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目的:建立一种体外有效富集、培养和鉴定具有肝癌干细胞特征的细胞亚群的方法。方法:采用成球培养法利用肿瘤干细胞样细胞(cancer stem cell,CSC)分化培养基对肝癌Huh7细胞进行富集培养,获得的干细胞样细胞于体外进一步扩增获得肝癌干细胞球。流式细胞术检测Huh7干细胞样细胞表面肿瘤干细胞标志物EpCAM、CD90和CD133的表达,平板克隆集落形成实验和裸鼠成瘤实验分别检测Huh7细胞和Huh7干细胞样细胞的克隆集落形成能力、体内成瘤能力。结果:Huh7细胞成球培养3~7 d后即可形成肝癌干细胞样细胞球,获得的干细胞样细胞具有自我更新和增殖能力,其EpCAM阳性细胞比例较Huh7细胞明显增加[(99.6%±0.31)%vs(0.12%±0.05)%,P<0.01],但两种细胞CD90[(0.11%±0.06)%vs(0.09%±0.07)%,P>0.05]和CD133[(0.17%±0.08)%vs(0.15%±0.05)%,P>0.05〗表达差异无统计学意义。Huh7干细胞样细胞克隆集落形成数量明显多于Huh7细胞[(188.67±12.5)vs(79±16.7)个,P<0.01];当接种量为5×104个细胞时,与接种Huh7细胞的对照裸鼠相比,接种Huh7干细胞样细胞的裸鼠成瘤时间更短(11 vs 30 d),成瘤率更高(100%vs 16.67%);接种5×105数量级的细胞时,实验组成瘤体积[(171.90±10.94)vs(86.39±11.21)mm3,P<0.01]和瘤体质量[(2.98±0.82)vs(0.32±0.17)g,P<0.01]均明显大于对照组。结论:利用成球培养法能够从Huh7肝癌细胞系中富集培养获得Huh7肝癌干细胞,其具有比Huh7细胞更强的成瘤能力。
OBJECTIVE: To establish a method for enriching, culturing and identifying cell subsets with hepatocellular carcinoma stem cells in vitro. Methods: The hepatocellular carcinoma Huh7 cells were enriched and cultured by using the culture medium of cancer stem cells (CSC) by using the method of ball formation culture. The obtained stem cell-like cells were further expanded in vitro to obtain the hepatoma stem cell spheres. Flow cytometry was used to detect the expression of EpCAM, CD90 and CD133 on Huh7 stem cell-like cell surface. Clonal colony formation assay and nude mouse tumorigenicity assay were used to detect the colony forming ability of Huh7 cells and Huh7 stem cell-like cells. Tumor capacity. Results: After culturing Huh7 cells for 3 ~ 7 d, hepatoma stem cell-like cytoplasm could be formed. The obtained stem cell-like cells had the ability of self-renewal and proliferation. The percentage of EpCAM positive cells was significantly higher than that of Huh7 cells [(99.6% ± 0.31) (0.11% ± 0.06)% vs (0.09% ± 0.07)%, P> 0.05] and CD133 [(0.17% ± 0.08)%, P <0.01] % vs (0.15% ± 0.05)%, P> 0.05 There was no significant difference in expression between the two groups. The number of colonies of Huh7 stem cell-like cells was significantly higher than that of Huh7 cells [(188.67 ± 12.5) vs (79 ± 16.7), P <0.01]. When the inoculum size was 5 × 104 cells, Nude mice inoculated with Huh7 stem cell-like cells had shorter tumorigenicity (11 vs 30 d) and higher tumor formation rate (100% vs 16.67%) than in control mice. When inoculated with 5 × 10 5 cells, the tumor volume [(171.90 ± 10.94) vs (86.39 ± 11.21) mm3, P <0.01] and the mass of the tumor [(2.98 ± 0.82) vs (0.32 ± 0.17) g, P <0.01] CONCLUSION: Huh7 hepatoma stem cells can be enriched and cultured from Huh7 hepatoma cell lines by using the culture-ball culture method, which has a stronger ability of tumorigenesis than Huh7 cells.