高、低侵袭性肝癌干细胞模型的建立及生物学特性鉴定

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摘要目的：采用人肝癌细胞HCCLM3建立高、低侵袭性肝癌干细胞（H/L-ILCSCs）模型，并与HCCLM3和正常肝细胞HL-7702相比较鉴定其生物学特性。方法：①利用Transwell小室迁移实验分离获得高、低迁移性HCCLM3细胞，再采用Transwell小室侵袭实验进一步分离获得高、低侵袭性HCCLM3细胞。②采用免疫磁珠分选法以CD133+为分子标志分离高、低侵袭性HCCLM3细胞，获得H/L-ILCSCs。③检测H/L-ILCSCs、HCCLM3细胞和正常肝细胞HL-7702干细胞表面标志分子CD133+、CD90+、CD44+的表达。④采用CCK-8法、Transwell侵袭、平板克隆形成及免疫缺陷动物成瘤实验，检测H-ILCSCs、L-ILCSCs、HCCLM3及HL-7702的生长增殖、体外侵袭、干细胞球形成及体内裸鼠成瘤能力。结果：表面标志分子CD133+、CD90+、CD44+在H-ILCSCs中高表达，而L-ILCSCs中CD133+高表达，CD90+和CD44+低表达。L-ILCSCs、HCCLM3和HL-7702细胞的生长增殖、体外侵袭、平板克隆形成及体内成瘤能力均低于H-ILCSCs。L-ILCSCs细胞的生长增殖和体外侵袭能力均低于HCCLM3细胞。此外，L-ILCSCs不能形成克隆球和移植瘤。结论：从正常人肝癌细胞HCCLM3中可以分离和富集H/L-ILCSCs，且二者生物学特性存在差别。

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	黎运呈
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关键词 ： HL-7702细胞, HCCLM3细胞, 侵袭性, 肝癌干细胞, 生物学特性

Abstract：Objective: To establish high/low invasion liver cancer stem cells (H/L-ILCSCs) model from human liver cancer cell line HCCLM3, and compare their biological properties with HCCLM3 and HL-7702 cells. Methods: ①Transwell immigration assay was used to separate high/low immigration HCCLM3 cells, then the high/low invasion HCCLM3 cells were isolated from high/low immigration HCCLM3 cells by Transwell invasion assay. ②H/L-ILCSCs were isolated from CD133+ high/low invasion HCCLM3 cells using magnetic activated cell sorting. ③The expression of cancer stem cell markers CD133+, CD90+ and CD44+ were detected in H-ILCSCs, L-ILCSCs, HCCLM3 and HL-7702 cells. ④The proliferation, invasion, clonal sphere formation and tumorigenicity were investigated by CCK-8 assay, Transwell invasion assay, monoclonal sphere formation and transplanted tumor formation assay in nude mice in H-ILCSCs, L-ILCSCs, HCCLM3 and HL-7702 cells. Results: High expression levels of cancer stem cell markers CD133+, CD90+ and CD44+ in H-ILCSCs, but high expression levels of CD133+ and low expression levels of CD90+ and CD44+ in L-ILCSCs. The abilities of proliferation, invasion, clonal sphere formation and tumorigenicity in L-ILCSCs, HCCLM3 and HL-7702 cells were lower than in H-ILCSCs. The abilities of proliferation and invasion in L-ILCSCs were lower than in HCCLM3 cells. Besides, stem cell spheres and nude mouse transplanted tumor couldn’t be established in L-ILCSCs cells. Conclusion: H/L-ILCSCs can be isolated and expanded from HCCLM3 cells, and both cells exhibit distinct biological properties.

Key words： HL-7702 cells HCCLM3 cells invasion liver cancer stem cell biological properties

基金资助:浙江省医药卫生科技计划项目（2013KYA211）。

作者简介: 黎运呈（1977-），男，浙江舟山人，副主任医师，博士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2018/V48/I8/572

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