转溶菌酶基因山羊乳腺上皮细胞的原代培养及特性分析

采用组织块法、胰酶差速消化法分离了人溶菌酶(hLYZ)转基因山羊的乳腺上皮细胞,建立了转基因羊乳腺上皮细胞(TGMEC)的体外培养体系,并对其生长与体外分泌特性进行了分析.研究显示其体外生长曲线符合典型的S型,传代期间二倍体染色体数正常;通过免疫荧光染色、RT-PCR发现该细胞能表达上皮细胞所特有的角蛋白18,并能表达内源性乳蛋白(酪蛋白与乳球蛋白);进一步通过酶联免疫试验发现该乳腺上皮细胞在体外培养中可持续稳定地分泌重组人溶菌酶.由此获得的人溶菌酶转基因羊乳腺上皮细胞系为乳腺生物反应器研究提供了一个重要细胞膜型.

Culture and Characterization of a Primary Mammary Epithelial Cell line of Lysozyme Transgenic Goat

The Culture and characterization of lysozyme transgenic goat mammary epithelial cell(TGMEC) were focused. The primary cell were isolated by using tissue culture method, and then purified by differential trypsin digestion. When cultured in the presence of insulin, hydrocortisone and prolactin, its growth curves comply with a typical S-type, and the chromosome number is normal when analyzed by Giemsa staining. The positive immunostaining to cytokeratin18 suggested that the resulting cell line exhibited the specific character of epithelial cells, and RT-PCR analysis showed the cell expressed endogenous milk protein (casein and lactoglobulin); Furthermore, the ELISA analysis demonstrated that this cell line also secreted recombinant lysozyme stably into the culture medium. The goat mammary epithelial cell line obtained provides an important cell model for mammary gland bioreactor studies.