两种荧光探针标记鹿茸提取物的稳定性和生物活性比较研究  

Comparison of the Stability and Biological Activity of Two Fluorescent Labeling Pilose Anlter Extracts

作　　者：李超华 王建农[1] 孙娅楠[2] 王毅[2] LI Chaohua;WANG Jiannong;SUN Yanan;WANG Yi(Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院西苑医院,北京100091 [2]中国中医科学院医学实验中心,北京100700

出　　处：《激光生物学报》2019年第1期46-53,共8页Acta Laser Biology Sinica

基　　金：国家自然科学基金面上项目(81373884);国家自然科学基金青年项目(81603285)

摘　　要：比较两种荧光探针异硫氰酸荧光素FITC和Cy5标记鹿茸提取物PAEs的最佳方法。用FITC和Cy5分别标记PAEs得到标记物FITC-PAEs和Cy5-PAEs;用SDS-PAGE凝胶电泳检测并比较两种荧光染料标记蛋白分子量的差异;用酶标仪检测在特定波长处OD值计算其标记率;用PC12细胞验证两种荧光染料的生物安全性;将荧光标记物分别与人工胃液和人工肠液共孵育,观察它们在其中的稳定性。结果表明,两种荧光染料标记PAEs蛋白分子量并无明显差异,但FITC-PAEs荧光强度稍强。通过计算标记率表明两种荧光染料均可充分标记PAEs,但Cy5价格昂贵。相比于FITC-PAEs,Cy5-PAEs在人工胃液和人工肠液中稳定性较差。因此,我们选择标记率高,在人工胃液和人工肠液中较为稳定且对机体无毒副作用,价格合理的FITC荧光染料标记PAEs并用于后续实验研究。这为建立蛋白类中药大分子的活性示踪方法,开发口服蛋白类药物奠定基础。To optimize the fluorescence labeling method of pilose anlter extracts(PAEs),PAEs was labeled by two fluorescent probes,fluorescein isothiocyanate(FITC)and Cyanine 5(Cy5).Molecular weight of the two fluorescent dyes-labeled proteins was identified by sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE).The cytotoxicity of the two fluorescent probes was detected in PC12 cells.To observe their stability in gastric fluids and intestinal fluids,FITC and Cy5 labeled PAEs were dissolved in artificial gastric fluids and artificial intestinal fluids.There was no significant difference between the molecular weight of FITC labeled PAEs and Cy5 labeled PAEs.The fluorescence intensity of FITC labeled PAEs was slightly stronger than that of Cy5 labeled PAEs.Compared with FITC labeled PAEs,Cy5 labeled PAEs was not stable in artificial gastric fluids or artificial intestinal fluids.In conclusion,FITC is well suited to label PAEs for further identification or in vivo study.

关 键 词：鹿茸提取物 荧光标记技术 异硫氰酸荧光素 Cy5 

分 类 号：Q51[生物学—生物化学]