牦牛乳腺上皮细胞体外培养及鉴定

Culture and Identification of Yak Mammary Epithelial Cells

本研究旨在建立牦牛乳腺上皮细胞系,并对建立的细胞系生物学特性进行鉴定,采用组织块种植法,分离培养乳腺上皮细胞;通过胰酶消化法纯化细胞;利用免疫荧光及Western blot技术对其进行鉴定;脂质体介导法将绿色荧光蛋白基因转染进乳腺上皮细胞中,荧光显微镜下检测绿色荧光蛋白基因表达;金黄色葡萄球菌侵染牦牛乳腺上皮细胞,流式细胞仪(AnnexinV/PI双染法)检测乳腺上皮细胞的凋亡,RT-PCR检测感染细胞中抗菌肽以及凋亡因子表达。结果表明,分离纯化获得牦牛乳腺上皮细胞,细胞角蛋白18表达呈阳性,波形蛋白表达呈阴性,证明培养的细胞是上皮细胞并且没有成纤维细胞污染;β-酪蛋白表达呈阳性,说明建立的乳腺上皮细胞具有一定的泌乳功能;荧光显微镜能够检测到绿色荧光蛋白表达,表明EGFP基因成功导入了乳腺上皮细胞;金黄色葡萄球菌感染牦牛乳腺上皮细胞3h后,Annexin V/PI双染法检测发现感染组细胞凋亡率明显升高,差异显著(P<0.05);感染细胞中TAP(P<0.01)、BNBD5(P<0.01)及BAX(P<0.01)表达量明显增高,BCL-2(P<0.05)表达量降低。综上表明,本研究成功建立了一株稳定的牦牛乳腺上皮细胞系,该细胞系能够作为研究牦牛乳腺发育及分化的体外研究模型。

The present study was carried out to establish yak mammary epithelial cell(YMEC)line and identify its biological characteristic,which was separated using tissue explants method,purified by selective trypsinization,characterized by immunofluorescence and Western blot assay.Transfecting EGFP-N1 gene into cells using lipofection to examine whether foreign genes could be transfected into YMECs,fluorescence microscope was used to detect the expression of EGFP gene.Lastly,flow cytometry was used to evaluate the apoptosis level of cells with Annexin V/PI double staining and RT-PCR was used to examine gene expression of antibacterial peptide and apoptosis factors to determine the effect of Staphylococcus aureus infection on YMECs.Immunofluorescence assay results proved established cell line could express cytokeratin 18 rather than vimentin,indicating no fibroblast existing in epithelial cells.Positive expression ofβ-casein in YMECs demonstrated cultured cells could synthesize and secrete mammary-specific milk protein.Moreover,we detected EGFP protein in transfected YMECs by fluorescence microscope,showing EGFP gene was successfully transfected into the YMECs.After YMECs were challenged with for Staphylococcus aureus for 3h,the apoptosis level of mammary epithelial cells was significantly up-regulated(P<0.05).Furthermore,the expression of TAP(P<0.01),BNBD5(P<0.01)andBAX(P<0.01)genes increased;whereas that of BCL-2gene decreased(P<0.05).To conclude,we successfully established a stable yak mammary epithelial cell line,which can be used as a useful model in vitro for study of yak mammary gland development and differentiation.