MMTV-PyMT转基因小鼠自发成瘤乳腺癌细胞的分离培养及鉴定

Isolation and identification of spontaneously tumorigenic primary breast cancer cells from MMTV-PyMT transgenic mice

目的:分离培养小鼠乳腺肿瘤病毒介导多瘤病毒中间T抗原(MMTV-PyMT)转基因小鼠自发成瘤的乳腺癌细胞,初步研究其生长特性、分子表型、体内成瘤和转移能力。方法:取MMTV-PyMT小鼠自发成瘤的乳腺癌组织(n=3),采用酶解贴壁培养法进行原代细胞分离并传代培养,倒置显微镜下观察细胞形态特征;采用CCK-8检测细胞的增殖能力;通过Western印迹检测细胞的雌激素受体(ER)、孕激素受体(PR)和人表皮生长因子受体2(HER2)表达以鉴定细胞分子亚型。将分离培养的细胞接种于C57BL/6雌性小鼠(n=4)乳腺脂肪垫,观察细胞成瘤能力和生长特性;取肿瘤组织和内脏器官,制备组织切片后采用苏木精-伊红(HE)染色,观察组织形态学特点;采用免疫组织化学(IHC)染色检测ER、PR、HER2及Ki-67蛋白表达,分析分子分型。结果:成功分离得到MMTV-PyMT转基因小鼠自发成瘤的乳腺癌细胞(MMTV-PyMT细胞)并可稳定生长和传代,细胞倍增时间为56.0 h;MMTV-PyMT细胞可在C57BL/6雌性小鼠乳腺脂肪垫成瘤并生长,但未见远处内脏转移。该细胞在细胞学和组织学中的分子分型皆为HER2过表达亚型,肿瘤组织中Ki-67阳性表达率为34%。结论:成功构建得到可体外稳定传代并具有体内成瘤能力的HER2过表达分子亚型小鼠乳腺癌细胞系,该细胞可作为HER2过表达亚型乳腺癌体内、外实验研究的细胞模型。

Objective:To isolate and identify primary breast cancer cells from MMTV-PyMT(mouse mammary tumor virus-polyoma middle T antigen)transgenic mice,explore its growth characteristics,molecular phenotype,tumorigenic and metastatic abilities in vivo preliminary. Methods:Breast cancer tissues from MMTV-PyMT transgenic mice(n=3)were collected,and primary breast cancer cells were isolated and passaged by enzymatic-adherence-culture method. An inverted microscope was used to observe the morphological characteristics of cells;Cell-Counting-Kit 8(CCK-8)was used to detect cell proliferation characteristics;Western blotting was used to detect estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2)protein expression to identify its subtypes. The cells were orthotopic injected into the mammary fat pads of C57BL/6 female mice(n=4),and the tumorigenic ability and growth characteristics of the cells were observed;tumor tissues and visceral organ tissues were taken and stained with hematoxylin-eosin staining(HE)to observe the morphological characteristics;Immunohistochemistry staining(IHC)was used to detect ER,PR,HER2 and Ki-67 protein expression,and the tumor molecular type was analyzed. Results:Tumor cells derived from MMTV-PyMT mice primary breast tumors(MMTV-Py MT cells)were successfully isolated,it could expand and be passaged stably in vitro,and the cell doubling time was 56.04 hours. It could grow in the mammary fat pad of C57BL/6 mice,but no distant visceral metastasis. The molecular subtype of MMTV-PyMT cells in both cytology and histology was HER2-overexpression subtyp e(ER-negative/PR-negative/HER2-positive),and the positive expression rate of Ki-67 in tumor tissue was 34%. Conclusion:A HER2-overexpressing molecular subtype mouse-derived breast cancer cell line that can be stably passaged in vitro and has tumorigenic ability in vivo is successfully constructed. It can be used as a HER2-overexpression cell model for breast cancer in vitro and in vivo experiments.