天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (12): 1233-1238.doi: 10.11958/20220771

• 细胞与分子生物学 •    下一篇

犀角地黄汤合方对DC激活ITP患者T细胞增殖分化的影响

杨武霞1(), 刘宝山2, 吴玉红3, 李润杰2, 王梦晓1, 王爱迪2,△()   

  1. 1 天津医科大学研究生院（邮编300041）
    2 天津医科大学总医院中医科
    3 血液科
  • 收稿日期:2022-05-21 修回日期:2022-07-16 出版日期:2022-12-15 发布日期:2022-12-30
  • 通讯作者: 王爱迪 E-mail:1782578835@qq.com;wangaidi@tmu.edu.cn
  • 作者简介:杨武霞（1995），女，硕士在读，主要从事中西医结合血液病方面研究。E-mail：1782578835@qq.com
  • 基金资助:
    国家自然科学基金资助项目(82004334);天津市卫生健康委员会，天津市中医重点专科建设项目

The effect of Xijiao Dihuang combined prescription on T cell differentiation induced by platelet antigen loaded DC in ITP patients

YANG Wuxia1(), LIU Baoshan2, WU Yuhong3, LI Runjie2, WANG Mengxiao1, WANG Aidi2,△()   

  1. 1 Graduate School of Tianjin Medical University, Tianjin 300041, China
    2 Department of Traditional Chinese Medicine
    3 Department of Hematology, Tianjin Medical University General Hospital
  • Received:2022-05-21 Revised:2022-07-16 Published:2022-12-15 Online:2022-12-30
  • Contact: WANG Aidi E-mail:1782578835@qq.com;wangaidi@tmu.edu.cn

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摘要：

目的 观察犀角地黄汤合方含药血清对树突状细胞（DC）激活T细胞增殖分化的影响及其对免疫性血小板减少症（ITP）的作用机制。方法 将10只雄性SD大鼠随机分为含药血清组和空白血清组，每组5只，分别使用犀角地黄汤合方和蒸馏水连续灌胃3 d，腹主动脉取血制备含药血清和空白血清。分选15例健康志愿者及8例ITP患者CD4+ T细胞，将荷载血小板抗原的DC与CD4+ T细胞共培养，采用随机数字表法分为对照组，模型组，犀角地黄汤合方低、中、高剂量组。对照组为荷载血小板抗原的DC与健康志愿者CD4+ T细胞，模型组和犀角地黄汤合方低、中、高剂量组为荷载血小板抗原的DC与ITP患者的CD4+ T细胞，其中对照组和模型组加入大鼠空白血清，犀角地黄汤合方低、中、高剂量组分别加入体积分数为5%、10%及20%大鼠含药血清。流式细胞术（FCM）检测CD4+ T细胞的增殖情况，各组调节性T细胞（Treg）和效应性T细胞（Teff）的比例以及CD4+ T表面程序性死亡受体-1（PD-1）的表达量；酶联免疫吸附试验检测各组细胞上清液中促炎因子白细胞介素（IL）-2、干扰素-γ（IFN-γ）、IL-17以及抑炎因子转化因子-β（TGF-β）、IL-10的表达量。结果 与对照组比较，模型组CD4+ T细胞增殖百分比、Teff细胞比例以及促炎因子IL-2、IFN-γ和IL-17升高（P<0.05），Treg细胞比例、CD4+ T细胞表面PD-1表达量以及抑炎因子TGF-β、IL-10水平降低（P<0.05）；与模型组比较，犀角地黄汤合方低、中、高剂量组CD4+ T细胞增殖百分比、Teff细胞比例依次降低，Treg细胞比例、CD4+ T细胞表面PD-1表达量和抑炎因子IL-10、TGF-β升高（P<0.05），促炎因子IL-2的量降低（P<0.05）；与模型组相比，犀角地黄汤合方中、高剂量组IFN-γ和IL-17降低（P<0.05）。结论 犀角地黄汤合方尤其是高剂量组含药血清能够在体外调节ITP患者CD4+ T细胞的过度增殖分化及细胞因子的分泌，这可能是犀角地黄汤合方治疗ITP机制之一。

关键词: 血小板减少, 树突状细胞, CD4阳性T淋巴细胞, 疾病模型，动物, 犀角地黄汤合方

Abstract:

Objective To observe the effect of drug-containing serum of Xijiao Dihuang combined prescription (XJDHCP) on the proliferation and differentiation of dendritic cell (DC)-activated T cells and its mechanism on immune thrombocytopenia (ITP). Methods Ten male SD rats were randomly divided into the drug-containing serum group and the blank serum group, with 5 rats in each group. Rats were gavaged with XJDHCF and distilled water for 3 days. Blood-extraction from the abdominal aorta was prepared for drug-containing serum and blank serum. Fifteen healthy volunteers and 8 ITP patients with CD4+ T cells were selected, and the DCs loaded with platelet antigens were co-cultured with CD4+ T cells and randomly divided into the normal control group, the model group and the low, medium and high (5%, 10% and 20%) dose groups of XJDHCF. The normal control group consisted of platelet antigen-loaded DCs and CD4+ T cells from healthy volunteers, and the model group and the low, medium and high dose groups of XJDHCF were platelet antigen-loaded DCs and CD4+ T cells from ITP patients, in which the normal control group and the model group were added with rat blank serum, and the low, medium and high dose groups of XJDHCF were added with 5%, 10% and 20% drug-containing serum. The proliferation of CD4+ T cells, the ratio of regulatory T cells (Treg), effector T cells (Teff) and the expression of programmed death receptor-1 (PD-1) on the surface of CD4+ T were detected by flow cytometry (FCM). Enzyme-linked immunosorbent assay was used to detect the expression levels of pro-inflammatory factors IL-2, IFN-γ, IL-17 and anti-inflammatory factors TGF-β and IL-10 in the supernatant of each group. Results Compared with the control group, the percentage of CD4+ T cell proliferation, the proportion of Teff cells and amounts of pro-inflammatory factors IL-2, IFN-γ and IL-17 were significantly higher in the model group (P<0.05), and the proportion of Treg cells, the amount of PD-1 expression on the surface of CD4+ T cells and amounts of anti-inflammatory factors TGF-β and IL-10 were greatly lower in the model group (P<0.05). Compared with the model group, the percentage of CD4+ T cell proliferation and the proportion of Teff cells decreased successively in the low, medium and high dose groups of XJDHCF (P<0.05), and the amount of PD-1 expression on the surface of CD4+ T cells and the amounts of TGF-β, an inflammatory factor were increased (P<0.05). The amount of IFN-γ was significantly lower in the high dose group of XJDHCF (P<0.05). Compared with the model group, the amounts of IFN-γ and IL-17 were significantly lower in the middle and high dose groups of XJDHCF (P<0.05). Conclusion XJDHCF can regulate the excessive proliferation and differentiation of CD4+ T cells and the secretion of cytokines in ITP patients in vitro, which may be one of the mechanisms of XJDHCF in the treatment of ITP.

Key words: thrombocytopenia, dendritic cells, CD4-positive T-lymphocytes, disease models, animal, Xijiao Dihuang combined prescription

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