华西口腔医学杂志

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舌癌单细胞培养建系与癌干细胞相关标志的检测

姚金光  解继胜  李俊  韦星 杨永荣 陈海波   

  1. 右江民族医学院 肿瘤分子生物学实验室, 百色 533000
  • 出版日期:2013-02-01 发布日期:2013-02-01
  • 通讯作者: 姚金光,Tel:0776-2848820
  • 作者简介:姚金光(1965—),男,广西人,教授,博士
  • 基金资助:

    国家自然科学基金资助项目(30860326);广西自然科学基金资助项目(桂科自0640202);广西科学研究与技术开发计划基金资助项目(桂科攻0816004);广西教育厅科研基金资助项目[桂教科研(2005)47号]

Establishment of subseries cell lines from tongue cancer single cell and detection of cancer stem cell markers

 Yao Jinguang, Xie Jisheng, Li Jun, Wei Xing, Yang Yongrong, Chen Haibo   

  1. Oncomolecularbiology Laboratory, You jiang Medical University for Nationalities, Baise 533000, China
  • Online:2013-02-01 Published:2013-02-01

摘要:

目的  以舌癌Tca8113M1细胞系单细胞培养建系为基础,观察Tca8113M1细胞系中舌癌干细胞存在的现象及其相关标志的变化规律。方法  选取Tca8113M1细胞系,以有限稀释法进行体外单细胞培养并建立细胞亚系,在证实其成瘤性的基础上,进一步采用流式细胞术检测癌干细胞相关标志CD44、CD184、细胞外可溶性抗原(ESA)的表达情况,着重观察单个细胞培养形成细胞克隆的形态与时间。结果  以有限稀释法获取192个Tca8113M1舌癌单个细胞,在96孔板中进行体外培养,获取12个细胞亚系(获取比例为6.25%),均有高成瘤性。癌干细胞相关标志CD44 与ESA均为高水平表达,而CD184表达则在12个细胞系之间有差异。在单个细胞培养中,形成完全克隆、部分克隆与旁克隆3种形态,12个细胞亚系均源于单个细胞形成的完全克隆,均可进行连续传代与扩增,而部分克隆与旁克隆则在后续培养中逐渐衰老与消亡。结论 Tca8113M1细胞系中可能存在癌干细胞,而单细胞培养可形成完全克隆并建立细胞亚系,是进行舌癌干细胞后续研究重要的细胞培养模式。

关键词: 单细胞, 有限稀释法, 舌癌干细胞, 完全克隆

Abstract:

Objective  To establish subseries cell lines from single cancer cell of Tca8113M1 cell line and detect the cancer stem cell markers in the different subseries cell lines. Methods  The subseries cell lines from single cancer cell of Tca8113M1 cell line were established by limiting dilution assay in vitro. The  characteristic of tumorigenicity and CD44, CD184, extracellular soluble antigen(ESA) of the cancer stem cell markers were detected by xenotransplantation and flow cytometry respectively. Results  Total 192 single cells of Tca8113M1 cell line were cultured and were deposited as one cell per well. There were 12 subpopulations origin from 192 single cells spheroid cultures. The ratio was 6.25%(12/192). In the different subpopulations, the tumorigenicity and expression of CD44 and ESA were at high levels, but the expression of CD184 was in different level. There were three kinds morphology of colonies derived from single cancer cells, holoclone, meroclone and paraclone. Cell line could be derived from carcinoma cell holoclones by cell culture. Meroclone and paraclone did not exist in cell culture in vitro. Conclusion  Tongue cancer stem cell may exist in Tca8113M1 cell line, cell line can be established and holoclone is the origin of cell line. This is a novel approach to the identification and enrichment for cancer stem cell.

Key words: single cell, limiting dilution assay, tongue cancer stem cell, holoclone