转基因小鼠乳腺上皮细胞的体外培养及其对催乳素反应的研究

实现转基因生物乳腺反应器对外源蛋白的高效表达是目前生物制药亟待解决的难题。催乳素对泌乳期乳蛋白的合成与分泌具有重要的调控功能。通过转基因小鼠乳腺上皮细胞模型的建立,研究催乳素如何调控乳蛋白的表达,为提高乳腺反应器高效表达外源蛋白提供技术及理论支撑。应用机械破碎及胶原酶消化法,经差速贴壁纯化,成功培养含人转铁蛋白基因的小鼠乳腺上皮细胞,细胞上清液中检测到人转铁蛋白表达。细胞经牛催乳素诱导后人转铁蛋白的表达水平明显升高。利用转基因小鼠乳腺上皮细胞模型,可以进行催乳素和环境因素等对乳腺上皮细胞合成及分泌蛋白能力影响的研究。

The Primary Cell Culture of Human Transferrin Transgenic Mouse Mammary Epithelia and the Study of the Cells Reponse to Bovine Prolactin

The aim of the construction of mammary gland bioreactor is to obtain the high-level expression of exogenous proteins in mammary gland.Prolactin plays an important role in the regulation of milk protein synthesis and secretion during mammalian lactation.The previous study demonstrated that bovine prolactin can efficiently promote the expression of exogenous gene(hTF) in milk of transgenic mice.To further explore the mechanism underlying this finding,the culture protocol were developed to cultivate transgenic mouse mammary epithelia and the biological effects of bovine prolactin on these cells were studied.Mammary tissue of mouse carrying human transferrin gene(hTF) were minced,digested with collagenase.Epithelial cells were then collected and cultured,subsequently purified by timing cells adherence to flask wall.Afterwards,purifed cells were transfected with pCMV-bPRL plasmids.The synthesis ability of the cells was tested by detecting the secretion of related proteins present in supernatant.Epithelial cells of transgenic mice carrying hTF were successfully cultured in vitro,hTF was shown to be presented in supernatants of cells.The level of hTF was elevated with the presence of bovine prolactin(1 208.55±20.07 pg/ml vs 3 161.32±71.62 pg/ml).The culture protocol could be employed in the near future experiments to investigate effects of bovine prolactin and environmental factors on the regulation of protein synthesis and secretion of mammary epithelial cells.