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目的:检测高转移潜能肝癌HCCLM3细胞株中侧群(side population,SP)细胞比例,分选SP细胞并探讨其生物学特性。方法:4种肝癌细胞株的转移潜能大小依次为HCCLM3>MHCC97H>MHCC97L>Huh7,分别予以荧光染料Hoechst33342染色,以加入维拉帕米的拮抗组为对照,流式细胞术检测SP细胞比例;从HCCLM3细胞中分选SP细胞和主群(main population,MP)细胞,采用悬浮球培养、Transwell侵袭实验及裸鼠成瘤实验判断HCCLM3 SP细胞是否具备肿瘤干细胞生物学特性;RT-PCR检测SP及MP细胞中干细胞相关基因的表达。结果:HCCLM3细胞中SP细胞比例为(16.9±1.8)%,显著多于MHCC97H、MHCC97L、Huh7中SP细胞比例[(8.4±0.7)%、(4.6±0.5)%、(1.0±0.2)%,均P<0.05]。HCCLM3细胞中,SP细胞悬浮球形成明显高于MP细胞[(25.3±5.1)%vs(11.2±2.6)%,P<0.05],SP细胞的侵袭能力显著高于MP细胞(P<0.05),SP细胞的裸鼠皮下成瘤能力也明显高于MP细胞。HCCLM3细胞的SP细胞中干细胞相关基因ABCG2、CD13、CD44、Nanog、Sox2及Klf4 mRNA平均表达水平分别是MP细胞的5.64、2.26、2.10、3.78、2.37、2.92倍(均P<0.05)。结论:肝癌HCCLM3细胞的SP细胞中富集了肝癌干细胞,这可能与其较高的转移潜能有关。
OBJECTIVE: To detect the proportion of side population (SP) cells in HCCLM3 cell line with high metastatic potential and to isolate the SP cells and to investigate their biological characteristics. Methods: The metastatic potentials of four hepatocellular carcinoma cell lines were HCCLM3> MHCC97H> MHCC97L> Huh7, respectively, and stained with fluorescent dye Hoechst33342. The antagonist group of verapamil was used as control, and the proportion of SP cells was detected by flow cytometry. SP cells and main population (MP) cells were sorted by HCCLM3 cells. The growth of HCCLM3 SP cells was assessed by Transwell invasion assay and nude mice tumorigenicity assay. RT-PCR was used to detect SP and Expression of Stem Cell Related Genes in MP Cells. Results: The proportion of SP cells in HCCLM3 cells was (16.9 ± 1.8)%, significantly higher than that in MHCC97H, MHCC97L and Huh7 cells (8.4 ± 0.7%, 4.6 ± 0.5%, 1.0 ± 0.2% All P <0.05]. In HCCLM3 cells, the spheroids of SP cells were significantly higher than that of MP cells [(25.3 ± 5.1)% vs (11.2 ± 2.6)%, P <0.05], and the invasive ability of SP cells was significantly higher than that of MP cells SP cells in nude mice subcutaneously tumorigenic ability was also significantly higher than the MP cells. The mean expression of stem cell related genes ABCG2, CD13, CD44, Nanog, Sox2 and Klf4 in HCCLM3 cells were 5.64,2.26,2.10,3.78,2.37,2.92 times that of MP cells respectively (all P <0.05). CONCLUSION: Liver cancer stem cells are enriched in SP cells of HCCLM3 cells, which may be related to their high metastatic potential.