小白鼠乳腺上皮细胞的体外培养及形态学变化

Study on the morphology of mouse mammary epithelial cells cultured in vitro

以无菌手术法取健康妊娠期、泌乳期昆明小白鼠的乳腺组织,用胰蛋白酶、胶原酶Ⅰ分段消化法获得小鼠乳腺细胞。生长培养液为DMEM/F12,含10%胎牛血清,添加胰岛素(5μg/mL)、EGF(5ng/mL)、氢化可的松(1μg/mL)、青霉素(200IU/mL)及链霉素(0.1mg/mL)。从乳腺组织中获得的细胞直接放入-80℃冰箱中进行冻存,冻存液为DMEM/F12,含20%胎牛血清、10%DMSO。用倒置显微镜观察细胞形态,发现乳腺组织消化后即可获得呈圆饼状的上皮细胞;培养过程中,乳腺上皮细胞的形态以鹅卵石型和多角型为主,增殖可形成乳头状结构,称之为乳球体,大量增殖后形成圆顶型结构。细胞核呈圆形或椭圆形,在传代过程中,乳腺上皮细胞可形成岛屿状的闭合型细胞群,边界清晰,细胞之间结合紧密,细胞间呈拉网结构。结果表明:获得典型上皮细胞形态的小鼠乳腺细胞原代培养物,从泌乳期小鼠获得的乳腺上皮细胞传至第1代时无法长成单层;而从妊娠期小鼠获得的乳腺上皮细胞传至第3代时才无法长成单层;冻存后复苏的乳腺细胞在培养过程中仍然具有典型的上皮细胞形态,并且增殖旺盛。

Mouse mammary were obtained from healthy kunming mouse during gestation and lactation and mouse mammary ceils（MMCs） were isolated by trypsin and collagenase 1 digestion. The MMCs were incubated with the growth medium containing DMEM/F12 and 10% FCS, and insulin（5 μg/mL）, EGF （5 ng/mL）, hydrocortisone（ 1 μg/mL）, penicillin（200 IU/mL） and streptomycin（0. 1 mg/mL）. Cells obtained from mammary tissue were cryopreserved （- 80℃） in 1 mL aliquots containing DMEM/F12, 20% FBS and 10% DMSO. The appearances of the MMCs were observed by inverted microscope ： cells looked like round flat cakes were obtained from mammary gland by trypsin and collagenase I digestion. During culture ,the major morphology of cells are characteristic cobblestone and polygonal-shaped, the nipple structure which called milk orb appears in the proliferated cells, the cell nucleus are circular or oval and formed dome-like construction. During sub-culture, the MMCs formed island monolayer aggregates as closed clusters whose boundary is clear and the cells combined closely and the netting structure has formed between cells. The results showed that the cells spread out to monolayers with typical epithelial organization. MMECs which obtained from the lactation mouse could not form monolayer in the first passage. However, MMECs which obtained from the pregnancy mouse could not form monolayer in the third passage. Aliquots of MMCs were thawed and grown in growth medium, the same characteristics of cell growth were seen after cryopreservation of cells, and proliferates vigorousely