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Screening of novel secondary bile acid-producing intestinal bacteria and clarification of the mechanism of formation of colon-cancer promoter

新型产次级胆汁酸肠道菌的筛选及结肠癌启动子形成机制的阐明

基本信息

批准号：
16380054
负责人：
YOKOTA Atsushi
金额：
$ 10.11万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

Isolation of novel intestinal bacteria converting primary bile acids such as cholic acid (CA) or chenodeoxycholic acid (CDCA) into colon cancer-promoting secondary bile acids such as deoxycholic acid (DCA) or lithocholic acid (LCA), respectively, was conducted from fecal samples of humans.Total 619 strains were isolated from fecal samples in anaerobic chamber. Their activities for the formation of secondary bile acids including DCA and LCA were examined by culturing them in the medium containing CA or CDCA followed by the detection of the reaction products by TLC, HPLC and GC-MS analyses. As the results, eight strains were obtained as the secondary bile-acid producers. Determination of 16SrRNA gene sequence revealed that two of them producing DCA or LCA were Clostridium scindens and C.leptum, both of which are known producers for DCA or LCA. The other six strains were found to produce 7-oxo-lithocholic acid (3α-hydroxy-7-oxo-5β-cholanoic acid, 7-keto-LCA) from CDCA. These strains consisted of already know producers for 7-keto-LCA including Escherichia coli(three strains) and Bacteroides fragilis (two strains) and one previously not described strain, Bacteroides intestinalis. Therefore, this strain was selected as a novel producer and designated B. intestinalis AM-1. This strain produced 7-keto-DCA from CA. The same activities were also detected in the type strain of B. intestinalis JCM13265^TConversion reaction of strain AM-1 was compared with those of E. coliHB101 and B. fragilis JCM11019^T as the reference strains. It was revealed that strain AM-1 showed conversion yield of more than 90% with lower growth level than the other two strains, resulting in higher activity of conversion per cell than the others. However, we did not observe significant differences in the activities of 7α-hydroxysteroid dehydrogenase, a responsible enzyme producing 7-keto-LCA, in these three strains.

从人粪便中分离到能将胆酸（CA）、鹅去氧胆酸（CDCA）等初级胆汁酸分别转化为脱氧胆酸（DCA）、石胆酸（LCA）等次级胆汁酸的肠道细菌619株。通过在含有CA或CDCA的培养基中培养它们，然后通过TLC、HPLC和GC-MS分析检测反应产物来检测它们形成二级胆汁酸（包括DCA和LCA）的活性。结果，获得了8株次级胆汁酸产生菌。16 SrRNA基因序列测定结果表明，产DCA和LCA的菌株中有两株为Clostridium dendens和C.leptum，这两株菌都是已知的DCA和LCA的产生菌。其余6株菌株均能从CDCA中产生7-氧代石胆酸（3α-羟基-7-氧代-5 β-胆烷酸，7-酮基-LCA）。这些菌株包括已知的7-酮基-LCA生产者，包括大肠杆菌（3株）和脆弱拟杆菌（2株）以及一种以前未描述的菌株，拟杆菌。因此，选择该菌株作为新的生产菌并命名为B。美国AM-1该菌株从CA产生7-酮-DCA。在B的模式菌株中也检测到相同的活性。对菌株AM-1和大肠杆菌JCM 13265的转化反应进行了比较。coliHB 101和B. fragilis JCM 11019 ^T作为参考菌株。结果表明，AM-1菌株的转化率可达90%以上，但其生长水平较低，单位细胞转化率较高。然而，我们没有观察到7α-羟基类固醇脱氢酶的活性在这三个菌株中的显著差异，7 α-羟基类固醇脱氢酶是一种产生7-酮-LCA的酶。