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目的:体外研究黄芪制剂对结直肠癌免疫抑制的逆转,初步分析其作用靶分子。方法:获取体外经黄芪作用后再传代的结直肠癌细胞(Colon26)培养上清,以不经黄芪作用的同步培养上清作对照,定量ELISA法测定上清中TGFβ-1、VEGF、IL-4、IL-6、IL-10、PGE2等六种免疫抑制分子的含量,分析其对MTT法测定的小鼠脾细胞NK杀伤和诱导转化以及流式细胞计数分析的IL-2Rα、CD3ε+ζ+和CD3ε-ζ+表达5项免疫功能的影响,多元相关分析黄芪下调Colon26分泌免疫抑制分子与免疫抑制逆转作用之间的关系。结果:结直肠癌细胞培养上清中可检测到免疫抑制分子,以TGFβ-1含量最高;对5项免疫功能指标均有显著抑制作用。黄芪作用后,其第一次再培养上清中TGFβ-1和IL-10含量及对除IL-2Rα以外的4项免疫功能指标的抑制均明显降低,IL-6、PGE2含量显著升高;与第一次再培养上清相比,第二次再培养上清中TGFβ-1含量及对CD3ε+ζ+表达的抑制继续降低,IL-6含量降低至无中药处理的对照水平,对IL-2Rα表达的抑制开始降低,IL-10含量显著回升。相关分析显示,诱导转化、CD3ε+ζ+、CD3ε-ζ+表达及NK杀伤功能抑制率与TGFβ-1含量呈正相关,与PGE2含量呈负相关;VEGF、IL-4、IL-6和IL-10与5项免疫功能抑制不相关。结论:黄芪可通过下调结直肠癌细胞分泌TGFβ-1等免疫抑制分子,影响其免疫抑制效应的发挥,这可能是黄芪的抗瘤新机制之一。
OBJECTIVE: To study the reversal of immunosuppression of Astragalus membranaceus in colorectal cancer in vitro and to analyze its target molecule in vitro. Methods: The supernatant of colorectal cancer cells (Colon26) cultured in vitro after the administration of Astragalus membranaceus was obtained. The supernatant of the culture medium without astragalus was used as a control. The levels of TGFβ-1, VEGF and IL- 4, IL-6, IL-10, PGE2 and other six kinds of immunosuppressive molecules, analyzed the MTT assay of mouse splenocytes NK killing and induction of transformation and flow cytometry analysis of IL-2Rα, CD3ε + ζ + And CD3ε-ζ + expression of five immune function, multivariate correlation analysis of Astragalus down Colon26 secretion of immunosuppressive molecules and immunosuppressive reversal effect between. Results: Immunosuppressive molecules could be detected in the culture supernatant of colorectal cancer cells, with the highest content of TGFβ-1, and significant inhibitory effect on all five immune function indexes. After the astragalus membranaceus was administered, the content of TGFβ-1 and IL-10 in the first re-culture supernatant and the inhibition of four immune functions except IL-2Rα were significantly decreased, while the contents of IL-6 and PGE2 were significantly increased. The level of TGFβ-1 and the inhibition of CD3ε + ζ + expression in the second re-culture supernatant continued to decrease compared with the first re-culture supernatant, the level of IL-6 decreased to the control level without traditional Chinese medicine treatment, The inhibition of -2Rα expression began to decrease, IL-10 content significantly increased. Correlation analysis showed that the expression of CD3ε + ζ +, CD3ε-ζ + and NK cytotoxicity were positively correlated with the content of TGFβ-1 and negatively correlated with the content of PGE2. The expressions of VEGF, IL-4, IL-6 and IL- 10 and 5 immune function is not related to inhibition. Conclusion: Astragalus can exert its immunosuppressive effect by down-regulating the secretion of TGF-β1 and other immunosuppressive molecules in colorectal cancer cells, which may be one of the new anti-tumor mechanisms of astragalus.