双荧光探针分子两步标记益生菌在肠道原位活性分析研究  

In situ activity analysis of two-step labeling probiotics with double fluorescent probes in intestinal tract

作　　者：陈荧 李琳 陈国平 李佩 谢智勇 廖琼峰[1] CHEN Ying;LI Lin;CHEN Guo-ping;LI Pei;XIE Zhi-yong;LIAO Qiong-feng(School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;School of Pharmaceutical Sciences(Shenzhen),Sun Yat-sen University,Guangzhou 510006,China)

机构地区：[1]广州中医药大学中药学院,广东广州510006 [2]中山大学药学院(深圳),广东广州510006

出　　处：《药学学报》2022年第5期1402-1410,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(82074142,82174104,U1903211,U1803123)。

摘　　要：有效补充益生菌可有益肠道健康,但是益生菌在肠道原位活性的分析却鲜有报道。本研究通过将异硫氰酸荧光素(FITC)和5(6)-羧基四甲基罗丹明琥珀酰亚胺酯[5(6)-TAMRA-SE]分别与D-赖氨酸进行偶联,获得了两种颜色的荧光D-氨基酸(fluorescent D-amino acids, FDAAs)探针:绿色探针(fluorescein-D-lysine, FDL)和红色探针(TAMRA-D-lysine, TDL),并尝试对嗜酸乳杆菌(Lactobacillus acidophilus, LA)、干酪乳杆菌(Lactobacillus casei, LC)和韦荣球菌(Veillonella atypica, VA) 3种益生菌进行了体外标记。随后将FDAAs应用到小鼠肠道菌群标记,建立方法考察了3种常用益生菌口服定植存活情况。所有动物实验方案经由广州中医药大学动物伦理委员会审查通过。结果表明,合成的两种FDAAs可以无毒且完全对3种益生菌进行体外标记;通过FDAAs两步标记口服益生菌可知,LA存活率较高为92.30%±1.67%, LC和VA的存活率相近,分别为84.13%±4.06%、82.27%±2.43%。本研究可通过快速比较不同益生菌在体内定植存活率的变化,为益生菌在肠道原位定植活性提供理论支撑,同时对临床益生菌合理用药具有指导作用。Effective supplementation of probiotics can be beneficial to intestinal health, but in situ analysis of probiotics activity has rarely been reported. In this study, by coupling fluorescein 5-isothiocyanate(FITC) and 5(6)-carboxytetramethylrhodamine N-succinimidyl ester(5(6)-TAMRA-SE) with D-lysine, two fluorescent D-amino acids(FDAAs) probes were obtained: green probe(fluorescein-D-lysine, FDL) and red probe(TAMRA-D-lysine,TDL). Then, we tried to label the three kinds of probiotics, Lactobacillus acidophilus(LA), Lactobacillus casei(LC) and Veillonella atypica(VA) in vitro. FDAAs was applied to the labeling of intestinal flora in mice, and a method was established to investigate the oral survival rate of three commonly used probiotics. All animal experiments were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine. The results show that the two synthetic FDAAs can be non-toxic and 100% for the in vitro labeling of the three probiotics.Known by FDAAs two-step labeling of oral probiotics, the high survival rate of LA was 92.30% ± 1.67%. The survival rates of VA and LC are similar, 84.13% ± 4.06% and 82.27% ± 2.43%, respectively. This study can quickly compare the changes of colonization survival rate of different probiotics in vivo, provide theoretical support for the in situ colonization activity of probiotics in the intestine, and guide the rational drug use of clinical probiotics.

关 键 词：荧光D-氨基酸 益生菌 肠道菌群 荧光成像 体内定植 

分 类 号：R917[医药卫生—药物分析学]