低DCAD上调小肠TRPV6表达促钙吸收：防治围产期奶牛低血钙作用机制的研究

Reducing dietary cation-anion difference(DCAD) has been reported the effective strategy for hypocalcemia prevention in transition dairy cows by increasing blood calcium (Ca) level.However,the precise mechanism behind it remains to be one of the urgent subjects in the field of dairy cows nutrition. Transient receptor potential vanilloid-6 (TRPV6) is the primary factor in the process of Ca entry into epithelial cells through transcellular membrane.Therefore, it is hypothesized that low DCAD would up-regulate TRPV6 expression level in the epithelial cells of small intestine, enhance Ca absorption, improve blood Ca level,and then prevent hypocalcemia. In order to test our hypothesis,2 parts of work will be conducted to evaluate the effect of TRPV6 expression level in small intestine on Ca absorption efficiency under the condition of low DCAD.The research methods are intergrated with melecular biology and feeding trial, and with extending 3 levels of body, tissue, and to cell. In part 1,transition dairy goats are used as experimental animals to model Ca absorption availability in the epithelial cells of small intestine segments when they are fed low DCAD. The research contents and techniques used in the study are as follows.(1) RT-PCR and Western blot are used to determine TRPV6 mRNA and TRPV6 protein expression level,respectively.(2)Ussing chamber is used to investigate the Ca2+ influx entry into blood through small intestine.(3)Blood Ca level will be evaluated to clarify the mechanism of low DCAD enhancing Ca absorption. In part 2,transition dairy cows will be served as experimental animals to test the mechanism which was concluded from the work of part 1. The contents are consisted of 2 works.(1)To determine blood TRPV6 and Ca levels of cows fed low DCAD.(2)To establish the mathematical model among DCAD, TRPV6, and blood Ca via SAS software. The present project is an extending and expanding proposal from our previous one. It is a systematic intergration of idea from microscopic-oriented mechanism clarifying to macroscopic-oriented testing.The results can provide scientific evidence on clarifying the mechanism of how low DCAD prevents hypocalcemia, and also,can be used as guidance in the field of dairy cows nutrition in the practice.

解析低日粮阴阳离子差（DCAD）增加血钙含量、防治围产期奶牛低血钙的机理，是奶牛营养研究的重要课题之一。瞬时性电位香草精受体(TRPV6)是Ca跨膜入胞的首要功能元件，由此假设：低DCAD可增强小肠TRPV6表达，促进钙吸收，提高血钙水平，防治奶牛低血钙。 拟以低DCAD为切入点，采用分子技术与饲养试验手段，开展"整体、组织、细胞"层次研究，旨在揭示低DCAD防治低血钙的机理。①以围产期奶山羊作机理研究：应用RT-PCR和Western blot检测小肠上皮细胞TRPV6表达水平，应用尤斯灌流仪检测小肠钙内流率；结合血钙水平，明确低DCAD促进小肠钙吸收的机理；以及②以围产期奶牛作理论验证：测定低DCAD处理下血TRPV6、钙水平，用SAS建立DCAD与血TRPV6、钙间的关联性。 本研究由"机理研究"的微观深入到"假设验证"的宏观拓展的系统思维，整体性强，可验证项目假说，指导奶牛生产。

解析低饲粮阴阳离子差（dietary cation-anion difference, DCAD, mmol/kg DM)防治低血钙的机理，是动物营养研究的重要课题之一。瞬时性电位香草精受体（TRPV6）大量分布于小肠，为Ca高选择吸收特异性通道蛋白，是调控食糜Ca跨膜入胞的关键功能元件，由此假设：低DCAD可增强小肠TRPV6表达，促进钙吸收，防治低血钙。. 项目从机理探讨和验证2方面开展了5个研究内容。首先，以围产期鼠开展组织TRPV6表达RT-PCR检测体系，发现TRPV6与校准蛋白β-actin的标准曲线相关系数（R2）为0.9985，建立了TRPV6基因的定量检测技术体系。第二，在围产期鼠上发现，与高DCAD水平相比（317、172），在产前5d和产仔0d，低DCAD水平（-56）提高了空肠、回肠TRPV6mRNA表达水平（P<0.05）和血Ca浓度（P<0.05）。第三，以肉羊为试验动物，发现低DCAD（-145.5）协同VD（0.5 g/d），与高DCAD（212.4）相比，血液TRPV6浓度提高（11.28:15.61，P<0.05）；低DCAD（-162）相对于高DCAD（157），瘤胃发酵指标没有差异（P>0.05），但低DCAD组羊血液TRPV6高于高DCAD组（13.88:16.77，P<0.05）。第四，将成果作用于围产期羊，发现低DCAD（-26）较高DCAD（420、187）相比，尿液pH降低、血Ca水平提高（P<0.05）；母羊产羔前10d、产羔0d、产羔后3d十二指肠、空肠、回肠、结肠TRPV6mRNA表达丰度增加（P<0.05）；TRPV6蛋白表达水平在产羔0d和产羔后3d上升（P<0.05），且空肠差异最为明显（P<0.05）。第五，在此基础上开展围产期奶牛DCAD试验，发现低DCAD（-110、-158）与高DCAD（103）相比，采食量、泌乳量、乳成分未受影响（P＞0.05）；尿液pH、乳体细胞数随DCAD水平的降低而降低（P＜0.05）；血钙水平显著提高（P＜0.05）。上述研究内容和结果，推进阐明了饲喂低DCAD日粮提高Ca吸收、防治低血钙的作用机制，验证了科学假说，体现了微观机理研究和宏观验证结合系统整体思维，可用于指导生产实践。. 项目共发表论文16篇，获省科技进步三等奖1项、发明专利1项，培养研究生12名。