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中华结直肠疾病电子杂志 ›› 2013, Vol. 02 ›› Issue (04) : 170 -176. doi: 10.3877/cma.j.issn.2095-3224.2013.04.06.

所属专题: 文献

论著

AC133抗原联合EpCAM识别转移性结直肠癌干细胞的研究
杜航向1, 杨治力1, 杨逸1, 盛能全1, 王志刚1,()   
  1. 1. 200233 上海交通大学附属第六人民医院普外科
  • 收稿日期:2013-06-07 出版日期:2013-08-25
  • 通信作者: 王志刚
  • 基金资助:
    上海市科委自然基金(11ZR1427500)

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

Hang-xiang DU1, Zhi-li YANG1, Yi YANG1, Neng-quan SHENG1, Zhi-gang WANG1,()   

  1. 1. General Surgery Department of Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200233, China
  • Received:2013-06-07 Published:2013-08-25
  • Corresponding author: Zhi-gang WANG
  • About author:
    Corresponding author: WANG Zhi-gang, Email:
引用本文:

杜航向, 杨治力, 杨逸, 盛能全, 王志刚. AC133抗原联合EpCAM识别转移性结直肠癌干细胞的研究[J]. 中华结直肠疾病电子杂志, 2013, 02(04): 170-176.

Hang-xiang DU, Zhi-li YANG, Yi YANG, Neng-quan SHENG, Zhi-gang WANG. The study of combining AC133 antigen and E-cadherin in the identifying metastatic colorectal cancer stem cells[J]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2013, 02(04): 170-176.

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AC133EpCAM结直肠癌干细胞,结直肠癌原代细胞中AC133EpCAM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AC133EpCAM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AC133EpCAM细胞移植在Balb/C裸鼠体内,表现出很强的致瘤性,移植瘤中AC133EpCAM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AC133EpCAM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 AC133+ 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 AC133+ EpCAM+ surface marker, which possess apparent feature of stem cells and stronger capacity of initiating tumors, moreover, generated spheroid in serum-free medium.These findings could provide foundation for the follow-up experiments.

图1 AC133EpCAM细胞和AC133-EpCAM-细胞体外培养倒置相差显微镜观察图像
图2 AC133EpCAM细胞和AC133-EpCAM-细胞克隆形成实验平皿在倒置显微镜下观察图像
图3 AC133EpCAM细胞和AC133-EpCAM-细胞经多细胞球培养后倒置相差显微镜下观察图像
图4 接种AC133EpCAM细胞和AC133EpCAM细胞的裸鼠肿瘤标本图像
表1 Balb-C裸鼠成瘤率的比较
图5 AC133EpCAM细胞和AC133-EpCAM-细胞及其微球体微孔膜下层显微镜观察图像
图6 AC133EpCAM细胞和AC133EpCAM细胞体外增殖能力生长曲线图
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