川芎嗪下调Colon26肿瘤细胞免疫抑制的体外研究

Study on the down-regulatory effects of Ligustrazine Hydrochloride on tumor-induced immunosuppression by Colon26 tumor cells in vitro

目的:体外研究川芎嗪对Colon26肿瘤细胞产生免疫抑制的影响。方法:获取经川芎嗪作用后再培养的Colon26及上清,以不经川芎嗪作用的同步培养细胞及上清作对照;分析川芎嗪作用后是否可下调Colon26肿瘤免疫抑制(MTT法检测NK活性和诱导转化,直接免疫荧光FACS法检测IL-2Rα、CD3ε+ζ+和CD3ε-ζ+表达),定量ELISA法测定上清中TGF-β1、VEGF、IL-4、IL-6和IL-10五种免疫抑制分子含量的变化,多元相关分析川芎嗪下调肿瘤免疫抑制与免疫抑制分子分泌变化之间的关系。结果:单纯Colon26培养上清中5种免疫抑制分子均可被测到,以TGF-β1含量最高,可显著抑制所测5项免疫功能。川芎嗪作用后,第一次传代再培养上清中TGF-β1、IL-6和IL-10含量,及5项免疫功能抑制均明显降低;第二次传代再培养上清中TGF-β1和IL-6含量,及转化抑制、CD3+ζ+和CD3-ζ+表达抑制均显著回升。相关分析显示,TGF-β1与除转化抑制以外的其他4项免疫功能抑制呈正相关,IL-6与转化抑制及CD3-ζ+表达抑制正相关;IL-10与NK杀伤抑制、IL-2Rα及CD3+ζ+表达抑制正相关。结论:川芎嗪作用Colon26肿瘤细胞后可明显下调其免疫抑制分子的分泌,影响其免疫抑制效应的发挥。通过下调肿瘤细胞分泌免疫抑制分子而阻碍肿瘤细胞产生免疫抑制,可能是川芎嗪的抗瘤效应机制之一。

Objective： To study the regulatory effects of Ligustrazine Hydrochloride （LHC）on tumor-induced immanosuppressian by Calon26 cells in vitro. Methods： Colon26 cells were cultured for 48 h in the presense of LHC and either the cell fraction or the cultural supernatants was collected, with the untreated Colon26 cells as control for the study. The down-regulating effects of LHC on tumor immunosuppressions （ including the suppressed NK killing and ConA induced transformation of murine spleen cells detected by MTF, and the reduced expression levels of IL-2Rα, CD3ε^＋ξ^＋ and CD3ε^-ξ^＋ detected by FCM） were determined. The concentrations of immunosuppressive cytokines, including TGF-β1, VEGF, IL-4, IL-6 and IL-10, in the supematants were analyzed by quantitative ELISA. The relationship among the down-regulatory effects of LHC on secretion immunosuppressive cytokines and tumor immunosuppressions were evaluated by multiple linear regression analysis. Results： All of the cytokines assayed were found in the supematant of Colon26 treated without LHC, in which TGF-β1 was the highest, and the significant inhibition of five immune functions mentioned above was showed. To the Colon26 treated by LHC, the concentrations of TGF-β1, IL- 6 and IL-10 in the first re-cultured supematant and its inhibition of five immunol functions decreased greatly. The concentrations of TGF-β1 and IL-6 in the second re-cultured supematant and its inhibitions of transformation, CD3^＋ξ^＋ and CD3^-ξ^＋ resumed highly. The positive correlations existed between TGF-β1 and inhibition of immunol functions except for transformation, between IL-6 and inhibition of transformation or CD3^-ξ^＋ ,between IL-10 and inhibition of NK killing or IL-2Rα or CD3^＋ξ^＋, respectively. Conclusion： LHC can exert down-regulate effects on Colon26 secretion of immunosuppressors and its tumor immunosuppression. Reducing tumor immunosuppression of Colon26 through decreasing its secretion of its should be one of anti-tumor mechanisms of LHC.