卵巢癌组织及腹腔积液中肿瘤干细胞的分离与培养

干细胞培养

906doc.docsou.com　　肿瘤2011年10月第31卷第10期　TUMOR Vol. 31, October, 2011

基础研究·Basic Research

DOI: 10.3781/j.issn.1000-7431.2011.10.006Copyright© 2011 by TUMOR

卵巢癌组织及腹腔积液中肿瘤干细胞的分离与培养

施敏凤1，谢　幸2，朱　

1

，吕卫国2，叶　枫2，焦　洁2

1．第二军医大学附属长海医院妇产科，上海　200433；2. 浙江大学医学院附属妇产科医院妇瘤科，杭州　310006[摘要]　目的：从人卵巢癌组织和腹腔积液中分离培养肿瘤干细胞，分析细胞表型和化疗药物敏感性。方法：取卵巢浆腺癌患者的原发肿瘤组织及腹腔积液，通过无血清培养形成悬浮生长的球体。采用FCM及免疫荧光法检测干细胞标志物的表达。体外药敏实验检测获得的球体细胞和分化细胞对紫杉醇和卡铂的药物敏感性。结果：卵巢癌组织及腹腔积液中的卵巢癌细胞经无血清悬浮培养可以形成球体，后者具有CD44＋CD24－表型并表达Oct4、Nanog及Sox2等干细胞标志物；腹腔积液及组织来源的球体细胞经血清培养液培养3周后发生分化，CD44＋结论：CD24－细胞比例分别下降至47%和3%；分化细胞对紫杉醇和卡铂的敏感性均显著高于球体细胞（P＜0.01）。卵巢癌的肿瘤干细胞属于CD44＋CD24–细胞，后者表达Oct4等干细胞标志并具有显著耐药性。[关键词]　卵巢肿瘤；干细胞；球体；药物筛选试验，抗肿瘤[中图分类号]　R737.31

[文献标志码] A

[文章编号]　1000-7431 (2011) 10-0906-05

Isolation and culture of cancer stem cells from ovarian cancer tissues and malignant ascites

SHI Min-feng1, XIE Xing2, ZHU Min1, LÜ Wei-guo2, YE Feng2, JIAO Jie2

1. Department of Obstetrics & Gynaecology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; 2. Department of Gynecologic Oncology, Obstetrics & Gynaecology Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China

[ABSTRACT]　Objective: To isolate and culture tumor stem cells from ovarian cancer tissues and malignant ascites, and to analyze the cellular phenotype and drug sensitivity. Methods: The primary collected, and the formations of oat-growing spheres was developed with serum-free medium culture. The expressions of stem cell markers were evaluated by ow cytometry (FCM) and immuno uorescent staining. The sensibilities of sphere cells and differentiated cells to paclitaxol and carboplatin were measured by in vitro drug sensitivity assay. Results: The ovarian cancer cells in both tumor tissues tumor tissues and the malignant ascites samples from one patient with ovarian adenocarcinoma were

and malignant ascites displayed an ability to generate spheres cultured in serum-free medium. These sphere-forming cells exhibited the phenotype of CD44+CD24- and also expressed stem cell markers including Oct4, Nanog and Sox2. These sphere cells from tumor tissues and malignant ascites became differentiated after culture in serum-containing medium for 3 weeks, and the fractions of CD44+ and CD24- cells reduced by 47% and 3%, respectively. The differentiated cells showed increased sensitivities to paclitaxol and carboplatin as compared with the sphere cells (P＜0.01). Conclusion: The ovarian cancer stem cells exhibit the phenotype of CD44+CD24-, which is characterized by expressions of several stem cell markers including Oct4 and a high level of drug resistance.

[KEY WORDS ]　Ovarian neoplasms; Stem cells; Sphere; Drug screening assays, antitumor

[TUMOR, 2011, 31 (10): 906-910]

卵巢癌是死亡率最高的妇科肿瘤之一，起病隐匿，缺乏早期症状和有效的早期诊断手段，病情进展快[1]。标准治疗方案为肿瘤减灭术辅以术后铂类/紫杉醇药物联合化疗。初次化疗有效率

Correspondence to: SHI Min-feng（施敏凤）　　　　　E-mail: minfengshi@163.com Received 2011-06-15 Accepted 2011-09-07

达80%，但大多数患者在2～3年内复发。对复发肿瘤即使选用作用机制不同的药物也不易奏效，从而影响患者预后，5年生存率一般在30%左右[2]。因此，复发、转移和耐药是威胁卵巢癌患者生存的主要原因。

近年来提出的肿瘤干细胞（cancer stem

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