亚临床低钙血症奶牛外周血中性粒细胞的糖异生作用下降  

Decreased gluconeogenesis pathway of peripheral blood neutrophils in subclinical hypocalcemia dairy cows

作　　者：张伟 何雨欣 麻芯茹 吕鑫泉 李铭 李思瑶 杨威[2] 徐闯[2] 张冰冰[1] ZHANG Wei;HE Yuxin;MA Xinru;LYU Xinquan;LI Ming;LI Siyao;YANG Wei;XU Chuang;ZHANG Bingbing(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang 163319,China;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang 163319,China)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国兽医学报》2023年第6期1315-1320,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金联合基金重点资助项目(U20A2062);黑龙江省自然科学基金优秀青年资助项目(YQ2020C035);黑龙江八一农垦大学校启动资助项目(XYB201809)。

摘　　要：为研究胞内Ca^(2+)对亚临床低钙血症奶牛外周血中性粒细胞糖异生作用的影响,用枸橼酸钠抗凝管采集奶牛尾静脉血液分离中性粒细胞;对中性粒细胞进行RNA测序及分析,利用qRT-PCR技术对其进行验证;通过流式细胞术检测胞内Ca^(2+)水平;Western blot技术检测钙池调控钙离子进入(store-operated calcium entry, SOCE)分子(ORAI1、ORAI2、ORAI3、STIM1、STIM2)的表达情况。添加SOCE激活剂Thapsigargin(TG)改变细胞内Ca^(2+)水平,探究Ca^(2+)水平对糖异生作用的影响。RNA测序结果分析发现,差异基因富集在Ca^(2+)信号通路和糖异生作用通路上,qRT-PCR结果显示亚临床低钙血症奶牛的糖异生作用(FBP1、PKM2、G-6-Pase、PFKFBF3、AKR1B1)与正常奶牛相比下降;流式细胞术结果显示,亚临床低钙血症奶牛胞内Ca^(2+)水平下降;Western blot结果显示,亚临床低钙血症奶牛SOCE分子表达下降。在TG处理后,胞内Ca^(2+)水平上升,糖异生作用显著上升。结果表明,中性粒细胞内Ca^(2+)水平影响糖异生作用。In order to study the effect of intracellular Ca^(2+)on the gluconeogenesis of peripheral blood neutrophils in subclinical hypocalcemia cows,blood was collected from the tail vein of cows using sodium citrate anticoagulation tubes to isolate neutrophils;RNA sequencing and analysis of neutrophils were performed and validated using qRT-PCR technology;and the level of intracellular Ca+was detected by flow cytometry,the expression of calcium pool regulated calcium ion entry(SOC)molecules(ORAI1,ORAI2,ORAI3,STIM1,STIM2)was detected by Western blot.The SOCE activator thapsigargin(TG)was added to change the intracellular Ca^(2+)level and the effect of Ca^(2+)on gluconeogenesis was investigated.RNA sequencing results showed that differential genes were enriched in Ca^(2+)signaling pathway and gluconeogenesis pathway.qRT-PCR results showed that the gluconeogenesis of subclinical hypocalcemia cows(FBP1,PKM2,G-6-Pase,PFKFBF3,AKR1B1)decreased compared with normal cows.Flow cytometry results showed that the intracellular Ca^(2+)of subclinical hypocalcemia cows decreased,and Western blot results showed that the SOCE molecular expression of subclinical hypocalcemia cows decreased.After TG treatment,intracellular Ca^(2+)level and gluconeogenesis increased significantly.The above data indicate that the level of Ca^(2+)in neutrophils affects gluconeogenesis.

关 键 词：CA^(2+) 亚临床低钙血症 中性粒细胞 糖异生 

分 类 号：S852[农业科学—基础兽医学]