清开灵注射液对小鼠T细胞行为和DTH的影响

Influence of Qingkailing Injection on Behaviors of T Lymphocytes and DTH of Mouse

【目的】研究清开灵注射液(QKL)对小鼠T细胞体外活化、增殖、细胞周期和迟发型超敏反应(DTH)的影响,探讨其免疫调节作用及机制。【方法】不同浓度的QKL作用于小鼠淋巴细胞4h后,加入刀豆蛋白A(ConA)刺激,于不同时间点收获细胞,利用流式细胞仪检测T细胞的活化、增殖和细胞周期分布情况;将小鼠分为正常对照组、二硝基氟苯(DNFB)诱导的DTH模型组和QKL治疗组,通过分析小鼠脾脏指数和耳朵肿胀率评价QKL对DTH的影响。【结果】ConA刺激12h后,T细胞表面CD69表达率为63.1%±1.3%,QKL下调CD69的表达,加入浓度为1/1280、1/320和1/80的QKL后,CD69表达率依次降为56.2%±2.4%、51.9%±2.8%和39.1%±1.4%,与ConA对照相比,差异有统计学意义(P<0.05);各浓度QKL明显降低ConA刺激的细胞增殖指数,且呈一定的浓度依赖性。对细胞周期分布的影响表现为:QKL增加ConA激活的细胞中处于S期和G2/M期的细胞数目,降低处于G0/G1期的细胞数目;体内应用QKL治疗小鼠DTH的结果为:QKL治疗组小鼠脾脏指数为4.96±0.90,与模型组(脾脏指数为5.78±0.85)相比,P<0.05;发病耳的肿胀率为28.4%±4.2%,与模型组(肿胀率为42.4%±4.6%)相比,P<0.01。【结论】QKL是一种有效的负向免疫调节剂,能抑制体内外的免疫反应,抑制T细胞的活化和增殖可能是其作用机制之一。

[ Objective ] To study the influence of Qingkailing injection （QKL） on activation, proliferation, and distribution of cell cycle of mouse T lymphocytes in vitro and delayed type hypersensitivity （DTH） in vivo, and to explore the regulatory effects and machenisms of QKL on immune system. [ Methods ] After different doses of QKL were used for mouse lymphocytes for 4 hours, concanavalin A was added to activate lymphocytes. Then activation, proliferation and distribution of cell cycle of mouse T lymphocytes were determined by flow cytomety analysis. Mice were divided into three groups： normal group, DTH model group established by stimulation with DNFB, and QKL group. Analyzing of spleen index and ear sweeling rate was used to evaluate the effects of QKL on DTH. [Results] The expression of CD69 on T cells activated by ConA for 12 hours was 63.1% ± 1,3%. While QKL groups showed a downregulation of expression of CD69 on T cells, 1/1280, 1/320 and 1/80 of QKL reduced to 56.2% ± 2,4%, 51.9% ± 2.8% and 39.1% ± 1.4% ,respectively.There was significant difference of expression of CD69 between QKL and ConA（P 〈 0.05）. QKL decreased the value of proliferation index of T cells activated by ConA in a dose dependent way. The distribution of cell-cycle revealed that the number of cells activated by ConA in S and G2/M was increased but in G0/G1 was decreased. The value of spleen index and ear sweeling rate of mice with DTH was 5.78 ±0.85 and 42.4% ± 4.6%, and both of them were reduced after QKL was used to care. The value was 4.96 ±0.90 and 28.4% ± 4.2%, respectively. There were significant difference of spleen index （F = 5.29, P 〈 0.05） and ear sweeling rate （F = 4.58,P 〈 0.01 ） between DTH and QKL group. [Conclusion] QKL was an effective immunsupressive agents which inhibit immune response in vitro and in vivo. Inhibition of activation and proliferation of T lymphoeytes may be one of mechanisms.