目的分离与鉴定肝癌细胞HepG2中CD133标记的肝癌干细胞（LCSC）,初步探讨曲格列酮（Tro）对HepG2及CD133⁺ LCSC的细胞毒性差异。方法利用流式细胞仪分选纯化HepG2中的CD133⁺和CD133^-细胞,采用悬浮微球形成法、平板克隆形成法、Transwell侵袭和迁移实验检测HepG2、CD133⁺和CD133^-细胞的自我更新能力；BALB/c裸鼠体内成瘤实验检测HepG2和CD133 LCSC细胞的成瘤能力；流式细胞术检测HepG2、CD133⁺ LCSC细胞周期,MTT法测定其对索菲替尼的耐药性；MTT法检测Tro对HepG2、CD133⁺ LCSC的毒性情况,全自动生化分析仪测定其对细胞上清天冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）、白蛋白（ALB）、尿素氮（BUN）和总蛋白（TP）水平的影响,荧光法检测Tro对细胞CYP450总活性和ROS水平的影响。结果 CD133标记的CSC在HepG2中占（0.72±0.05）%,CD133⁺细胞分选后纯度为98.7%；CD133+细胞微球形成能力、克隆形成能力以及Transwell迁移与侵袭能力、肿瘤形成能力明显高于亲本（P<0.05、0.01）；CD133⁺细胞群大多处于G₀/G₁期,G₂/M期未阻滞,并且对索菲替尼表现较大耐药性（P<0.05、0.01）；Tro处理12、24、48、72 h后,CD133⁺ LCSC半数抑制浓度（IC₅₀）显著低于HepG2（P<0.05、0.01）；80 μmol/L Tro处理48 h时,LCSC上清AST、TP、LDH、ALB、BUN生化指标不同程度升高,CYP450总活性和ROS水平出现明显抑制（P<0.05、0.01）。结论成功筛选和鉴定具有高增殖能力的CD133+HepG2 CSC,其在Tro引起肝细胞毒性方面较HepG2细胞更敏感,细胞毒性差异显著,为Tro靶向CD133+LCSC的细胞毒性研究提供新思路。

Objective To isolate and identify CD133 liver cancer stem cells (LCSC) in HepG2 cells, and to investigate the cytotoxicity of troglitazone (Tro) on HepG2 and its liver stem cells. Methods The CD133⁺ and CD133^- population in the HepG2 cell line were sorted by flow cytometry. Cells of CD133 sphere formation, colony formation assay, Transwell invasion migration assay, and BALB/c nude mice in vivo tumor formation were used to identify proliferative capacity of HepG2 cells and CD133 LCSC. Flow cytometry was used to detect the cell cycle of HepG2 and CD133⁺ LCSC, and LCSC drug resistance to sorafenlb was evaluated by MTT. The toxicity of Tro to CD133 LCSC was detected by MTT assay, automatic biochemical analyzer was used to detect the contents of AST, LDH, TP, ALB and BUN changes in cell supernatants after administration, and the effect of Tro on the total activity of CYP450 and level of ROS was examined by fluorescence method to compare the cytotoxicity differences. Results CD133 expression in LCSC were found to be (0.72 ±0.05)% in HepG2 and 98.7% in CD133⁺ cells. The ability of sphere formation, colony formation and Transwell invasion migration of CD133⁺ cells were higher than those of parental cells (P < 0.05 and 0.01). Compared with HepG2 cell, the ability of CD133⁺ cell tumor formation was significantly increased. At the same time, CD133⁺ cells were mostly in G₀/G₁ phase, G₂/M phase was not blocked, and the drug resistance of sorafenlb was higher than HepG2 cell (P < 0.05 and 0.01). IC₅₀ of CD133⁺ cell was significantly lower than that of HepG2 after treated with Tro for 12, 24, 48 and 72 h (P < 0.01). TP, LDH, ALB and BUN in LCSC treated with 80 μmol/L Tro for 48 h increased significantly, and the total activity of CYP450 and ROS were significantly inhibited (P < 0.05 and 0.01). Conclusion Highly proliferative CD133⁺ HepG2 tumor stem cells was successfully isolated and identified, which was more sensitive than HepG2 cells in liver cytotoxicity induced by Tro, showing significant cytotoxicity difference. This study provides a new idea for the study of CD133⁺ HepG2 cells specific toxicity.

国家重大新药创制科技重大专项（2013ZX09302303，2012ZX09301-001-008）；北京市科委基金项目（Z131100006513010）