ-氨基丁酸对小鼠B淋巴细胞分化和抗体生成的影响  

Effect of γ-aminobutyric acid on differentiation and antibody production of B cells of mice

作　　者：宋广平 吴敏[1] 梁丽云 李慧艳[1] 张学敏[1] 李平 张宇程 SONG Guangping;WU Min;LIANG Liyun;LI Huiyan;ZHANG Xuemin;LI Ping;ZHANG Yucheng(National Center of Biomedical Analysis,Beijing 100850,China;Department of Nephrology,the First Medical Center of Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]国家生物医学分析中心 [2]中国人民解放军总医院第一医学中心肾脏病医学部

出　　处：《中国药理学与毒理学杂志》2023年第9期671-679,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81790252)。

摘　　要：目的 探索神经递质γ-氨基丁酸(GABA)对B细胞分化和抗体生成的影响,为提高疫苗有效性提供新的靶点和策略。方法 流式分选经绵羊红细胞免疫小鼠的脾幼稚B细胞、生发中心B细胞和浆细胞,荧光定量PCR检测GABA A1型受体(Gabra1)和Gabra2、GABA B1型受体(Gabbr1)和Gabbr2、溶质运载蛋白6A家族成员1(Slc6a1)、Slc6a11、Slc6a12和Slc6a13 mRNA表达。建立B细胞体外分化体系,分离小鼠脾B细胞与NB-21饲养层细胞共培养,流式细胞术检测GABA 0.2,1.0和2.0 mmol·L^(-1)对浆细胞分化百分比的影响。每只小鼠ip给予100μg乙酰化4-羟基-3硝基苯基偶联钥孔血蓝蛋白(NP-KLH)和0.1 mL铝佐剂构建经典的抗原免疫小鼠模型,分为免疫+PBS和免疫+GABA组。正常对照组注射0.1 mL双蒸水和0.1 mL铝佐剂。免疫+PBS和免疫+GABA组分别ip给予PBS和GABA 20 mg·kg^(-1),每天1次,共12 d。第13天流式细胞术分析小鼠脾生发中心B细胞、总浆细胞和IgG_(1)^(+)浆细胞百分比,ELISA检测血清中抗原特异性抗体水平;第27天后流式细胞术分析骨髓中抗原特异性浆细胞百分比。结果 绵羊红细胞免疫小鼠幼稚B细胞、生发中心B细胞和浆细胞均表达Gabbr1。在B细胞体外分化过程中,与细胞对照组相比,GABA1.0和2.0 mmol·L^(-1)处理均显著促进幼稚B细胞向CD138^(+)浆细胞分化(P<0.01)。与正常对照组相比,免疫+PBS组小鼠脾生发中心B细胞百分比增加(P<0.01),总浆细胞和IgG_(1)^(+)浆细胞百分比增加(P<0.05,P<0.01);骨髓中抗原特异性浆细胞百分比增加(P<0.01);血清抗原特异性抗体水平升高(P<0.01)。与免疫+PBS组相比,免疫+GABA组总浆细胞(P<0.01)和IgG_(1)^(+)浆细胞百分比(P<0.05)增加,骨髓中抗原特异性浆细胞百分比增加(P<0.05),血清抗原特异性抗体水平升高(P<0.01)。结论 神经递质GABA可促进浆细胞分化和高亲和力抗体生成,其在调控B细胞分化过程中发挥重要作用。OBJECTIVE To explore the effect of neurotransmitterγ-aminobutyric acid(GABA)on differentiation and antibody production of B cells,and to recommend a new potential strategy to enhance humoral immune response after vaccination.METHODS Naïve B cells,germinal center(GC)B cells and splenic plasma cells(SPPCs)were sorted from the spleen of mice on the 7^(th) day after immunization with sheep red blood cells(SRBCs),and RT-qPCR was used to detect the mRNA expressions of GABA A receptor,subunitα1(Gabra1),Gabra2,GABA B receptor 1(Gabbr1),Gabbr2,solute carrier family 6 member 1(Slc6a1),Slc6a11,Slc6a12 and Slc6a13.A plasma cell differentiation system in vitro was constructed.Purified naïve B cells were co-cultured with NB-21 feeder cells in the presence of mouse interleukin-4,and the production of CD138^(+)SPPCs was analyzed by flow cytometry with or without the treatment with GABA 0.2,1.0 and 2.0 mmol·L^(-1).A classical antigen-immunized mouse model was prepared by ip injecting 4-hydroxy-3-nitrophenylacetyl conjugated to keyhole limpet hemocyanin(NP-KLH)100μg per C57BL/6 wild-type mouse mixed with 0.1 mL alum.The normal control group was injected with 0.1 mL double distillated water mixed with 0.1 mL alum.The NP-KLH immunized mice were ip injected with PBS or GABA 20 mg·kg^(-1),once a day,for 12 d after immunization before the percentages of GC B cells,SPPCs and IgG_(1)^(+)SPPCs were tested by flow cytometry.The titer of antigen-specific IgG in serum was measured by ELISA on the 13rd day,and the abundance of antigen-specific bone-marrow plasma cells was tested by flow cytometry on the 27^(th) day after immunization.RESULTS Gabbr1 mRNA was highly expressed in naïve B cells,GC B cells and SPPCs in SRBC immunized mice.In the in vitro plasma cell differentiation system,naïve B cells could differentiate into CD138+SPPCs constantly.Compared with the cell control group,the percentage of SPPCs was increased after GABA 1.0 and 2.0 mmol·L^(-1) treatment(P<0.01).Compared with the normal control group,the percentage of GC B c

关 键 词：Γ-氨基丁酸 B细胞 生发中心 浆细胞 高亲和力抗体 

分 类 号：R963[医药卫生—微生物与生化药学] R967[医药卫生—药理学]