荧光素酶报告系统检测大肠杆菌代谢产物调控STC-1细胞表达胆囊收缩素研究  

Detection of Escherichia coli metabolites by luciferase reporter system to regulate secretion of cholecystokinin by STC-1 cells

作　　者：余倩 张玉宇 张曾娣 朱伟云[1] 黄赞 YU Qian;ZHANG Yuyu;ZHANG Zengdi;ZHU Weiyun;HUANG Zan(National Center for International Research on Animal Gut Nutrition/Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health/College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China;Nanjing Children’s Hospital,Nanjing 210029,China)

机构地区：[1]南京农业大学国家动物消化道国际联合研究中心/江苏省消化道营养与动物健康重点实验室/动物科技学院,江苏南京210095 [2]南京市儿童医院,江苏南京210029

出　　处：《南京农业大学学报》2019年第6期1133-1140,共8页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31500944);江苏省自然科学基金项目(BK20150687,BK20170147)

摘　　要：[目的]本文旨在筛选出可调控肠道Ⅰ细胞表达胆囊收缩素(cholecystokinin,CCK)的大肠杆菌代谢物,建立可方便检测CCK表达的STC-1细胞模型。[方法]利用分子克隆技术构建含有CCK启动子调控荧光素酶的慢病毒载体,包装慢病毒并感染STC-1细胞,获得STC-1/CCK-Luc细胞系。用大肠杆菌基因敲除株培养液上清处理上述细胞,检测荧光素酶活性,确定可影响CCK表达的大肠杆菌基因及代谢产物。使用纯化的代谢产物处理STC-1细胞及肠道类器官,通过Western blot及免疫荧光法检测CCK表达水平的变化。[结果]酶切鉴定及DNA测序表明慢病毒载体构建成功,荧光素酶活性检测发现大肠杆菌ΔSapD株菌液上清可显著抑制荧光素酶的表达。敲除SapD基因可以使培养液中腐胺水平下降,而腐胺可以上调STC-1细胞及肠道类器官中CCK的表达。[结论]成功构建了能够体外检测CCK表达的细胞系,筛选出1个能够调控CCK表达的大肠杆菌突变体。[Objectives]The purpose of this study was to screen Escherichia coli metabolites that regulate intestinal Ⅰ cells expressing cholecystokinin(CCK),and an in vitro model for detecting CCK expression in STC-1 cells was establised. [Methods]Molecular cloning technique was used to construct the lentiviral vector containing CCK promoter-regulated luciferase,and STC-1/CCK-Luc cell line was obtained by packing lentivirus and infecting STC-1 cells. The supernatant of the E.coli mutants was treated the above cells,then the luciferase activity was measured to determine the genes and metabolites of E.coli which may affect CCK expression. STC-1 cells and intestinal organoid were treated with purified E.coli metabolites,and the expression level of CCK was confirmed by Western blot and immunofluorescence. [Results]The lentiviral vector was proved to be successfully conducted by enzyme digestion analysis and DNA sequencing. The luciferase activity assay showed that the supernatant of E.coli mutant ΔSapD could significantly inhibit the expression of luciferase. SapD knockout gene could decrease the level of putrescine in the culture medium,but putrescine could up-regulate the expression of CCK in STC-1 cells and intestinal organoid. [Conclusions]In this experiment,the cell line that could detect CCK expression in vitro was constructed,and an E.coli mutant which could regulate CCK expression was successfully screened. This laid a foundation for the further screening experiments.

关 键 词：慢病毒载体 胆囊收缩素(CCK) 荧光素酶 大肠杆菌敲除株 

分 类 号：R593.22[医药卫生—内科学]