利用牛乳汁体细胞研究乳腺免疫相关因子的表达  

Use of somatic cell in cow′s milk to study the expression of mammary immune-associated factores

作　　者：王月影[1] 李平[1] 李宏基[1] 郭豫杰[1] 汪新建[1] 杨国宇[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2011年第9期1313-1317,共5页Chinese Journal of Veterinary Science

基　　金：国家"十一五"科技攻关计划资助项目(2006BAD-04A03-10);河南省科技攻关资助项目(072102140002)

摘　　要：利用乳汁体细胞分子生物学技术平台,以奶牛初乳和末乳为研究对象,看家基因GAPDH为内参,对乳汁中IL-8、CCL5、CXCL14、κ-CN和α-LA进行半定量RT-PCR分析。结果显示,在209、202、376、151、233bp位置分别出现5种基因预期大小的特异性扩增条带。乳中IL-8、CCL5、CXCL14、κ-CN和α-LA mRNA转录水平的相对定量结果表明,与初乳相比,末乳中IL-8和α-LA mRNA的转录水平显著降低(P<0.05),CCL5、CXCL14和κ-CN mR-NA的转录水平分别下降21%、40%和37%,差异不显著(P>0.05)。结果提示,末乳期奶牛乳腺免疫功能下降,应加强此阶段的管理,降低乳房炎的发生。The research and application of somatic cell in the breast secretions have exceeded its determinations as mastitis indications and have wide application prospects in mammary cell culture and gene expression.For this purpose,the study was to use somatic cell in bovine to research on the expressions of immune-associated factors in mammary gland.Colostrum was as an object of study,we optimized the centrifugal conditions of milk somatic cell and identificated the SCC by directoscope method,blood cell countion method and trypan blue exclusion staining method.And then extracted RNA from milk somatic cell according to the Puissant＇s method and determined the purity integrity and content of RNA by electrophoresis and UV spectrophotometer,and housekeeping gene GAPDH by RT-PCR.The result showed that：the best centrifugal condition is 4℃,1 500×g 15 min;SCC is about 70%;RNA D260/D280 is 1.84,0.776 g/L can be used in the cDNA synthesis.The result of RT-PCR showed that specific amplification strap of housekeeping gene GAPDH existed at 452 bp and showed that the milk somatic cell were successfully prepared.Somatic cell in colostrums and ending milk was selected as object and housekeeping gene GAPDH was selected as internal standard,analysised IL-8,CCL5,CXCL14,κ-CN and α-LA in milk by semi-quantitive RT-PCR.The results showed that three specific amplification straps existed at 209,202,376,151 and 233 bp respectively.The transcription level of the relative quantitative results of IL-8 and α-LA mRNA in ending milk decreased significantly（P 0.05） compared with the colostrums.CCL5 mRNA、CXCL14 mRNA and κ-CN mRNA transcription levels decreased by 21%、40% and 37%,respectively（P0.05）.

关 键 词：奶牛 乳汁体细胞 免疫相关因子表达 RT-PCR 

分 类 号：S852.4[农业科学—基础兽医学]