目的分離與鑒定肝癌細(xì)胞HepG2中CD133標(biāo)記的肝癌干細(xì)胞（LCSC）,初步探討曲格列酮（Tro）對HepG2及CD133⁺ LCSC的細(xì)胞毒性差異。方法利用流式細(xì)胞儀分選純化HepG2中的CD133⁺和CD133^-細(xì)胞,采用懸浮微球形成法、平板克隆形成法、Transwell侵襲和遷移實驗檢測HepG2、CD133⁺和CD133^-細(xì)胞的自我更新能力；BALB/c裸鼠體內(nèi)成瘤實驗檢測HepG2和CD133 LCSC細(xì)胞的成瘤能力；流式細(xì)胞術(shù)檢測HepG2、CD133⁺ LCSC細(xì)胞周期,MTT法測定其對索菲替尼的耐藥性；MTT法檢測Tro對HepG2、CD133⁺ LCSC的毒性情況,全自動生化分析儀測定其對細(xì)胞上清天冬氨酸氨基轉(zhuǎn)移酶（AST）、乳酸脫氫酶（LDH）、白蛋白（ALB）、尿素氮（BUN）和總蛋白（TP）水平的影響,熒光法檢測Tro對細(xì)胞CYP450總活性和ROS水平的影響。結(jié)果 CD133標(biāo)記的CSC在HepG2中占（0.72±0.05）%,CD133⁺細(xì)胞分選后純度為98.7%；CD133+細(xì)胞微球形成能力、克隆形成能力以及Transwell遷移與侵襲能力、腫瘤形成能力明顯高于親本（P<0.05、0.01）；CD133⁺細(xì)胞群大多處于G₀/G₁期,G₂/M期未阻滯,并且對索菲替尼表現(xiàn)較大耐藥性（P<0.05、0.01）；Tro處理12、24、48、72 h后,CD133⁺ LCSC半數(shù)抑制濃度（IC₅₀）顯著低于HepG2（P<0.05、0.01）；80 μmol/L Tro處理48 h時,LCSC上清AST、TP、LDH、ALB、BUN生化指標(biāo)不同程度升高,CYP450總活性和ROS水平出現(xiàn)明顯抑制（P<0.05、0.01）。結(jié)論成功篩選和鑒定具有高增殖能力的CD133+HepG2 CSC,其在Tro引起肝細(xì)胞毒性方面較HepG2細(xì)胞更敏感,細(xì)胞毒性差異顯著,為Tro靶向CD133+LCSC的細(xì)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.

國家重大新藥創(chuàng)制科技重大專項（2013ZX09302303，2012ZX09301-001-008）；北京市科委基金項目（Z131100006513010）