乳腺干细胞的体外培养及免疫磁珠法分离

目的：乳腺干细胞可以从正常乳腺腺体、良性或恶性乳腺肿瘤的瘤旁组织以及乳汁分离的乳腺上皮细胞中获得。实验选取乳腺癌旁正常组织，体外培养并利用免疫磁珠法分离出具有干细胞特性的乳腺细胞，拟进一步验证在成人乳腺组织中含有可为组织工程所用的种子细胞。方法：实验于2007—03／10在吉林大学公共卫生学院放射生物学教研室完成。①细胞来源：标本取材于吉林大学第一医院乳腺外科同期收治的20例女性乳腺癌患者手术切除的癌旁组织（〉3cm），经病理证实为正常乳腺腺体，患者对治疗及实验均知情同意。②实验方法：去除乳腺腺体周围脂肪组织及毛细血管，剪碎成约为0．3cm^3的组织块，离心，去上清，Ⅰ型胶原酶消化，加入含10％胎牛血清的DMEM／F12基础培养基进行原代培养。2周后待细胞接近铺满整个培养瓶底部时，用胰蛋白酶＋D—HANKS液将贴壁细胞消化分离，按1：2或1：3传代。利用免疫磁珠系统分离筛选乳腺成体干细胞。③实验评估：观察原代与传代培养的乳腺细胞生长特点，以及免疫磁珠分选后的乳腺干细胞形态、生长特性。采用免疫组化法对分选结果进行鉴定。结果：①体外培养的乳腺细胞生长特点：原代培养72h后可见乳腺细胞贴壁，5d后细胞呈纺锤形、多角形生长，10d后细胞生长旺盛且排列紧密，随着培养时间的延长梭形细胞逐渐占据优势。传代后细胞增殖速度加快，细胞更加均匀有序的生长。②乳腺干细胞的形态特点及生长特性：分离出的人乳腺成体干细胞生长状态良好，呈类圆形，培养3d后呈多角形分化生长。③分选结果免疫组化鉴定：免疫磁珠分选出的乳腺干细胞均表达上皮特异性抗原，而不表达唾液黏蛋白。结论：成人乳腺组织中存在唾液黏蛋白1^-上皮特异性抗原^＋且具有干细胞特性的乳

In vitro culture and isolation of human mammary stem cells by magnetic-activated cell sorting

AIM： Mammary stem cells can be isolated successfully from the normal mammary gland, tissue beside the benign or malignant breast tumor and mammary epithelium separated from latex. This experiment chose the normal mammary para-cancer tissue, which was cultured in vitro. And human mammary cells that had characteristic of stem cells were isolated by magnetic-activated cell sorting （MACS）. Then we were going to validate that the adult mammary tissue contains seed cells that can be used in the tissue engineering.
METHODS： The experiment was carried out in the Department of Radiobiology, School of Public Health of Jilin University from March to October in 2007.①The cell samples were from normal mammary para-cancer tissue （〉 3 cm） identified as the normal mammary gland by pathological methods in the resected specimens of twenty female patients with breast cancer in the Department of Mammary Surgery of the First Hospital of Jilin University. Informed consent was obtained from each patient.②The fat and capillary vessels around the mammary gland were removed and cut into pieces about 0.3 cm^3 by scissors. After centrifugation, superuatant removal and digestion by I collagenase, the DMEM/F12 basic culture medium that contained 10% fetal bovine serum was added for perimary culture. Two weeks later, the cells coated the bottom of culture flask. Then trypsinase and D-HANKS were used to digest and separate the cells that adhered to the culture plate. We passaged the cells according to the ratio of 1：2 or 1：3. The adult mammary stem cells were isolated by MACS.③The growth characteristics of the mammary cells after primary culture and passage were observed. The shape and growth characteristics of the mammary stem cells after isolation by MACS were also observed. Immunohistochemistry was used to identify the results after isolation.
RESULTS：①Growth characteristic of the mammary cells cultured in vitro： The cells adhered to the culture plate 72 hours after isolation, and showed fusiform and polygon