基底样型乳腺癌肿瘤干/祖细胞的长时程无血清培养  

Long-term serum-free culture of cancer stem/progenitor cells from basal-like breast carcinoma

作　　者：董华英[1] 王伟[1] 陈元文[2] 包俊杰[2] 陈鑫[2] 吴诚义[2] Dong Hua-ying;Wang Wei;Chen Yuan-wen;Bao Jun-jie;Chen Xin;Wu Cheng-yi(Department of Breast Surgery, Hainan General Hospital, Haikou 570311, Hainan Province, China;Department of Endocrine and Breast Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China)

机构地区：[1]海南省人民医院乳腺外科,海南省海口市570311 [2]重庆医科大学附属第一医院内分泌乳腺外科,重庆市400016

出　　处：《中国组织工程研究》2017年第33期5313-5319,共7页Chinese Journal of Tissue Engineering Research

基　　金：海南省卫生计生科教项目(02A2150014P1)~~

摘　　要：背景:乳腺癌干细胞是乳腺癌细胞中的亚群,具有类似干细胞的自我更新和多系分化潜能,具有放化疗和缺氧抵抗性、高致瘤性和高侵袭转移性等特征,在乳腺癌的发生发展以及复发转移中发挥重要作用。乳腺癌干细胞学说在一定程度上阐明了乳腺癌的发病机制,在治疗方案选择、新的治疗靶点的发现和乳腺癌预后的判断等方面有很大的应用价值。目的:从基底样型乳腺癌组织中分离培养肿瘤干/祖细胞,并通过无血清悬浮技术长时程培养,研究其自我更新能力、分化潜能和细胞表型等生物学特性。方法:20例基底样型乳腺癌患者,其中10例患者未经过任何治疗,另外10例患者术前均行新辅助化疗(≥4周期),术前于肿瘤边缘切取1 cm3新鲜肿瘤组织,立即送实验室,经机械分离后获取肿瘤细胞。应用无血清悬浮培养技术富集乳腺癌干/祖细胞微球体后,通过单克隆实验检测乳腺癌干/祖细胞微球体细胞的克隆形成和自我更新能力,在添加血清的培养液中诱导乳腺癌干/祖细胞微球体细胞分化,观察其分化能力。通过流式细胞术检测CD44^+/CD24^-和ALDH1^+表型细胞的比例。结果与结论:(1)在含生长因子的无血清培养基中,未行新辅助化疗的基底样型乳腺癌细胞均可生成乳腺癌干/祖细胞微球体,而大部分新辅助化疗后的乳腺癌组织中可直接分离获得微球体样的细胞团;(2)乳腺癌干/祖细胞微球体细胞可连续传代形成新生的乳腺癌干/祖细胞微球体,随着培养传代次数的增加,贴壁细胞逐渐增多,新产生的乳腺癌干/祖细胞微球体数量逐渐减少,球体直径变小,且乳腺癌干/祖细胞微球体也更加容易贴壁。乳腺癌干/祖细胞微球体细胞在添加血清的培养基中培养可贴壁分化;(3)乳腺癌干/祖细胞微球体中富集了CD44^+/CD24^-和ALDH1^+表型细胞,诱导分化后两种表型细胞的含量大幅下降或缺失;(4)大部分�BACKGROUND:Breast cancer stem cells,a subgroup of breast cancer cells,have self-renewal and multilineage differentiation potential and are characterized as resistance to chemotherapy,radiotherapy and hypoxia,and high propensity for invasiveness and metastasis,which play an important role in the recurrence and metastasis of breast cancer.The breast cancer stem cell theory can elucidate the pathogenesis of breast cancer to a certain extent,and has great values in the selection of treatment options,the discovery of new therapeutic targets and the prognosis of breast cancer.OBJECTIVE:To isolate the breast cancer stem/progenitor cells from basal-like breast carcinoma and study their self-renewal ability,phenotype and differentiation potential over long-term serum-free suspension culture in vitro.METHODS:Twenty patients with basal-like breast cancer,including10patients without any treatment and10with neoadjuvant chemotherapy(≥4cycles),were enrolled.A1cm3fresh tumor tissue sample from each patient was taken and immediately sent to the laboratory to mechanically isolate tumor cells.Then,the stem/progenitor cells of basal-like breast carcinoma were enriched in ultra low attachment plates in serum free media as nonadherent mammospheres.Serial sphere formation assay was performed to determine colony formation and self-renewal ability of mammosphere-derived cells.Differentiation was induced by culturing mammosphere-derived cells in DMEM-F12supplemented with serum but without growth factors.The proportion of CD44+/CD24-and ALDH1+cell population was evaluated by flow cytometry.RESULTS AND CONCLUSION:The mammospheres formed after inoculation of primary basal-like breast cancer cells isolated from the tumor tissues of breast cancer patients who did not receive neoadjuvant chemotherapy cultured in the serum-free medium with growth factors,while the mammospheres could be directly isolated from the tumor specimens of patients with neoadjuvant chemotherapy.The mammosphere-derived cells could be passaged continuously to form new ma

关 键 词：干细胞 肿瘤干细胞 乳腺癌 分子分型 新辅助化疗 细胞培养 

分 类 号：R394.2[医药卫生—医学遗传学]