奶中两种共轭亚油酸异构体的特异性合成机制的体外研究

Conjugated linoleic acid (CLA) is a collective term for geometric and positional isomers of linoleic acid with conjugated double bonds. They have been found to be responsible for many biological properities that related to human health. c9,t11-CLA has the anticarcinogenic activity, and t10,c12-CLA seems to be specifically responsible for reducing body fat and enhancement of energy metabolism. CLA occurs naturally in the fat of milk in a relatively low level. There are several CLA isomers in milk, such as c9,t11-CLA and t10,c12-CLA. These CLA isomers are mainly biosynthesized by some bacteria in the rumen of cow and goat. .Ultraviolet spectral scan and capillary electrophoresis methods will be used to screen and identify bacteria which can biosynthesize c9,t11-CLA and t10,c12-CLA, respectively, from goat's rumen bacteria. The isolated bacteria will be identified to species by classical methods and 16S rRNA analysis. The gene of linoleic acid isomerase, which converts linoleic acid to CLA, will be amplified by polymerase chain reaction (PCR) and analyzed the gene polymorphism by restriction fragment length polymorphism (RFLP). The reason of isomer specific biosynthesis can be clarified with the find of the gene polymorphism of linoleic acid isomerase. The fermentation and metabolism of these CLA biosynthesis bacteria will be tested in an artifical rumen in vitro. Denaturing gradient gel electrophoresis (DGGE) will be used to analyse the changes of the biodiversity in the rumen. Thus the metabolism of c9,t11-CLA and t10,c12-CLA can be disclosed in the level of rumen microecology. The mechanism of the isomer specific biosynthesis of c9,t11-CLA and t10,c12-CLA in milk will be clearly explained with the carrying out of this project.

共轭亚油酸（CLA）具有多种重要的生理活性，其中c9,t11-CLA具有很强的抗癌作用，t10,c12-CLA具有减肥和防治2型糖尿病的功能。在自然界，CLA主要存在于牛羊的奶中，通常包含c9,t11-CLA和t10,c12-CLA等异构体，牛羊瘤胃中的一些细菌参与了不同CLA异构体的生物合成。本项目拟通过UV扫描、毛细管电泳等方法从山羊瘤胃细菌中筛选分离出能合成c9,t11-CLA和t10,c12-CLA的菌株，通过生化特性和基因学分析，了解这些菌株的属种和同源性关系。再通过PCR-RFLP技术，分析菌株中亚油酸异构酶基的多态性，从亚油酸异构酶的基因水平阐明奶中c9,t11-CLA和t10,c12-CLA的特异性合成机理。最后研究CLA合成菌株在人工模拟瘤胃环境中的生长代谢，通过PCR-DGGE技术，从瘤胃微生态的群体水平进一步阐明c9,t11-CLA和t10,c12-CLA的代谢机理。

共轭亚油酸（CLA）具有多种重要的生理活性，其中c9,t11-CLA具有很强的抗癌作用，t10,c12-CLA具有减肥和防治2型糖尿病的功能。在自然界，CLA主要存在于牛羊的奶中，通常包含c9,t11-CLA和t10,c12-CLA等异构体，牛羊瘤胃中的一些细菌参与了不同CLA异构体的生物合成。本项目通过UV扫描、毛细管电泳等方法从山羊瘤胃细菌中筛选分离出能合成c9,t11-CLA和t10,c12-CLA的菌株，通过生化特性和基因测序，鉴定菌株的属种。建立部分重叠引物PCR的染色体步移技术，对菌株中亚油酸异构酶基进行克隆表达，对四株菌的亚油酸异构酶基因测序结果进行比对，发现两株植物乳杆菌的相似性高达99.5%，两株干酪乳杆菌相似性高达99.8%，而植物乳杆菌与干酪乳杆菌的相似性仅为40.9%，从亚油酸异构酶的基因多态性阐明了奶中c9,t11-CLA和t10,c12-CLA的特异性合成机理。将分离到的能生物合成CLA的乳酸菌与德氏乳杆菌保加利亚亚种和嗜热链球菌进行混合菌株发酵，利用DGGE分析菌群变化与CLA合成关系，从群体水平进一步阐明c9,t11-CLA和t10,c12-CLA的代谢机理。采用人乳腺癌MCF-7细胞为体外模型，通过MTT法、荧光染色及流式细胞术比较了3种CLA异构体(c9,t11-CLA，t10,c12-CLA，t9,t11-CLA）及2种反式脂肪酸（反油酸、异油酸）对乳腺癌细胞MCF-7细胞凋亡的影响。运用RT-PCR和Western-blot方法研究3种CLA单体激活PPARγ的能力与其调控凋亡相关基因和蛋白表达之间的相互关系，从基因和蛋白水平阐明CLA单体诱导人乳腺癌细胞MCF-7凋亡的可能机制。