不同培养条件对CD133^(+)原代口腔鳞癌细胞干性维持的影响

Maintenance of the stemness in CD133^(+) primary oral squamous cell carcinoma cells under different culture conditions

目的:通过将CD133^(+)/-细胞从原代口腔鳞癌细胞中分离纯化,探索不同培养条件对CD133^(+)原代口腔鳞癌细胞的干性维持及生物学特性的影响。方法:应用CCK-8法检测CD133^(+)/-细胞亚群体外增殖能力以及对顺铂抵抗耐受能力,利用Transwell法检测顺铂对CD133^(+)/-细胞亚群侵袭能力的影响。以无血清培养法(含或不含白血病抑制因子LIF)和含血清培养法分别培养CD133^(+)细胞亚群,流式细胞仪分析CD133^(+)细胞的比例变化。在动物实验模型中验证CD133^(+)和CD133-细胞致瘤能力的差异,最后将移植瘤取出,行H-E染色和免疫组织化学染色。采用SPSS 25.0软件包对数据进行统计学分析。结果:与CD133-细胞亚群相比,CD133^(+)细胞具备较强的体外增殖能力(P<0.05)和顺铂化疗耐受能力(P<0.001)。顺铂对CD133-细胞亚群侵袭能力的影响更强(P<0.01)。无血清培养法更能维持CD133的比例(P<0.05),无血清培养基是否添加LIF对CD133的比例维持无显著差异(P>0.05)。在裸鼠体内成瘤实验中,CD133^(+)细胞亚群以较少的数量级表现出较强的致瘤能力(P<0.05)。结论:无血清培养法可较好地维持原代口腔鳞癌细胞干细胞特性,添加LIF对原代口腔鳞癌细胞的干性维持无显著影响。

PURPOSE:CD133^(+)/-cells were isolated and purified from primary oral squamous cell carcinoma(OSCC)to explore the effects of different culture conditions on the maintenance and biological characteristics of CD133^(+)primary OSCC.METHODS:CCK-8 was used to detect the ability of proliferation and cisplatin resistance between CD133^(+)/-cell subsets.Transwell assay was used to compare the invasive ability of two cell subsets under the action of cisplatin.Flow cytometry was used to detect the proportion of CD133^(+)cells cultured by serum free medium(SFM)(with or without leukemia inhibitory factor,LIF)or serum supplied medium(SSM).Subcutaneous tumor model in nude mice was used to verify the difference in tumorigenicity of CD133^(+)/-cell subsets.The transplanted tumor was removed for H-E staining and immunohistochemistry(IHC).SPSS 25.0 software package was used for statistical analysis.RESULTS:Compared with CD133-cell subsets,CD133^(+)cell subsets had stronger ability of proliferation in vitro(P<0.05)and cisplatin tolerance(P<0.001).Cisplatin had a stronger effect on the invasive ability of CD133-cell subsets than CD133^(+)cell subsets(P<0.01).No significant difference in the proportion of CD133^(+)cell between LIF-SFM and no-LIF-SFM was found(P>0.05);but compared with SSM culture method,SFM culture method could maintain the proportion of CD133^(+)cell better(P<0.05).CD133^(+)cell subsets showed stronger tumorigenic ability with fewer cells than CD133-cell subsets in nude mice(P<0.05).CONCLUSIONS:Serum free culture method can better maintain the characteristics of primary OSCC stem cells,but the addition of LIF has no significant effect on the maintenance of stemness of primary OSCC cells.