NIX和LC3-Ⅱ在成年牦牛不同脑组织中的表达与定位分析  

Expression and Localization of NIX and LC3-Ⅱin the Different Brain Tissues of Adult Yaks(Bos grunniens)

作　　者：冯法铭 张倩[1] 崔燕[1] 何俊峰[1] 靳宏亮 FENG Faming;ZHANG Qian;CUI Yan;HE Junfeng;JIN Hongliang(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou,Gansu 730070)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《核农学报》2023年第12期2378-2386,共9页Journal of Nuclear Agricultural Sciences

基　　金：甘肃农业大学青年导师扶持基金(GAU-QDFC-2022-10);国家自然基金(32002241);甘肃省自然基金(22JR5RA859)。

摘　　要：为了探讨牦牛(Bos grunniens)脑组织低氧适应性,选择成年(3~5岁)牦牛脑组织,利用实时荧光定量PCR(qRT-PCR)、免疫组织化学技术及免疫荧光技术检测低氧诱导因子2α(HIF2α)与促凋亡BNIP3L蛋白(BNIP3L/NIX)和微管相关蛋白轻链3(LC3-Ⅱ)在大脑皮质、海马、丘脑、延髓和小脑中的表达与定位情况,并用半胱氨酸天冬氨酸酶3(Caspase-3)检测脑组织中细胞的凋亡水平。qRT-PCR和免疫组织化学光密度分析结果显示,成年牦牛大脑皮质和海马的HIF2α、NIX、LC3-ⅡmRNA和蛋白水平显著高于丘脑、延髓及小脑(P<0.05),另外Caspase-3 mRNA和蛋白水平在五个部位之间无显著差异。免疫组化镜下观察发现HIF2α阳性反应表达于神经元胞核及胞质,而NIX、LC3-Ⅱ和Caspase-3阳性反应均集中在细胞质,且上述四个因子主要分布于大脑皮质的多形细胞层和海马的CA区锥体细胞层,另外,在丘脑、延髓和小脑神经元中均有表达。免疫荧光结果显示,HIF2α、NIX、LC3-Ⅱ与神经元核抗原抗体NeuN在上述各部位神经元细胞共定位,Caspase-3荧光强度较弱。以上结果表明,海马和大脑皮质可能更容易受到低氧刺激;另外,HIF2α的表达可能促进NIX和LC3-Ⅱ的表达,降低Caspase-3表达水平,从而使牦牛大脑和海马适应高原环境。本试验为深入研究牦牛中枢神经系统的低氧适应机制提供了基础数据。In order to investigate the hypoxia adaptability of Bos grunniens brain tissue,the adult yak brain tissue(3-5 years old)was selected.Real-time quantitative fluorescent PCR(qRT-PCR),immunohistochemical techniques and immunofluorescence techniques were used to detect the expression and localization of HIF2α,BNIP3L/NIX and LC3-Ⅱin the cerebral cortex,hippocampus,thalamus,medulla oblongata and cerebellum.Caspase-3 was used to detect the cell apoptosis in brain tissue.The results of qRT-PCR and immunohistochemical density analysis showed that the mRNA and protein levels of HIF2α,NIX and LC3-Ⅱin cerebral cortex and hippocampus of adult yaks were significantly higher than those in thalamus,medulla oblongata and cerebellum(P<0.05),while the mRNA and protein levels of Caspase-3 were not significantly different among the five regions.Immunohistochemical observation showed that HIF2αpositive reaction was expressed in the nucleus and cytoplasm of neurons,while the positive reactions of NIX,LC3-Ⅱand Caspase-3 were concentrated in the cytoplasm,and the above four factors were mainly distributed in the polymorphic cell layer of cerebral cortex and the pyramidal cell layer of the CA region of hippocampus.In addition,they were expressed in the neurons of thalamus,medulla oblongata and cerebellum.The results of immunofluorescence showed that HIF2α,NIX,LC3-Ⅱand NeuN were co-located in the neurons of the above sites,and the fluorescence intensity of Caspase-3 was weak.These results suggest that hippocampus and cerebral cortex may be more susceptible to hypoxia stimulation.In addition,the expression of HIF2αmay promote the expression of NIX and LC3-Ⅱ,and reduce the expression level of Caspase-3,so as to adapt the brain and hippocampus of yaks to the plateau environment.This study provided basic data for further study on the hypoxia adaptive mechanism of yak central nervous system.

关 键 词：牦牛 低氧 脑组织 NIX LC3-Ⅱ 

分 类 号：S823.85[农业科学—畜牧学]