手术切除的结直肠癌组织中的肿瘤干细胞的培养及鉴定  

Culture and identification of tumor stem cells from surgically resected colorectal cancer tissues

作　　者：唐娜 周略 成志强 邓永键[4,5] 丁彦青[4,5] TANG Na;ZHOU Lue;CHENG Zhiqiang;DENG Yongjian;DING Yanqing(Department of Pathology, Shenzhen People's Hospital/ First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518020, China;Department of Pathology, Shenzhen People's Hospital/Second Clinical College of Jinan University, Shenzhen 518020, China;Department of Radiation Oncology, Shenzhen People's Hospital/First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518020, China;Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China)

机构地区：[1]南方科技大学第一附属医院//深圳市人民医院病理科,广东深圳518020 [2]暨南大学第二临床医学院//深圳市人民医院病理科,广东深圳518020 [3]南方科技大学第一附属医院//深圳市人民医院放疗科,广东深圳518020 [4]南方医科大学南方医院病理科,广东广州510515 [5]南方医科大学基础医学院病理学系,广东广州510515

出　　处：《南方医科大学学报》2019年第4期415-421,共7页Journal of Southern Medical University

基　　金：国家重点研发计划(973项目;2016YFC1201801);国家自然科学基金(81702359;81672453);广东省自然科学基金(2015A030310089)资助;深圳市科技计划项目(JCYJ20180301170035531)~~

摘　　要：目的获得结直肠癌患者手术切除癌组织的肿瘤干细胞(CSC),培养并鉴定其干细胞特性。方法通过原代培养手术切除结直肠癌组织获得结直肠癌细胞,再通过有限稀释法和无血清培养法筛选结直肠癌CSC,软琼脂集落实验检测CSC和对照组人结直肠癌细胞株SW480的增殖能力;用低数量级细胞裸鼠皮下成瘤实验,检测结直肠癌CSC和SW480的成瘤能力;并运用Western blot及免疫荧光方法,检测CSC和SW480的免疫表型。结果临床结直肠癌标本原代培养的CSC,加入血清培养可使CSC分化。软琼脂集落形成实验结果显示,原代培养的CSC克隆形成率为56.64%和54.45%,对照组人结直肠癌细胞株SW480的克隆形成率为4.41%,CSC与SW480集落形成率的差异具有统计学意义(P<0.01)。裸鼠皮下成瘤实验中,原代培养的CSC皮下注射500个细胞可形成皮下瘤,而SW480皮下注射500个细胞不形成皮下瘤。CSC表达CD133、CD44,不表达CK7;SW480细胞不表达CD133、CD44,表达CK7。结论原代培养的手术切除结直肠癌组织标本获得的肿瘤细胞,再行无血清培养可形成CSC,鉴定具有CSC的自我更新及分化能力。Objective To obtain cancer stem cells(CSCs) from surgically resected colorectal cancer specimens and identify their stem cell characteristics. Methods Colorectal cancer tissue specimen obtained from a patient undergoing radical resection of colorectal cancer were implanted in nude mice, and the xenograft was harvested 1 month later to obtain purified tumor cells by enzyme digestion and adherent culture. The CSCs were screened by limiting dilution method and serum-free culture to identify their phenotypes. Soft agar colony assay was used to assess the proliferative ability of the CSCs and human colorectal cancer cell line SW480. The tumorigenic ability of the isolated CSCs and SW480 cells was evaluated by observing their subcutaneous tumor formation in nude mice. Western blotting and immunofluorescence assay were used to detect the immunophenotype of the CSCs and SW480 cells. Results The primary cultured CSCs from clinical specimens of colorectal cancer underwent differentiation in the presence of serum in the culture. Soft agar colony formation assay showed that the CSCs had a colony formation rate above 50%, significantly higher than the rate of colorectal cancer SW480 cells(4.41%;P<0.01).In nude mice, subcutaneous injection of 500 CSCs was sufficient to result in subcutaneous tumor formation, while the injection of 500 SW480 cells failed to form any subcutaneous tumors. The CSCs expressed CD133 and CD44 but not CK7, while SW480 cells expressed CK7 but not CD133 or CD44. Conclusion CSCs can be derived by primary culture of cancer cells obtained from surgically resected colorectal cancer tissue followed by serum-free culture, and the CSCs obtained have self-renewal and differentiation abilities.

关 键 词：肿瘤干细胞 结直肠癌 原代培养 

分 类 号：R735.34[医药卫生—肿瘤]