基于高内涵分析的山药增强巨噬细胞吞噬活性及质量评价研究

Enhancement Study of Macrophage Phagocytic Activity and the Quality Evaluation of Dioscorea opposita Thunb. Based on the Analysis of High Content Screening

目的比较铁棍山药与非铁棍山药促进巨噬细胞吞噬活性的差异。方法采用CCK-8法,考察不同山药在不同浓度下的细胞毒性;在无毒剂量的基础上,建立高内涵结合荧光标记技术检测RAW264.7细胞吞噬GFP-E.coli细菌的体外生物模型,计算不同山药在不同浓度下的细胞吞噬率;采用"质反应平行线法"将测定的细胞吞噬率转化为生物效价,更好地检测铁棍山药与其他山药生物效价的区别。结果在0.695~5.56 mg(生药)·m L^(-1)内,11个批次的山药样品都不存在细胞毒性;高内涵分析细胞吞噬强度结果显示,在此浓度范围内,各种山药都能促进细胞吞噬率的增高;高内涵图像能够清晰地显示出RAW264.7细胞吞噬绿色荧光蛋白标记细菌的情况。生物效价结果显示,不同批次山药的免疫生物效价介于39.56~100 U·mg^(-1)之间,以铁棍山药的生物效价为高,且平均效价值明显高于非铁棍山药(P<0.05)。结论体外实验表明,铁棍山药及非铁棍山药都能不同程度地促进RAW264.7巨噬细胞的吞噬活性;且铁棍山药的生物效价明显高于非铁棍山药,这与普遍认为的"铁棍山药质量最佳"也相符。

OBJECTIVE To compare the differences of macrophages phagocytic activities between Dioscorea opposita Thunb.cv.Tiegun（abbreviated as TG）and Dioscorea opposita Thunb.but not Tiegun（abbreviated as NTG）.METHODS CCK-8 assay was used to test the cytotoxicity.On the basis of non-toxic dose,the high content screening（HCS）cell imaging analysis was applied to test the phagocytic ability of RAW264.7 cells engulfing GFP-E.coli in vitro,and the phagocytic rates between different kinds of samples at various concentrations were calculated.Furthermore,the biological potency was measured according to the qualitative response parallel method to better evaluate the difference of TG and NTG,by translating phagocytic rates into biological potency.RESULTS At the range of 0.695-5.56 mg（crude drug）·mL-1,all 11 batches samples have no toxicity.The results of HCS displayed that every sample could promote cell phagocytic activity in varying degrees at a certain concentration.RAW264.7 cells could engulf the GFP-E.coli,and the images of HCS reflected the situation of phagocytosis clearly.In addition,the results of biopotency showed that the biopotency value of different samples was between 39.56 to 100,and the average biopotency value of TG was significantly higher than NTG（P〈0.05）.CONCLUSION In vitro experiments showed that all the samples could promote the phagocytic activity of RAW264.7 cells at different degrees.And the average biopotency value of TG was significantly higher than NTG,which is consistent with the saying that “TG has a better quality”.