铁棍山药促进巨噬细胞吞噬细菌的高内涵分析方法研究  

High content screening for promoting effect of Dioscorea opposita on macrophage phagocytosis

作　　者：杨文华[1,2] 史志龙[3] 柏兆方[3] 王伽伯[3] 张玉君[1] 樊冬鹤 张孟捷 李朝峰[1] 戚姝娅 肖小河[3] 曹俊岭[4,5] 黄璐琦[5] 

机构地区：[1]北京中医药大学中药学院,北京100102 [2]中国中医科学院中药研究所,北京100700 [3]解放军第三0二医院全军中医药研究所,北京100039 [4]北京中医药大学东直门医院,北京100700 [5]中国中医科学院中药资源中心,北京100700

出　　处：《中草药》2017年第8期1604-1610,共7页Chinese Traditional and Herbal Drugs

基　　金：中央本级重大增减支项目:名贵中药资源可持续利用能力建设(2060302);国家自然科学基金项目:“道地指数”的构建及其在中药材品质评控中的应用研究(81274026);国家公益性行业专项:20种道地药材优良品种质量生物评价技术研究(201507002)

摘　　要：目的建立巨噬细胞系RAW 264.7对绿色荧光蛋白标记的大肠杆菌(GFP-E.coli)吞噬活性的高内涵分析方法,评价铁棍山药在体外对巨噬细胞增殖和吞噬活性的促进作用。方法采用CCK-8法测定铁棍山药作用于RAW 264.7巨噬细胞的增殖活性。采用高内涵技术,建立铁棍山药调节巨噬细胞RAW 264.7吞噬细菌活性的评价方法。结果通过考察给药时间、细菌加入量、细菌刺激时间等因素,确定所建立方法的最佳方案为给药12 h、细菌加入量为50倍、细菌刺激时间1.5 h;方法重复性考察RSD值为2.31%。与不加药组相比,在铁棍山药质量浓度为0.156~1.25 mg/m L都能促进巨噬细胞的增殖,以及单个细胞的平均吞噬率;在1.25 mg/m L时铁棍山药促进巨噬细胞的增殖及吞噬活性的能力最强。结论首次建立铁棍山药促进巨噬细胞吞噬活性的高内涵分析方法,该方法具有准确、直观、高通量等优势;铁棍山药在体外既能促进巨噬细胞增殖,又能提高单个细胞的吞噬能力,为进一步分析山药等补益类中药的免疫活性及增强免疫机制的研究提供新思路和新方法。Objective To establish a high content screening（HCS） method by testing the phagocytic function of RAW 264.7, and to evaluate the effect of Dioscorea opposita on cell proliferation and phagocytosis in vitro. Methods CCK-8 was used to measure the proliferation of RAW 264.7, and HCS was helpful to determine the ability of RAW 264.7 to engulf GFP-E.coli. Results The best method was established by examining the time of administration, the amount of bacteria added, and the time of bacterial stimulation： administration time of 12 h, 50 times of bacteria, and stimulation time of 1.5 h. Then, the repeatability of this method was tested, and the RSD value was 2.31%. Compared with the group without drugs, at the concentration of 0.156—1.25 mg/m L, the water extract of D. opposita could promote the proliferation, and improve the average phagocytosis rate of every single RAW264.7 cell. And the samples had the strongest effect on proliferation and phagocytosis when the concentration was 1.25 mg/m L. Conclusion An HCS method is established and firstly used in D. opposita. HCS has the advantage of accurate, intuitive, and high throughput. D. opposita can not only promote cell proliferation, but also improve the phagocytic ability of each single RAW264.7 in vitro. A new method is provided for further study on immune activity and on the mechanism of enhancing the immune activity that some tonic herbs may have, such as D. opposita.

关 键 词：铁棍山药 高内涵分析 巨噬细胞 吞噬作用 细胞增殖 大肠杆菌 

分 类 号：R285.5[医药卫生—中药学]