OE19肿瘤干细胞的无血清分离培养以及肿瘤干细胞特性的鉴定  

Serum-free suspension culture and identification of cancer stem cells from human OE19 cell line

作　　者：尹晓然[1] 冯诚[2] 马一楠[1] 高晓燕[1] 刘欣[2] 贾丽君[1] 张寅斌[1] 张军[2] 

机构地区：[1]西安交通大学医学院第二附属医院肿瘤科,西安710004 [2]西安交通大学医学院第二附属医院消化科

出　　处：《山西医科大学学报》2017年第8期800-807,共8页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(81274136);陕西省社发攻关基金资助项目(2011K13-02-05)

摘　　要：目的从人Ⅱ型食管胃交界处腺癌OE19细胞中分离富集肿瘤干细胞的悬浮肿瘤细胞球,鉴定该悬浮肿瘤球细胞的肿瘤干细胞生物学特性。方法使用无血清培养法从OE19细胞株中富集得到悬浮肿瘤细胞球,并将其传代扩增;同时常规培养OE19亲本细胞。采用MTT实验、单细胞克隆形成实验、血清分化实验以及Transwell实验,研究OE19悬浮肿瘤球细胞与OE19亲本细胞的增殖、更新、分化和侵袭能力的差异;使用real-time PCR以及Western blot法检测两者肿瘤干细胞标志ABCG2和CD133的表达情况;使用裸鼠成瘤实验研究OE19悬浮肿瘤球细胞以及OE19亲本细胞的体内成瘤能力。由此鉴定悬浮肿瘤球细胞的肿瘤干细胞特性。结果在无血清培养条件下,OE19细胞能够形成悬浮肿瘤细胞球。悬浮肿瘤球细胞具有稳定传代的特性以及多向分化的潜能。OE19悬浮肿瘤球细胞与OE19亲本细胞相比较高表达肿瘤干细胞标志ABCG2和CD133(P<0.05),其体外增殖、自我更新、克隆形成、侵袭转移以及裸鼠体内成瘤能力均高于OE19亲本细胞(P<0.05)。结论通过无血清非黏附培养法可以有效富集OE19悬浮肿瘤细胞球。该悬浮肿瘤球细胞高表达肿瘤干细胞标志分子ABCG2和CD133,具有肿瘤干细胞特性。Objective To isolate the floating tumor spheres from human type Ⅱesophageal-gastric junction adenocarcinoma cell line OE19,and to identify their cancer stem cell characteristics. Methods OE19 cells were cultured in the serum-free medium to form the floating tumor spheres,and OE19 parent cells were cultured in serum medium with 10% FBS. MTT assay,single cell clone formation experiment,serum differentiation test and Transwell assay were employed to observe the proliferation,self-renewal,differentiation and invasive abilities of OE19 tumor spheres and OE19 parent cells. Real-time PCR and Western blot assays were performed to detect the expression of stem cell markers（ ABCG2 and CD133） in OE19 tumor sphere cells and OE19 parent cells. OE19 tumor sphere cells and OE19 parent cells were inoculated subcutaneously in nude mice and the tumor growth was assessed to identify the cancer stem cell property. Results In the serum-free medium,the cancer stem cells isolated from OE19 cells grew as floating tumor spheres,which had stable transmission and multi-directional differentiation characteristics. The expression of stem cell markers such as ABCG2 and CD133 in OE19 tumor sphere cells was higher than that in OE19 parent cells（ P〈0. 05）. Moreover,the proliferation,self-renewal,proliferation and invasive abilities of OE19 tumor sphere cells,and the tumorigenicity in nude mice of OE19 tumor sphere cells were stronger than that of OE19 parent cells（ P〈0. 05）. Conclusion Isolating tumor spheres without adhesion from OE19 cells in serumfree medium can be a quick and easy method. The floating tumor spheres are of high expression of ABCG2 and CD133,and possess cancer stem cell characteristics.

关 键 词：食管胃交界处腺癌 OE19细胞 无血清培养 肿瘤干细胞 肿瘤细胞球 

分 类 号：R735[医药卫生—肿瘤]