【作者】 秦君；

【导师】 高学军；

【作者基本信息】 东北农业大学 ， 动物生物化学与分子生物学， 2007， 硕士

【摘要】 我国奶牛业普遍存在产乳量低、奶牛发病率高等问题。长期以来，人们主要用抗生素及一些化学合成物作为饲料添加剂来防治奶牛疾病，提高奶牛生产性能。但这些添加剂具有毒副作用大、残留高，易产生抗、耐药性和致畸、致癌、致突变等弊端，严重危害了人体健康。中药以其源自天然，具有毒副作用小、无残留、不易产生耐药性的优点，已广泛应用于我国奶牛业。许多具有补益气血、通经活络、调和脾胃的中草药及其复方制剂均具有显著的催乳作用。如党参、白术、黄芪、王不留行、当归、川芎、丝瓜络、路路通、甘草等中药含有多种营养物质和生物活性物质，能够补充机体营养，提高动物食欲，促进动物消化吸收，增强机体代谢，改善机体的造血机能和血液循环，以及杀灭微生物，增强机体免疫力，防治疾病发生，从而提高奶牛泌乳性能。传统中药王不留行广泛应用于我国奶牛饲料添加剂中，具有行血通经，催生下乳，消肿敛疮等功效。其主要成分为多种皂甙类、黄酮甙类、香豆素类、生物碱类等成分。本实验采用溶剂萃取法与酸碱法对王不留行三类主要成分(皂甙类、黄酮甙类及香豆素类)进行提取，并用化学方法及薄层层析方法予以鉴定。将三种成分分别以不同浓度添加入细胞培养液，一方面，采用MTT法检测这三类成分对体外培养的小白鼠乳腺上皮细胞增殖的作用，并运用激光共聚焦显微技术对乳腺上皮细胞中细胞周期蛋白cyclin D1的表达进行鉴定；另一方面，运用荧光定量RT-PCR方法对细胞中β-酪蛋白的表达进行检测，并用激光共聚焦显微技术对细胞信号通路中磷酸化stat5因子的表达进行鉴定，最后通过动物实验加以辅证。实验结果表明，王不留行中的黄酮甙类成分对小鼠乳腺上皮细胞的增殖有明显的促进作用(P＜0.05)，并随其浓度(100～200mg／L)的增加，其促进作用增强；皂甙类成分则对小鼠乳腺上皮细胞的增殖有明显的抑制作用，并随浓度(50～200mg／L)的增加而抑制作用增强；香豆素类成分(20～200mg／L)则对小鼠乳腺上皮细胞的增殖无明显作用(P＞0.05)。荧光定量RT-PCR结果表明，黄酮甙类成分可促进乳腺上皮细胞中β-酪蛋白基因的表达(P＜0.05)，皂甙类和香豆素类成分对β-酪蛋白基因的表达无明显作用(P＞0.05)。免疫荧光定量结果显示黄酮甙类成分可促进细胞周期蛋白cyclin D1的表达，表明黄酮甙类成分是通过激活cyclin D1的表达而促进乳腺上皮细胞增殖的。同时，黄酮甙类成分能够促进磷酸化的stat5因子的表达(P＜0.05)，表明黄酮甙类成分是通过激活Jak-stat5信号转导通路调控β-酪蛋白基因表达的。动物实验结果黄酮甙类成分能够提高小鼠泌乳量(P＜0.05)也证明了这一点。更多还原

【Abstract】 Disadvantages such as low lactating, high incidence of diseases are existing in milk cowbreeding of our country. For a long time, people use antibiotic and some chemic compound as feedadditive to prevent and cure diseases of milk cows, so as to improve lactating of milk cows.Nevertheless, these additives have great side-effect, a lot of remainder. It is easy to bring drug-fastand cause abnormality, cancer and mutation, which jeopardize body health. Chinese traditionalmedicine is derived from nature. It has a lot of advantages such as low side-effect, little remainder,and hardly bring drug-fast, tt has applied abroad in milk cow breeding of our country. ManyChinese traditional medicine ant its compound which have the function of unblocking blood vessel,recruiting vigor and accommodating spleen and stomach can improve lactating remarkably. SuchChinese traditional medicine as liquorice, Semen Vaccariae, and etc contain a lot of nutritioussubstance and biologically active substance. They can supply organism with nutrition, improvecreatural appetite, digesting and assimilating, metabolism, blood circulating of organism. They alsocan kill microorganism, improve immunity of organism, prevent and cure diseases, accordinglyimprove lactating capability of milk cows.The Chinese traditional medicine Semen Vaccariae applied widely in milk cows breeding ofour country as additive. It has the function of unblocking blood vessel, inducing lactating,detumescence an dwindling scar and so on. Its main components are saponin, flavonoid glycosideand coumarin.This test studied both the extracting methods of three main components from SemenVaccariae—saponin, flavonoid glycoside and coumarin and the identification using chemicmethods and chromatogram methods. Add these components to cell cultural medium with severalconcentration. Then detect their effects on multiplication of mouse mammary epithelial cells usingthe mothod MTT and the expression of beta-casein of mouse mammary epithelial cells usingReal-time PCR. Furthermore, this test put up pilot study of their mechanism. We detected theexpression of cyclin D1 and phosphatic stat5 using laser scanning confocal microscope. Finally, the function of the effective component was validated through animal experiment.The result indicated that the component of saponin could restrain the multiplication of mousemammary epithelial cells (P＜0.05), and its function was stronger as its concentration(50～200mg/L) increasing. The component of flavonoid glycoside could promote themultiplication of mouse mammary epithelial cells (P＜0.05), and its function was stronger as itsconcentration (100～200mg/L) increasing. Whereas the component of coumarin had no influenceon the multiplication of mouse mammary epithelial cells (P＞0.05).The component of flavonoid glycoside was able to promote the expression of beta-casein (P＜0.05). The component of saponin and coumarin had no influence on the expression ofbeta-casein (P＞0.05). The component of flavonoid glycoside could promote the expression ofcyclin D1 and phosphatic stat5 (P＜0.05). This indicated that the component of flavonid glycosidecould promote the multiplication of mouse mammary epithelial cells by activating the expressionof cyclin D1 and regulate expression of beta-casein by activating Jak-stat5. Animal experimentindicated that the component of flavonoid glycoside could increase lactating of mouse (P＜0.05).更多还原