基于巨噬细胞吞噬模型的阿胶品质生物评价方法构建
|
摘要
目的:建立基于小鼠巨噬细胞系RAW 264.7吞噬模型的阿胶生物活性评价方法。方法:采用CCK-8法考察细胞密度、孵育时间以及阿胶对巨噬细胞增殖活性的影响,确定合适的阿胶给药浓度。采用高内涵结合荧光成像技术,通过考察其感染复数(multiplicity of infection,MOI,MOI=细菌数/细胞数)、给药剂量等影响因素,分析阿胶影响巨噬细胞的整体吞噬率及单个细胞吞噬指数。结果:确定最佳检测方法,即给药时间为24 h、MOI值为200倍、细菌刺激时间为2 h。与Control组相比,在阿胶质量浓度为0.125～2.0 mg·mL~(-1)时,可促进巨噬细胞的增殖,在0.5 mg·mL~(-1)时阿胶促进巨噬细胞的增殖能力达到最强;在阿胶质量浓度为0.25～1.0 mg·mL~(-1)时,对单个细胞的吞噬能力没有明显作用,但是对巨噬细胞的整体吞噬率具有剂量依赖的促进作用,并在0.25 mg·mL~(-1)时阿胶促进巨噬细胞吞噬率的能力达到最强。结论:本研究建立了以巨噬细胞吞噬绿色荧光标记的大肠杆菌(Green Fluorescent Protein-Escherichia coli,GFP-E.coli)为生物模型的高内涵荧光成像筛选技术的阿胶品质评价生物方法,可用于对不同品质阿胶的检测。
Objective:To establish a method for evaluating the biological activity of Asini Corii Colla(ACC)based on the macrophage phagocytosis model.Methods:The murine RAW 264.7 macrophage cell line and GFP-Escherichia coli were used to construct the model.The cell concentration and incubation time,as well as the appropriate concentration of ACC,were determined by CCK-8 method.The macrophage phagocytosis rate and single cell phagocytosis index affected by CAA were also analyzed by investigating the multiplicity of infection(MOI)and the dosage of ACC with high-content screening(HCS)technology.Results:To investigate the effect of CCA on macrophage cells,the cells were cultured with ACC solution for 24 hours,and then 200-fold GFP-Escherichia coli(MOI=200)were added and stimulated for 2 hours before HCS analyzing.Compared with the control group,the proliferation of macrophages could be promoted by ACC at an increased concentration from 0.125 to 2.0 mg·mL~(-1),and the strongest proliferation effect was shown with 0.5 mg·mL~(-1).Furthermore,there was no obvious promotion on the phagocytic ability in single cell for the CCA concentration of 0.25-1.0 mg·mL~(-1),but it had a dose-response effect on the holistic phagocytic rate of macrophages and had the strongest ability to promote the phagocytosis rate of macrophages at 0.25 mg·mL~(-1).Conclusion:This study established an in vitro biological method for evaluating the quality of ACC with high-content screening technology,which has the potential to detect the quality and consistence of different ACC.
Objective:To establish a method for evaluating the biological activity of Asini Corii Colla(ACC)based on the macrophage phagocytosis model.Methods:The murine RAW 264.7 macrophage cell line and GFP-Escherichia coli were used to construct the model.The cell concentration and incubation time,as well as the appropriate concentration of ACC,were determined by CCK-8 method.The macrophage phagocytosis rate and single cell phagocytosis index affected by CAA were also analyzed by investigating the multiplicity of infection(MOI)and the dosage of ACC with high-content screening(HCS)technology.Results:To investigate the effect of CCA on macrophage cells,the cells were cultured with ACC solution for 24 hours,and then 200-fold GFP-Escherichia coli(MOI=200)were added and stimulated for 2 hours before HCS analyzing.Compared with the control group,the proliferation of macrophages could be promoted by ACC at an increased concentration from 0.125 to 2.0 mg·mL~(-1),and the strongest proliferation effect was shown with 0.5 mg·mL~(-1).Furthermore,there was no obvious promotion on the phagocytic ability in single cell for the CCA concentration of 0.25-1.0 mg·mL~(-1),but it had a dose-response effect on the holistic phagocytic rate of macrophages and had the strongest ability to promote the phagocytosis rate of macrophages at 0.25 mg·mL~(-1).Conclusion:This study established an in vitro biological method for evaluating the quality of ACC with high-content screening technology,which has the potential to detect the quality and consistence of different ACC.
引文
[1]张贵锋,刘涛,王前,等.中药阿胶的质量控制方法研究[J].药物生物技术,2009,16(3):250-254.
[2]EDITOR GROUP of TMR.Is Colla Corii Asini just boiled donkey skin?[J/OL].Trad Med Res,2018,3(3).[2018-08-09].https://www.tmrjournals.com/tmr/EN/Y2018/V3/I3/0.
[3]WANG D,RU W,XU Y,et al.Chemical constituents and bioactivities of Colla corii asini[J].Drug Discov Ther,2014,8(5):201-207.
[4]国家药典委员会.中华人民共和国药典:一部[M].北京:中国医药科技出版社,2015:189
[5]LI X,SHI F,GONG L,et al.Species-specific identification of collagen components in Colla corii asini using a nano-liquid chromatography tandem mass spectrometry proteomics approach[J].Int J Nanomedicine,2017,12:4443-4454.
[6]葛重宇,庞慧,李楠,等.18家企业阿胶中氨基酸的含量分析与比较研究[J].中国药房,2017,28(1):122-126.
[7]伊娜,杨铧,武勇,等.阿胶药理药效研究进展[J].世界最新医学信息文摘,2017,17(54):12-15.
[8]周旋,张海珠,周海燕,等.基于高内涵筛选的铜皮石斛促进巨噬细胞吞噬作用评价方法的建立[J].药学学报,2017,52(5):737-744.
[9]杨文华,史志龙,柏兆方,等.铁棍山药促进巨噬细胞吞噬细菌的高内涵分析方法研究[J].中草药,2017,48(8):1604-1610.
[10]周玉环,刘树迎,平苏宁,等.Hoechst 33342与DAPI标记细胞核对胞内活性氧检测效果的比较[J].中国动脉硬化杂志,2014,22(1):13-17.
[11]刘锡娟,丁慧荣,张宏.用DAPI和Hoechst33342染色法检测DNA的流式细胞方法探讨[J].北京大学学报(医学版),2010,42(4):480-484.
[12]曹雨诞,颜晓静,张丽,等.醋制降低京大戟对大鼠小肠隐窝上皮细胞IEC-6毒性研究[J].中国中药杂志,2014,39(6):1069-1074.
[13]张珣,李冰,田守生,等.阿胶对小鼠免疫功能的影响[J].食品工业科技,2011,32(11):400-402.
[14]陈东亚,陆罗定,俞萍,等.流式细胞术检测小鼠腹腔巨噬细胞吞噬能力的方法学探讨[J].中国免疫学杂志,2014,30(8):1074-1077.
[15]邹同祥,张翼冠,张海港.青藤碱对巨噬细胞吞噬能力的影响及机制探讨[J].中药药理与临床,2008,24(6):15-17.
[16]张晊瑞,刘素素,张芬芬,等.小鼠巨噬细胞体外吞噬功能方法比较[J].中国卫生检验杂志,2017,23(12):41-43.
[17]郑筱祥,杨勇,叶剑锋,等.东阿阿胶的升白作用及机制研究[J].中国现代应用药学,2005,22(2):102-105.
[18]庞萌萌,李敏,田晨颖,等.阿胶酶解液相对分子质量分布及其补血升白作用[J].中国实验方剂学杂志,2017,23(12):21-25.
[19]葛帅,汤纳平,付立杰,等.毒理学研究的高内涵筛选及其在药物肝毒性研究中的应用[J].中国药理学与毒理学杂志,2017,31(6):689-695.
[20]李朋彦,李春雨,陆小华,等.基于类器官3D培养和高内涵成像的药物肝毒性评价模型研究[J].药学学报,2017,52(7):43-50.
[2]EDITOR GROUP of TMR.Is Colla Corii Asini just boiled donkey skin?[J/OL].Trad Med Res,2018,3(3).[2018-08-09].https://www.tmrjournals.com/tmr/EN/Y2018/V3/I3/0.
[3]WANG D,RU W,XU Y,et al.Chemical constituents and bioactivities of Colla corii asini[J].Drug Discov Ther,2014,8(5):201-207.
[4]国家药典委员会.中华人民共和国药典:一部[M].北京:中国医药科技出版社,2015:189
[5]LI X,SHI F,GONG L,et al.Species-specific identification of collagen components in Colla corii asini using a nano-liquid chromatography tandem mass spectrometry proteomics approach[J].Int J Nanomedicine,2017,12:4443-4454.
[6]葛重宇,庞慧,李楠,等.18家企业阿胶中氨基酸的含量分析与比较研究[J].中国药房,2017,28(1):122-126.
[7]伊娜,杨铧,武勇,等.阿胶药理药效研究进展[J].世界最新医学信息文摘,2017,17(54):12-15.
[8]周旋,张海珠,周海燕,等.基于高内涵筛选的铜皮石斛促进巨噬细胞吞噬作用评价方法的建立[J].药学学报,2017,52(5):737-744.
[9]杨文华,史志龙,柏兆方,等.铁棍山药促进巨噬细胞吞噬细菌的高内涵分析方法研究[J].中草药,2017,48(8):1604-1610.
[10]周玉环,刘树迎,平苏宁,等.Hoechst 33342与DAPI标记细胞核对胞内活性氧检测效果的比较[J].中国动脉硬化杂志,2014,22(1):13-17.
[11]刘锡娟,丁慧荣,张宏.用DAPI和Hoechst33342染色法检测DNA的流式细胞方法探讨[J].北京大学学报(医学版),2010,42(4):480-484.
[12]曹雨诞,颜晓静,张丽,等.醋制降低京大戟对大鼠小肠隐窝上皮细胞IEC-6毒性研究[J].中国中药杂志,2014,39(6):1069-1074.
[13]张珣,李冰,田守生,等.阿胶对小鼠免疫功能的影响[J].食品工业科技,2011,32(11):400-402.
[14]陈东亚,陆罗定,俞萍,等.流式细胞术检测小鼠腹腔巨噬细胞吞噬能力的方法学探讨[J].中国免疫学杂志,2014,30(8):1074-1077.
[15]邹同祥,张翼冠,张海港.青藤碱对巨噬细胞吞噬能力的影响及机制探讨[J].中药药理与临床,2008,24(6):15-17.
[16]张晊瑞,刘素素,张芬芬,等.小鼠巨噬细胞体外吞噬功能方法比较[J].中国卫生检验杂志,2017,23(12):41-43.
[17]郑筱祥,杨勇,叶剑锋,等.东阿阿胶的升白作用及机制研究[J].中国现代应用药学,2005,22(2):102-105.
[18]庞萌萌,李敏,田晨颖,等.阿胶酶解液相对分子质量分布及其补血升白作用[J].中国实验方剂学杂志,2017,23(12):21-25.
[19]葛帅,汤纳平,付立杰,等.毒理学研究的高内涵筛选及其在药物肝毒性研究中的应用[J].中国药理学与毒理学杂志,2017,31(6):689-695.
[20]李朋彦,李春雨,陆小华,等.基于类器官3D培养和高内涵成像的药物肝毒性评价模型研究[J].药学学报,2017,52(7):43-50.