改良型混合酶消化法培养小鼠乳腺上皮细胞

Cultivation of mouse mammary epithelial cells by using improved collagenase/trypsin digestion method

背景:目前采用的A型胶原酶和胰蛋白酶混合酶分离乳腺细胞团的方法操作复杂,实验条件要求高。目的:观察改良型混合酶消化法能否成功进行乳腺上皮细胞的体外培养。方法:采用Ⅰ型、Ⅱ型胶原酶和胰蛋白酶(1:1:1)混合消化直接用眼科剪剪碎的小鼠乳腺组织,37℃振荡消化获取乳腺细胞团,并采用差速贴壁法去除成纤维细胞,将细胞团接种于细胞培养瓶进行培养。结果与结论:倒置显微镜下观察显示,改良型混合酶消化法能获得较多的细胞团,且培养12h后细胞团绝大多数贴在细胞瓶壁,培养72h后细胞团完全铺开融合成片,细胞呈典型的铺路石样。免疫荧光细胞化学染色结果显示,培养细胞角蛋白18呈阳性反应。说明应用改良型混合酶消化法能在短时间内获得大量较纯的乳腺上皮细胞。

BACKGROUND： The mixture of collagenase A and trypsin that used to isolate the mammary epithelial cells is complex and strict to operate. OBJECTIVE： To clarify whether mammary epithelial cells can be successively cultured with improved collagenase/trypsin digestion method in vitro. METHODS： The mixture of type I and ]l collagenase and trypsin （1：1：1 ） was used to digest breast tissue, which was shredded directly with ophthalmic scissors. The organoids were obtained by 37 ＇C oscillation extraction and the differential adhesion method was used to remove the fibroblasts. Then the organoids were inoculated in the plastic dish. RESULTS AND CONCLUSION： Inverted microscope observation showed that epithelial organoids could be mostly acquired and had attached to the plastic dish after 12 hours, and then cells were spreading out from them and forming confluent monolayer of cobblestone after cultured for 72 hours. In addition, the tissue-specific expression of cytokeratin 18 in mammary epithelial cells was identified by cytokeratin immunohistochemistry. It indicated that plenty of pure mammary epithelial cells could be harvested by using improved collagenase/trypsin digestion.