牦牛乳腺上皮细胞的体外分离培养及鉴定  

Isolation,Culture and Identification of Yak Mammary Epithelial Cells in Vitro

作　　者：杨玉莹 荆海霞[1] 董宝霞 张勤文[1] YANG Yu-ying;JING Hai-xia;DONG Bao-xia;ZHANG Qin-wen(Department of Veterinary Medicine,College of Agriculture and Animal Husbandry,Qinghai University,Xining,Qinghai 810016,China)

机构地区：[1]青海大学农牧学院动物医学系,青海西宁810016

出　　处：《动物医学进展》2022年第2期64-69,共6页Progress In Veterinary Medicine

基　　金：青海省科学技术厅应用基础研究项目(2019-ZJ-7025)。

摘　　要：为提高牦牛乳腺上皮细胞体外培养成功率,实现高度可重复性,试验采用2.5 g/L(含0.5 g/L EDTA)胰酶和Ⅰ型胶原酶(1 mg/mL)分段消化牦牛乳腺组织块以分离细胞,原代培养时于DMEM/F12培养体系内添加氢化可的松(1μg/mL)、表皮生长因子(50 ng/mL)及胰岛素-转铁蛋白-硒(5μg/mL),传代时用牦牛乳腺上皮细胞与成纤维细胞对胰酶敏感度及贴壁时间的差异特性进行纯化。采用CCK8法测定细胞生长曲线,细胞免疫荧光技术鉴定细胞分子表型,Western blot技术对细胞进行功能鉴定。结果显示,组合酶分段消化法可获得较多细胞量,激素添加有利于促进细胞贴壁及增殖,分离纯化后的细胞具有正常生长特性,经分子表型及功能鉴定纯化后的细胞可阳性表达角蛋白18及β-酪蛋白,阴性表达角蛋白14及波形蛋白,上述3种激素存在时,细胞可在蛋白质水平表达β-酪蛋白,催乳素可促进其表达,说明成功培养了具有泌乳功能的牦牛乳腺上皮细胞,建立了有效的牦牛乳腺上皮细胞体外培养技术体系,可为以后的相关研究提供参考。In order to establish the technical system of in vitro culture of yak mammary epithelial cells and improve the success rate and reproducibility of cell culture,the yak mammary gland tissue was digested with 2.5 g/L(containing 0.5 g/L EDTA)trypsin and type I collagenase(1 mg/mL)to isolate cells.In the initial stage,hydrocortisone(1μg/mL),epidermal growth factor(50 ng/mL)and insulin-transferin-selenium(5μg/mL)were added into DMEM/F12 culture system,the differences of trypsin sensitivity and adhesion time between yak mammary epithelial cells and fibroblasts were used for purification,CCK8 was used to determine the growth curve of purified cells,cell immunofluorescence staining and Western blot were used to identify the molecular phenotypes and functions.The results showed that the combined enzyme digestion method could obtain more cells;the addition of hormone can effectively promote cell adhesion and proliferation,and the cells have normal growth characteristics;the purified cells expressed keratin 18 andβ-casein,but not keratin 14 and vimentin;under the action of three hormones,the cells could expressβ-casein at protein level whether stimulated by prolactin or not.The results showed that this experiment established an effective method for isolation and culture of yak mammary epithelial cells,which could provide some reference for the later immortalization of yak mammary epithelial cells and the study of yak mammary gland development,milk protein synthesis and lactation related gene expression.

关 键 词：牦牛 乳腺上皮细胞 体外培养 纯化 生长曲线 

分 类 号：S852.6[农业科学—基础兽医学]