小鼠和奶牛乳腺上皮细胞的分离及培养特性比较

Comparison the Isolation and Culture Characteristics Between Mouse Mammary Epithelial Cells and Bovine Mammary Epithelial Cells

为建立小鼠乳腺上皮细胞(MMECs)及奶牛乳腺上皮细胞(BMECs)分离培养体系,并比较其生长特性。分别无菌采集健康妊娠后期昆明鼠及泌乳中期荷斯坦奶牛乳腺组织,采用改良混合酶消化结合差速贴壁法分离原代MMECs及BMECs,并进行细胞角蛋白18的免疫荧光鉴定;通过细胞形态跟踪观察评价传代、冻存和复苏后MMECs及BMECs生物学特性;连续培养及细胞计数绘制MMECs及BMECs生长曲线。结果显示,从小鼠和奶牛乳腺组织分离的目的细胞,经角蛋白18免疫荧光鉴定证实为MMECs及BMECs;汇合的单层MMECs多角形,呈铺路石样外观,BMECs呈鹅卵石样;MMECs在传至第2代时细胞特有形状消失,而BMECs传至第9代时仍保持其鹅卵石样外观;MMECs只能复苏冻存原代细胞,传代BMECs均可冻存和复苏。采用改良混合酶消化结合差速贴壁法可获得高纯度的MMECs和BMECs,两种细胞在生长培养特性上存在显著差异。

The objective of the study was the comparison of culture characteristics between mouse mammary epithelial cells（MMECs）and bovine mammary epithelial cells（BMECs）,and establishing the isolation systems.The breast tissues from late pregnancy KM mice and mid-lactation Holstein cows were collected aseptically,then modified enzyme mixture combined with differential adherence time was used to digest and separate primary MMECs and BMECs.Finally,the MMECs and BMECs were identified via cytokeratin18 expression by immunofluorescence staining.The biological characteristics of MMECs and BMECs were evaluated by cell morphology observation after cell passage,cryopreservation and resuscitation.Growth curves of MMECs and BMECs were made by cell culture and counting continuously.The results showed that those cells separated from breast tissues were MMECs and BMECs;The confluent monolayer MMECs were polygonal and paving stone appearance,while BMECs were cobblestone appearance;MMECs＇ specific morphology disappeared when passage 2nd generation,in contrast to MMECs,BMECs were holding cobblestone appearance until the passage 9th generation;Compared to MMECs who can only be cryopreservated and resuscitated once,each passage BMECs can be cryopreservated and resuscitated.In conclusion,there are significant differences in the growth and culture characteristics of these isolated highpurity MMECs and BMECs.