【作者】 孙灵芝；

【导师】 沈赞明；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 为了探讨山羊瓣胃上皮是否表达NHE mRNA及日粮营养对其表达的影响，本试验利用Genebank中牛的同种亚型NHE1、NHE3 mRNA序列分别设计引物(NHE1、NHE3)，利用RT-PCR试验方法检测NHE1、NHE3 mRNA在山羊瓣胃上皮的存在；并进一步分析不同日粮营养水平、日粮因子半胱胺盐酸盐对山羊瘤胃、瓣胃上皮NHE mRNA表达的影响，为NHE在反刍动物上的研究提供基础资料。1 NHE1、NHE3 mRNA在山羊瓣胃上皮的表达采取山羊瓣胃(瓣页)组织样品，利用GeneBank中牛NHE3 mRNA(AJ131764.1)和NHE1 mRNA(U49432)的序列，分别设计出NHE3和NHE1引物序列，通过RT-PCR的方法，首次检测到NHE1、NHE3 mRNA在山羊瓣胃上皮组织中表达。NHE1、NHE3序列片段测定显示其与牛同种亚型之间的同源性分别达到91％、95％。2不同日粮营养水平对山羊瘤胃、瓣胃上皮NHE1、NHE3 mRNA表达影响的体内研究将8头断奶山羊随机分为试验和对照两组，在日粮中添加100g／d和0g／d精料，试验期为42天。RT-PCR结果显示，提高日粮营养对瘤胃、瓣胃上皮NHE1、NHE3 mRNA表达有一定促进作用(0.05＜P＜0.1)。3 IGF-I、丁酸对山羊瘤胃上皮NHE3 mRNA表达影响的体外研究采集山羊瘤胃后腹盲囊(caudal blind sac)靠近后沟处组织，分离上皮，经过IGF-I(25ng／mL)、丁酸钠(5mmol／L)处理24h，收集组织样。RT-PCR结果显示，IGF-I、丁酸显著促进瘤胃上皮NHE3 mRNA表达(P＜0.05)。4日粮因子半胱胺盐酸盐对瘤胃、瓣胃上皮NHE3 mRNA表达影响的体内研究将8头2～3月龄断奶山羊随机分为试验和对照两组，在日粮中分别添加45mg／kg．BW．d和0 mg／kg．BW．d半胱胺盐酸盐(CT2000)，试验期为35天。RT-PCR结果显示，CT2000显著促进瘤胃、瓣胃上皮NHE3 mRNA的表达(P＜0.05)。综上所述，山羊瓣胃上皮存在NHE1 mRNA、NHE3 mRNA。动物饲养试验显示增加日粮营养对山羊瘤胃、瓣胃上皮NHE1、NHE3 mRNA的表达有一定的促进作用；体外试验进一步表明IGF-I、丁酸钠处理可以直接上调瘤胃上皮NHE mRNA表达；口服CT2000亦上调山羊瘤胃上皮NHE mRNA表达。更多还原

【Abstract】 The objectives of the present studies were to detect whether NHE1, NHE3 mRNA were expressed in goat omasum epithelium and the effects of nutrition and Cysteamine on NHE mRNA expression in rumen and omasum epithelium.1. Detection of Na~+/H~+ exchanger mRNA in omasum by RT-PCROmasum epitheliums were collected from goats. Reverse transcription polymerase chain reaction (RT-PCR) were applied by specific primers. The results showed NHE1、NHE3 mRNA expressed in goat omasum epithelium. And the sequence homology analysis between the bovine NHE3、NHE1 sequence and the PCR products from goat omasum epithelium samples were 95% and 91% resPectively.2 Dietary nutritional levels on mRNA of Na＋/H＋exchanger in rumen and omasum epithelium of weanling Goats in vivoGoats were randomly allocated into EXP and CON two groups (n=4) and fed with 0g/d (CON) or 100g/d concentrate (EXP) for 42 days. The expressions of NHE1 mRNA and NHE3 mRNA in rumen or omasum epithelium were higer in rumen epithelium of goats fed a concentrate diet (0.05＜P＜0.1).3 Effect of IGF-Ⅰand NaB on NHE1、NHE3 mRNA expression in rumen epithelium of goats in vitroRumen epitheliums were collected from ventral ruminal sac and ventral blind sac of four goats. Papillae from the atrium rumen were cut into small pieces. IGF-I(25 ng/L) and NAB(5 mmol/L) were administrated respectively. After incubation for 24 h, the samples were collected for RT-PCR analysis.The results showed that rumen epithelium expression of NHE mRNA was up-regulated by IGF-Ⅰor NaB treatment, compared with those in the medium without IGF-Ⅰ. or NaB.4 Effect of cysteamine on expression of NHE3 mRNA in rumen and omasum epithelium of goat in vivoGoats (n=4) were randomly allocated to EXP and CON groups and fed with CT2000 45mg/kg.BW.d(EXP) or 0mg/kg.BW.d (CON1) for 35 days. RT-PCR analysis showed that NHE3 mRNA in rumen and omasum epithelium were up-regulated after supplemented with CT2000.In conclusion, NHE1 and NHE3 mRNA were expressed in goat omasum epithelium. The expressions of NHE1 mRNA and NHE3 mRNA were higer in rumen and omasum epithelium of goats fed a concentrate diet. Furthermore, the expression of NHE mRNA in rumen epithelium was up-regulated by IGF-Ⅰor NaB(sodium butyrate) treatment in vitro. And the cysteamine increased the expression of NHE mRNA in vivo too.更多还原