黄芪皂苷Ⅱ调控CD45分子介导CD4^+T细胞活化效应的研究  

Study on ASI Ⅱ triggered CD4^+T cells activation through regulating CD45 molecular

作　　者：赵榕慧 顾瑛媛[2] 张霞 普晓佳 郑喜 刘蓓 万春平 Zhao Ronghui;Gu Yingyuan;Zhang Xia;Pu Xiaojia;Zheng Xi;Liu Bei;Wan Chunping(Department of Pharmacy,Characteristic Medical Center of PAP,Tianjing 300162,China;Department of Pharmacy,Changzhou Traditional Chinese Medicine Hospital,Changzhou 213003,China;School of Pharmacy,Yunnan University of Traditional Chinese Medicine,Kunming 650021,China;The NO.1 Affiliated Hospital of Yunnan University of Traditional Chinese Medicine,Kunming 650021,China)

机构地区：[1]武警部队特色医学中心药剂科,天津300162 [2]江苏省常州市中医医院药剂科,213003 [3]云南中医药大学中药学院,昆明650021 [4]云南中医药大学第一附属医院中心实验室,昆明650021

出　　处：《国际中医中药杂志》2019年第5期481-486,共6页International Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81460624);云南省自然科学基金(2015FB199);云南省科技厅-云南中医学院-应用基础研究联合专项(2017FF117-041);常州市科技计划应用基础研究项目(CJ20179041).

摘　　要：目的研究黄芪皂苷Ⅱ调控CD45分子介导CD4^+T细胞活化效应的机制。方法将细胞按随机数字表法分为阴性对照组、刺激对照组、黄芪皂苷Ⅱ组、CD45抑制剂组、联合组。除阴性对照组外,其余各组采用CD3/CD28抗体构建CD4^+T细胞活化效应细胞模型;黄芪皂苷Ⅱ组加入10 nmol/L黄芪皂苷Ⅱ进行干预,CD45抑制剂组加入CD45PTPase抑制剂0.8 μmol/L进行干预,联合组加入10 nmol/L黄芪皂苷Ⅱ和CD45PTPase抑制剂0.8 μmol/L进行干预。干预36 h后,采用Ki67胞内染色法测定活化CD4^+T淋巴细胞增殖功能;ELISA法检测CD4^+T细胞活化模型IFN-γ、IL-4、IL-2水平;流式细胞术检测T淋巴细胞表面标志CD44、CD25和胞内细胞因子IFN-γ表达。结果与刺激对照组比较,黄芪皂苷Ⅱ组T细胞CD4^+Ki67^+[(5.37±0.92)%比(1.19±0.23)%]、CD4^+CD25^+[(50.23±4.65)%比(15.89±1.13)%]、CD4^+CD44^+[(33.16±6.08)%比(15.36±1.45)%]、CD4^+IFN-γ^+[(1.42±0.44)%比(0.38±0.06)%]阳性比例增加(P<0.01),IFN-γ、IL-4、IL-2水平升高(P<0.01)。CD45抑制剂可抑制黄芪皂苷Ⅱ的促增殖和细胞因子产生作用,阻断黄芪皂苷Ⅱ促T淋巴细胞表面标志CD44、CD25表达上调。结论黄芪皂苷Ⅱ可能通过激活CD45蛋白酪氨酸磷酸酯酶介导Th1淋巴细胞克隆性增殖、活化及Th1细胞因子的产生。Objective This paper was designed to reveal the new mechanism on ASI Ⅱ triggered CD4^+T cells activation via regulating CD45 molecular and provide a basis for the theoretical foundation of antitumor immunotherapy of Astragalus.Methods The CD4^+T cells were randomly divided into negative group,stimulated control group,ASIⅡgroup,CD45 inhibitor group,and the combination of ASIⅡ and CD45 inhibitor group.Besides negative group,the cells from other groups were activated by anti-CD3/CD28 antibody.ASIⅡgroup was treated with 10 nmol/L ASIⅡ,CD45 inhibitor group was treated with 0.8 μmol/L CD45 inhibitor,and the combination group were treated with 10 nmol/L ASIⅡ and 0.8 μmol/L CD45 inhibitor.After 36h culture,the proliferation of CD4^+T cells was detected by Ki-67 intracellular staining assay.Cytokine production of Th1 and Th2 were examined ELISA method.The proportion of surface marker (CD44 and CD25) and Th1 intracellular cytokines (IFN-γ) were detected by flow cytometry.Results Compared with stimulated group,Astragaloside Ⅱ group in CD4^+Ki67^+T positive proportion (5.37%± 0.92% vs.1.19%± 0.23%),in CD4^+CD25^+ positive proportion (50.23%± 4.65 % vs.15.89%± 1.13%),in CD4^+CD44^+ positive proportion (33.16%± 6.08% vs.15.36%± 1.45%),in CD4^+IFN-γ^+ positive proportion (1.42%± 0.44 % vs.0.38%± 0.06%) were significntly increased.And the secretion of IFN-γ,IL-4 and IL-2 in ASI Ⅱ group were higher than stimulated group.The anti-mouse CD45 Ab treatment markedly blocked the proliferation and Th1 cytokines production which induced by ASI Ⅱ.Furthermore,the anti-mouse CD45 Ab treatment significantly decreased the expression of surface marker (CD44 and CD25).Conclusions Activating CD45 protein tyrosine phosphatase may be involved in ASI Ⅱ triggered CD4^+T cells activation.This study will provide a basis for antitumor immunotherapy of Astragalus.

关 键 词：黄芪皂苷 CD45分子 CD45抑制剂 细胞增殖 干扰素-Γ 小鼠 

分 类 号：R285[医药卫生—中药学]