槐耳清膏在人结肠癌HCT116细胞株中对肿瘤干细胞的作用研究

Screening,Culture and Identification of Tumor Stem Cells in Human Colon Cancer HCT116 Cell Line and Effects of Huaier Soft Extract on Tumor Stem Cells

目的从人结肠癌细胞系HCT116中筛选鉴定肿瘤干细胞,观察分析其生物学特性,研究其耐药性以及了解槐耳清膏对肿瘤干细胞的抑制作用。方法通过无血清有限稀释法筛选肿瘤干细胞样克隆球,通过流式细胞术检测肿瘤干细胞标志物的阳性表达率和细胞周期,软琼脂集落形成实验以及药物干预实验来鉴定克隆球的肿瘤干性,通过CCK-8实验研究槐耳清膏对肿瘤干细胞的抑制作用。结果与HCT116细胞相比,克隆球细胞具有更高的成球率(43.2%vs 28.6%),肿瘤标志物CD133+CD44+占93.87%,处于静止期的克隆球细胞比例更高(77.18%vs 49.00%),表现出更强的耐药性;经无血清有限稀释法获得的细胞克隆球表现出明显的肿瘤干细胞特性,证明筛得的细胞即为肿瘤干细胞;槐耳清膏及氟尿嘧啶干预实验表明,肿瘤干细胞较原代细胞具有更强的耐药性,槐耳清膏对肿瘤干细胞均有抑制作用,且较氟尿嘧啶抑制作用强。结论:采用本实验方法可筛选得到肿瘤干细胞;槐耳清膏对肿瘤干细胞具有抑制作用可能更强,且不易产生耐药性。

Objective To screen and identify tumor stem cells from human colon cancer cell line HCT116 as well as observing their biological characteristics and drug resistance,and to investigate the inhibitory effect of Huaier Soft Extract(the concentrated preparation of extract from Trametes robiniophila Murr.)on tumor stem cells.Methods Tumor stem cell-like clones were screened by serum-free limited dilution method.The positive expression rate and cell cycle of tumor stem cell markers were detected by flow cytometry,and the tumor stem cells were identified by soft agar assay for colony formation and drug intervention experiment.CCK-8 test was used to study the inhibitory effect of Huaier Soft Extract on tumor stem cells.Results Compared with HCT116 cells,colony cells had higher conglomeration rate(43.2%vs 28.6%),tumor marker CD133+CD44+accounted for93.87%,and colony cells in stationary stage accounted for higher percentage(77.18%vs 49.00%)and showed stronger drug resistance.The colony cells obtained by serum-free limited dilution method showed were confirmed as the tumor stem cells for their biological characteristics.The intervention experiments of Huaier Soft Extract and fluorouracil showed that cancer stem cells had stronger drug resistance than primary cells,Huaier Soft Extract had inhibitory effect on cancer stem cells and the effect was stronger than fluorouracil.Conclusion Tumor stem cells can be obtained by the established tests.Huaier Soft Extract exerts inhibitory effect on cancer stem cells and with good anti-drug resistance.