绵羊下丘脑神经元细胞培养与鉴定  

In vitro cultivation and identification of hypothalamic neurons in sheep

作　　者：梁慧慧[1] 赵宗胜[1] 杨恒[1] 翟曼君[1] 梁艳萍 解一凡 LIANG Huihui;ZHAO Zongsheng;YANG Heng;ZHAI Manjun;LIANG Yanping;XIE Yifan(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院

出　　处：《畜牧与兽医》2018年第8期1-5,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金资助项目(31260537)

摘　　要：通过改良神经元细胞体外培养体系,建立一种高效、可行的绵羊下丘脑神经元体外培养方法,为研究绵羊神经内分泌调节体系及季节性发情机理奠定基础。通过Ⅳ型胶原酶将90 d左右的胎羊下丘脑组织消化成单细胞悬液,接种到预先用多聚赖氨酸包被的培养皿中,2 d后更换为无血清培养基进行培养,并采用两种方法对神经元细胞进行鉴定。结果显示：培养2 d的下丘脑神经细胞,胞体较小且单个独立分布。培养4~6 d神经元的突起明显增多增长,神经元胞体变大。培养8d的神经元细胞更加成熟,突起联系更加紧密,形成密集的神经细胞网络。培养12d后,下丘脑神经元细胞活性减弱,部分细胞开始凋亡。通过RT-PCR检测发现,培养的下丘脑细胞能够表达神经元特异基因Noggin、TUBA4A,经免疫荧光鉴定下丘脑神经元细胞纯度为95.6%。这些结果表明,通过此细胞培养方法能够获得纯度较高的绵羊下丘脑神经元细胞,为进一步研究绵羊神经内分泌调节体系及季节性发情机理提供目标细胞。An efficient and stable method of cultivating hypothalamus neurons in sheep by improving the in vitro neuronal cell cultivating system so as to lay the foundation for study of the neuroendocrine regulation system and the mechanism of seasonal estrus in sheep. The hypo- thalamus of fetal sheep of about 90 days was dissected and cell suspension was made by collagenase IV. The cells were centrifugalized, culti-vated with serum-free medium and seeded in the petri dishes which had been coated with polylysine. Neuronal cells were identified in two ways. The results showed that after in vitro cultivation for two days, the hypothalamic neurons were small in size, singly and evenly distribu- ted. After four to six days of cultivation, the number of neurons increased significantly and the cell bodies of neurons became larger. After eight days of cultivation, the neurons were more mature and the neurites were more closely connected to form a dense network of nerve cells. After twelve days of cultivation, the cells were weakened in activity and some of them started their apoptosis. It was found that the cultivated cells could express Noggin and TUBA4A by RT-PCR. The purity of hypothalamic neurons was identified as 95.6% by anti-MAP2-FITC. These results indicated that cultured hypothalamic neurons of higher purity could be obtained by the present cell cultivation method, providing target cells for further study of the mechanism of ovine neuroendocrine regulation and seasonal estrus in sheep.

关 键 词：绵羊 下丘脑 神经元细胞 体外培养 鉴定 

分 类 号：S826[农业科学—畜牧学]