肝癌高转移细胞株的肿瘤干细胞生物学特性研究

摘要

目的：建立高转移肝癌细胞株，研究其肿瘤干细胞生物学特征，为靶向人肝癌干细胞治疗提供有价值的细胞模型。方法：肝癌细胞系 Bel7402接种裸鼠皮下，成瘤后，取小鼠肺部转移灶，通过机械分离法，获取肺部肝转移细胞，体外扩大培养后，再次接种裸鼠。如此反复接种裸鼠，获得稳定肺转移肝癌细胞株 Bel7402- V13。采用无血清悬浮培养及 PKH26染色确定 Bel7402- V13中肿瘤干细胞的存在。流式细胞术分析亲本Bel7402和 Bel7402- V13中肿瘤干细胞标志物 ESA 的表达情况，分选 Bel7402和 Bel7402- V13中 ESA +细胞并进行体外生物学特征研究及裸鼠致瘤实验。结果：建立高转移 Bel7402- V13细胞株，Bel7402- V13细胞无血清悬浮培养7d 后形成的细胞球体中存在单个 PKH26阳性细胞。与 Bel7402中 ESA +细胞相比，流式分析显示干细胞标志物 ESA 比例高转移 Bel7402- V13显著提高5.67倍（17.6±0.4 vs 3.1±1.5）。Bel7402-V13中 ESA +细胞具有更强的自我更新能力[成球率：（94.8±7.5）% vs（52.3±6.9）%，P <0.01]，侵袭能力提高1.51倍（397.7±79.4 vs 262.0±40.1，P <0.01），耐药能力也显著增强（IC50：0.286 vs 0.196，P <0.01）， ESA +细胞 Bel7402- V13在裸鼠皮下接种2×102个细胞3月即可致瘤（3/6），而接种2×102个 ESA +细胞Bel7402细胞3月才能致瘤（1/6）。结论：获得高转移肝癌细胞株 Bel7402- V13，伴随 ESA +细胞增加，其体内外功能显著增强，为肝癌干细胞的靶向治疗提供有价值的细胞模型。%Objective:To establish a high metastatic cell line derived from human hepatic carcinoma cell line Bel7402 and study the characteristics of cancer stem cells. Methods:Bel7402 inoculated into subcutaneous tumor, nude mice lung metastases by mechanical separation was repeated inoculation in nude mice to obtain Bel7402 - V13. Non - adherent culture and PKH26 staining was used to determine whether there were cancer stem cells in Bel7402 -V13. Flow cytometry analysis was performed for stem marker ESA within Bel7402 and Bel7402 - V13. ESA + cells of Bel7402 and Bel7402 - V13 were sorted to identify its biological characteristics in vivo and tumorigenicity in vitro. Results:The single PKH26 positive cell was observed in spheroids after Bel7402 - V13 cells were cultured for 7 days. Compared with Bel7402,the proportion of stem cell marker ESA was significantly increased in Bel7402 - V13 [(72. 9 ± 1. 5)% vs(8. 96 ± 1. 2)% ]. The sorted ESA + cells of Bel7402 - V13 presented higher self - renewal a-bility[sphere formation rate:(27. 8 ± 1. 7)% vs(20. 4 ± 1. 0)% ,P < 0. 01],invasion ability enhanced 1. 64 times (329 . 5 ± 7 . 5 vs 200 . 0 ± 2 . 0 ),resistance ability enhancement and the IC50 increased 1. 46 times(0. 286 vs 0. 196),tumorigenicity also was significantly enhanced. Conclusion:The high metastatic cell line Bel7402 - V13 was obtained,and in vivo and in vitro function of ESA + cells was significantly enhanced,which provided a valuable cell model for the targeted therapy of human hepatic carcinoma stem cells.