曲格列酮对HepG2及其CD133肝癌干细胞的细胞毒性差异研究

发布时间：2018-01-26 12:05

本文关键词： 肝癌干细胞 CD133 曲格列酮细胞选择毒性　出处：《广东药科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的分离与鉴定肝癌细胞HepG2中CD133标记的肝癌干细胞;初步探讨曲格列酮(troglitazone,Tro)对HepG2及其肝癌干细胞的细胞毒性差异。方法利用流式细胞分选仪分选纯化HepG2中的CD133阳性和阴性细胞,无血清培养基培养分选后细胞亚群;通过利用流式细胞仪和Western blot实验检测分选前后CD133表达和干性相关蛋白Oct4和c-Myc的表达;采用悬浮微球形成法,平板克隆形成法和Transwell侵袭和迁移,BALB/c裸鼠体内成瘤实验及其肿瘤组织的免疫组化和HE染色,细胞周期的检测增殖能力和MTT法测定肝癌干细胞的耐药性来共同鉴定肝癌干细胞;通过CCK8法对比Tro与盐霉素对CD133肝癌干细胞毒性情况;全自动生化分析仪测定Tro对细胞上清AST、ALT、LDH、ALB、BUN、TP生化指标的变化,荧光法检测Tro对CYP酶总活性和ROS水平的影响,Western blot法分析药物处理后CYP 1A2的变化;流式细胞仪分析Tro处理后的细胞周期和细胞凋亡变化来研究Tro显示的细胞毒性差异。结果CD133型肿瘤干细胞在HepG2中占(0.72±0.05)%,CD133+细胞分选后纯度为98.7%;CD133+细胞中CD133、c-Myc和Oct4表达不同程度高于亲本细胞;CD133+细胞微球形成力,克隆形成力以及Transwell迁移与侵袭能力等明显高于亲本细胞(P0.05,P0.01),且CD133+细胞肿瘤形成能力显著升高(P0.01)。同时,CD133+细胞群大多处于G0/G1期,G2/M期未得到阻滞,并且对索菲替尼表现较大耐药性(P0.01);Tro处理12 h,24 h,48 h,72 h后,肝癌干细胞IC50为(150.52±1.25)μmol/L,(99.08±1.90)μmol/L,(43.96±0.71)μmol/L和(14.81±1.30)μmol/L,显著低于HepG2,具有明显量效-关系,显示有统计学差异(P0.01);Tro处理48 h时,肝癌干细胞上清AST、TP、LDH、ALB、BUN生化指标不同程度升高,其中LDH升高水平较亲本细胞高(P0.05);CYP450总活性(P0.05)和ROS水平(P0.05)出现明显抑制,CYP 1A2抑制程度随浓度增加。在细胞凋亡中Tro能够引起CD133+细胞发生早期凋亡和坏死,显著高于亲本细胞,显示较大Tro的细胞毒性差异(P0.01);在对细胞周期影响中,Tro能较少GO/G1期细胞比重(P0.01),增加S期和G1/M期,显示较大Tro的细胞选择毒性差异(P0.01)。结论成功筛选和鉴定具有高增殖能力的CD133+HepG2肿瘤干细胞,在Tro引起肝细胞毒性方面较HepG2细胞更为敏感性,显示显著细胞选择毒性差异,为Tro靶向CD133+HepG2的细胞毒性研究提供新思路。
[Abstract]:Objective to isolate and identify hepatoma stem cells labeled with CD133 in HepG2. Preliminary study on troglitazone with trioglitazone. Methods HepG2 positive and negative cells were purified by flow cytometry. The cell subsets were cultured in serum-free medium. The expression of CD133, Oct4 and c-Myc were detected by flow cytometry and Western blot assay before and after sorting. Suspension microsphere formation, plate clone formation and Transwell invasion and migration in nude mice were used for tumorigenesis and immunohistochemical and HE staining. The proliferative ability of cell cycle and the resistance of hepatocellular carcinoma stem cells were determined by MTT assay to identify the liver cancer stem cells. CCK8 method was used to compare the toxicity of Tro and salinomycin on CD133 hepatoma stem cells. Automatic biochemical analyzer was used to determine the changes of the biochemical indexes of the supernatant of ASTH, LDHH, ALBUNT and BUNTP in the supernatant of ASTH. The effect of Tro on the total activity of CYP and the level of ROS was detected by fluorescence assay. The changes of CYP 1A2 after drug treatment were analyzed by Western blot. The changes of cell cycle and apoptosis after Tro treatment were analyzed by flow cytometry to study the difference of cytotoxicity in Tro. Results CD133 type tumor stem cells accounted for (1) in HepG2 (. 0.72 卤0.05%. The purity of CD133 cells was 98.7%. The expression of CD133c-Myc and Oct4 in CD133 cells was higher than that in parental cells. The ability of CD133 cells to form microspheres, clone formation and Transwell migration and invasion were significantly higher than that of their parent cells (P0.05, P0.01). Moreover, the tumor formation ability of CD133 cells was significantly increased (P 0.01). Meanwhile, most of CD133 cells were not blocked in G _ 0 / G _ 1 phase and G _ 2 / M phase. And showed greater resistance to sofetinib (P 0.01); The IC50 of hepatoma stem cells was 150.52 卤1.25 渭 mol/L after Tro treatment for 12 h and 24 h and 48 h for 72 h. 99.08 卤1.90 渭 mol / L, 43.96 卤0.71 渭 mol / L and 14.81 卤1.30 渭 mol / L, significantly lower than HepG2. There was a significant dose-effect relationship, showing a statistical difference (P 0.01). After 48 h of Tro treatment, the biochemical indexes of the supernatant of liver cancer stem cell ASTX, Tro, LDHH, Albun were increased to varying degrees, and the level of LDH was higher than that of parent cells (P 0.05). The total activity of CYP450 (P0.05) and the level of ROS (P0.05) were significantly inhibited. The degree of inhibition of CYP 1A2 increased with the concentration. During apoptosis, Tro could induce early apoptosis and necrosis of CD133 cells, which was significantly higher than that of parent cells. The cytotoxicity of Tro was different from that of P0.01C; In the effect on cell cycle, Tro can increase S phase and G 1 / M phase with less specific gravity of GO/G1 phase. Conclusion the tumor stem cells with high proliferative ability of CD133 HepG2 were successfully screened and identified. The hepatocyte toxicity induced by Tro was more sensitive than that of HepG2 cells, which showed significant cytotoxicity difference and provided a new idea for the study of cytotoxicity of Tro targeting CD133 HepG2.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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