青蒿琥酯、苦参素对Colon26肿瘤细胞免疫抑制的影响

为了研究青蒿琥酯(ART)、苦参素(MOX)是否可逆转Colon26肿瘤细胞对免疫细胞功能的抑制,分别制备经这两种中药作用后的Colon26再培养上清,同时以不经中药作用的Colon26相应上清作对照,研究对小鼠脾细胞功能的影响,包括以MTT法测定NK杀伤和ConA诱导转化,以及流式细胞术分析的CD4+及CD8+表达的影响,定量ELISA测定TGF-β1、VEGF、IL-4、IL-6和IL-10五种免疫抑制物质的含量,并分析这两种中药下调Colon26分泌免疫抑制物质与上清免疫抑制之间的关系。结果显示,不经中药作用的Colon26培养上清中五种免疫抑制物质均可被测到,以TGF-β1含量最高;该上清对所测的小鼠脾细胞免疫功能均有显著抑制。ART作用后的Colon26,其第一次再培养上清的TGF-β1、VEGF、IL-10含量,及对小鼠脾细胞ConA诱导转化、CD4+CD8-表达及NK细胞杀伤的抑制均明显降低;其第二次再培养上清的TGF-β1和IL-10含量,及对ConA诱导转化和CD4+CD8-表达的抑制继续保持较低,VEGF含量及NK细胞杀伤的抑制恢复至对照水平。MOX作用后的Colon26,其第一次再培养上清的TGF-β1和IL-10含量,及对小鼠脾细胞ConA诱导转化的抑制均明显降低;其第二次再培养上清TGF-β1和IL-10含量继续降低,对ConA诱导转化的抑制恢复至对照水平。相关分析显示,ART及MOX作用后,Colon26培养上清对小鼠脾细胞转化的抑制均与TGF-β1含量正相关;ART作用后,Co-lon26培养上清对NK细胞杀伤的抑制与IL-10含量正相关。以上结果表明,ART和MOX可通过抑制免疫抑制物质的分泌而逆转Colon26肿瘤细胞的免疫抑制作用,这可能是ART和MOX的抗瘤机制之一。

Influence of Artesunate and Oxymatrine on the immunosuppressive effect of mouse colon-rectal carcinoma cell line Colon 26

In order to determine whether Artestinate(ART) and Oxymatrine(MOX) could reverse the immunosuppressive effect of mouse colon-rectal carcinoma cell line Colon26,recultured supernatants of the Colon26 cell line cultures were collected after treated with either ART or MOX respectively,with the corresponding supernatants without treatment of any herbs as control.The effects of different supernatants on the immune functions of mouse splenocytes including the killing activity of NK cells and ConA induced transformation,detected by MTT assay,the expressions of CD4+and CD8+ cells assayed by flow cytometry and the levels of 5 immunosuppressive agents,such as TGF-β1,VEGF,IL-4,IL-6 and IL-10 determined by quantitative ELISA,were investigated accordingly.Meanwhile,the relationship between the down-regulating effects of ART and MOX on the Colon26 cell line to secrte immunosuppressive molecules and create immunosuppression was evaluated.It was demonstrated that the 5 immunosuppressive agents all could be detected in the supernatants of the Colon26 cell cultures without treatment of any herbs with highest level of TGF-β1.The immune functions of mouse splenocytes were remarkebly inhibited by the supernatants of Colon26 cell cultures.In case of the Colon26 cell cultures treated with ART,the levels of TGF-β1,VEGF and IL-10 in the first recultured supernatants and its inhibition of immune function except the expressions of CD4+and CD8+cells were greatly decreased;and the levels of TGF-β1 and IL-10 in the second recultured supernatants remained decreasing,and its inhibition on the ConA induced transformation as well as the expressions of CD4+ CD8-cells still kept decreasing.In case of the Colon26 cells treated with MOX,the levels of TGF-β1 and IL-10 in the first recultured supernatants and its inhibition of transformation also decreased greatly,while the levels of TGF-β1 and IL-10 in the second recultured supernantants kept decresing,and its inhibition of transformation increased was just the same as control.The positive correlations existed between the level of TGF-β1 and inhibition of transformation after treatment with ART and MOX,as well as between level of IL-10 and inhibition of NK killing after treatment with ART alone.It is concluded that the results in the present study demonstrate the reversing effect of the immunosuppression induced by the Colon26 cells through the decrease of the secreted immunosuppressors,and this effect may be the new antitumor mechanism afforded by ART and MOX.