基于1H NMR技术的乳热奶牛血清代谢组学分析  

1H NMR-based Serum Metabolomics Analysis of Dairy Cows with Milk Fever

作　　者：孙雨航[1] 许楚楚[1] 李昌盛[1] 夏成[1] 徐闯[1] 吴凌[1] 张洪友[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国农业科学》2015年第2期362-369,共8页Scientia Agricultura Sinica

基　　金：国家自然基金青年基金(31001094);"十二五"农村领域国家科技支撑计划(2012BAD12B05-2;2012BAD12B03-2);国家科技支撑计划(2013BAD21B01);国家自然科学基金面上项目(31672625);科技部星火计划(2012GA670001);黑龙江省新世纪优秀人才项目(1252-NCET-003)

摘　　要：【目的】应用1H谱核磁共振(1H NMR)技术筛选乳热奶牛血清内差异表现的小分子代谢物,从小分子水平和物质、能量代谢的角度探究奶牛发生乳热时其体内的代谢变化。【方法】选取年龄、胎次、体况和泌乳量相近的分娩当天的荷斯坦高产奶牛共32头,根据其血清中钙离子浓度及其有无临床症状分为两组。其中,24头奶牛为健康对照组(Group1,血钙浓度>2.5 mmol·L-1,无其他任何症状)和8头乳热组(Group2,血钙浓度<1.4mmol·L-1,伴有明显乳热临床症状)。32头奶牛分别于清晨饲喂和榨乳前从颈静脉采集血液10 m L,置于离心管中,4℃下以1 500×g离心20 min,将离心得到的血清装入1.5 m L的EP管中,置于-80℃下待测。待样品解冻后,分别从每个EP管中抽取400μL血清,加入200μL缓冲盐溶液,充分混合、离心后,提取550μL上清液置于5mm核磁管中,在500 MHz的核磁共振波谱仪下采集信号。然后利用Topspin和Mest Re Nova等软件将采集到的信号进行傅里叶转换,同时进行调零、校正基线和相位等预处理,去除水峰和尿素峰信号,将一维图谱进行积分分段,并将图谱信息转换为TXT格式文件,以便于后续数据分析,然后使用Chenomx软件进行化合物指认。最后应用SIMCA-P软件对得到的图谱数据进行多元统计分析,包括主成分分析(PCA)和正交修正-偏最小二乘法-判别分析(OSC-PLS-DA),同时结合SPSS软件对核磁数据进行单因素方差分析得到的P值以及Loading图,最终筛选出表现差异的小分子代谢物。【结果】本试验成功得到了乳热组和健康对照组奶牛的血清代谢图谱及差异表达代谢物的Loading图;PCA结果显示每组样品均在95%置信区间内,无需剔除,主成分贡献率较低,其中PC1=26.2%,PC2=16.7%,组间差异变量不能被选择;OSC-PLS-DA结果显示经过5次正交信号修正,与分组无关的变量被去除,组间差异达到最大化;与健康对照组相比,共筛选出9种血清[ Objective ] This trial was designed to screen small molecule metabolites with differences in quantity in the serum of cows with milk fever （MF）, and to clarify the changes of metabolites in cows suffering from MF at the molecular level and from the angle of material, energy metabolism. [Method] Thirty-two multiple holstein cows with similar age, parity, body condition and milk yield at O day after parturition were selected, and then assigned into two groups according to their serum calcium concentration and the clinical symptoms. Among them, 24 cows were assigned to the healthy control group （Groupl, serum calcium concentration〉 2.5 mmol.L-1, without any other symptoms） and 8 MF group （Group2, serum calcium concentrations 〈l.4mmol＇L-1, with obvious clinical symptoms of MF）. Blood at 10mL was collected from the jugular vein from each of the 32 cows before feeding and miking in the morning, placed in a centrifugal tube, centrifuged at 4℃, 1 500μg for 20min, and then the serum obtained by centrifugation was separately loaded in a 1.5 mL EP tube at -80℃ until the test. After thawing, 400 μL serums was taken from every EP tube, added 200 μL buffered salt solution, mixed well, and then 550 μL supernatant was extracted into 5 mm NMR tube after centrifugation to collect signals using the 500 MHz NMR spectrometer. Then MestReNova and Topspin softwares were applied on the Fourier transform of the signal, the correction of zero, the baseline and phase, removed water and urea peaks, and the integration of one-dimensional map for segmentation, and mapping information into a TXT file format to facilitate subsequent data analysis, and then all compounds were identified using the Chenomx software. Finally, the SIMCA-P software was used for the multivariate statistical analysis, including principal component analysis （PCA） and orthogonal signal correction-partial least squares-discriminant analysis （OSC-PLS-DA）, combined with the P values obtained from one-way ANOVA of NMR data performed in SPS

关 键 词：奶牛 乳热 代谢组学 1H核磁共振 能量代谢障碍 

分 类 号：S823[农业科学—畜牧学]