AC133抗原联合EpCAM识别转移性结直肠癌干细胞的研究  

The study of combining AC133 antigen and E-cadherin in the identifying metastatic colorectal cancer stem cells

作　　者：杜航向[1] 杨治力[1] 杨逸[1] 盛能全 王志刚[1] 

机构地区：[1]上海交通大学附属第六人民医院普外科,200233

出　　处：《中华结直肠疾病电子杂志》2013年第4期170-176,共7页Chinese Journal of Colorectal Diseases(Electronic Edition)

基　　金：上海市科委自然基金(11ZR1427500)

摘　　要：目的利用AC133抗原联合EpCAM检测人结直肠癌组织中结直肠癌干细胞,并初步观察其生物学特性,为后续结直肠癌干细胞研究提供实验基础。方法利用新鲜结直肠癌组织,无血清悬浮成球培养,流式细胞检测AC133、EpCAM的表达情况,Balb/C小鼠移植观察其成瘤情况。组间比较采用t检验,以P<0.05为差异有统计学意义。结果从人原代结肠腺癌中分离、纯化AC133+EpCAM+结直肠癌干细胞,结直肠癌原代细胞中AC133+EpCAM+细胞比例为1.5%~35%(平均4.8%)。单克隆形成实验证实结直肠癌组织中存在肿瘤干细胞。其比例为2.18±0.15%,分离获得的AC133+EpCAM+细胞能在无血清培养基中'成球',在血清诱导下能贴壁分化;将AC133+EpCAM+细胞移植在Balb/C裸鼠体内,表现出很强的致瘤性,移植瘤中AC133+EpCAM+细胞比例为4.9%~40.2%(平均17.2%)。结论人结肠腺癌组织中存在AC133+EpCAM+细胞群,具有和普通干细胞相类似的无限增殖、自我更新和分化能力,可为人结直肠癌干细胞的后续研究提供实验基础。Objective To isolate colorectal cancer stem cells from human fresh carcinoma samples obtained at surgical operation and to study their biological characteristics .Then to provide foundation for the follow-up experiments .Methods Colorectal cancer tissues obtained from surgically resected specimens of colorectal cancer patients were fully chopped ,trypsinized,and filtered to prepare single cell suspensions .The cells were cultured in serum-free DMEM/F12 medium,with LIF,EGF,bFGF and 2% fetal bovine serum weekly.The stem cell-markers AC133 and EpCAM on the isolated cells were detected by using flow cytometry.The tumorigenic potent of the isolated cells was evaluated via the transplantation into Balb /C nude mice.All the variables were tested with T-test.P<0.05 was considered to be statistically significant .Results AC133 +EpCAM + colorectal cancer stem cells were isolated and purified from the human primary colorectal carcinoma.The percentage of AC133 +EpCAM +colorectal cancer stem cells in primary colorectal cancer was 1.5%~35%( mean 4.8%).There were colon cancer stem cells in colon cancer by limited dilution assay.The percentage was 2.18 ±0.15%.The suspension spheres were generated by the isolated AC 133 +EpCAM +Co-CSCs in serum-free medium.Their differentiation were induced in medium containing 10%FBS.AC133 +EpCAM +CCSCs were implanted into Balb/C nude mice ,and observed stronger capacity of tumor initiation.The percentage of AC133 +EpCAM + cells in xenografts was 4.9% ~40.2%( mean 17.2%).Human CRC xenografts established in Balb/C nude mice grew as differentiated carcinomas ,which formed gland-like structures and scored positive for expression of colon-specific differentiation markers ,such as cytokeratin-20(CK20),and neutral epithelial mucins .Conclusions There are colon cancer stem cells in human colorectal carcinoma .These cells are characterized by AC 133 +EpCAM + surface marker , which possess apparent feature of stem cells and stronger capacity of initiating tumors ,moreover ,generated spheroid in serum-fr

关 键 词：AC133 上皮细胞黏附分子 结直肠癌干细胞 

分 类 号：R735.34[医药卫生—肿瘤]