本文關(guān)鍵詞： 肝癌干細胞 CD133 曲格列酮 細胞選擇毒性　出處：《廣東藥科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的分離與鑒定肝癌細胞HepG2中CD133標記的肝癌干細胞;初步探討曲格列酮(troglitazone,Tro)對HepG2及其肝癌干細胞的細胞毒性差異。方法利用流式細胞分選儀分選純化HepG2中的CD133陽性和陰性細胞,無血清培養(yǎng)基培養(yǎng)分選后細胞亞群;通過利用流式細胞儀和Western blot實驗檢測分選前后CD133表達和干性相關(guān)蛋白Oct4和c-Myc的表達;采用懸浮微球形成法,平板克隆形成法和Transwell侵襲和遷移,BALB/c裸鼠體內(nèi)成瘤實驗及其腫瘤組織的免疫組化和HE染色,細胞周期的檢測增殖能力和MTT法測定肝癌干細胞的耐藥性來共同鑒定肝癌干細胞;通過CCK8法對比Tro與鹽霉素對CD133肝癌干細胞毒性情況;全自動生化分析儀測定Tro對細胞上清AST、ALT、LDH、ALB、BUN、TP生化指標的變化,熒光法檢測Tro對CYP酶總活性和ROS水平的影響,Western blot法分析藥物處理后CYP 1A2的變化;流式細胞儀分析Tro處理后的細胞周期和細胞凋亡變化來研究Tro顯示的細胞毒性差異。結(jié)果CD133型腫瘤干細胞在HepG2中占(0.72±0.05)%,CD133+細胞分選后純度為98.7%;CD133+細胞中CD133、c-Myc和Oct4表達不同程度高于親本細胞;CD133+細胞微球形成力,克隆形成力以及Transwell遷移與侵襲能力等明顯高于親本細胞(P0.05,P0.01),且CD133+細胞腫瘤形成能力顯著升高(P0.01)。同時,CD133+細胞群大多處于G0/G1期,G2/M期未得到阻滯,并且對索菲替尼表現(xiàn)較大耐藥性(P0.01);Tro處理12 h,24 h,48 h,72 h后,肝癌干細胞IC50為(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,顯著低于HepG2,具有明顯量效-關(guān)系,顯示有統(tǒng)計學差異(P0.01);Tro處理48 h時,肝癌干細胞上清AST、TP、LDH、ALB、BUN生化指標不同程度升高,其中LDH升高水平較親本細胞高(P0.05);CYP450總活性(P0.05)和ROS水平(P0.05)出現(xiàn)明顯抑制,CYP 1A2抑制程度隨濃度增加。在細胞凋亡中Tro能夠引起CD133+細胞發(fā)生早期凋亡和壞死,顯著高于親本細胞,顯示較大Tro的細胞毒性差異(P0.01);在對細胞周期影響中,Tro能較少GO/G1期細胞比重(P0.01),增加S期和G1/M期,顯示較大Tro的細胞選擇毒性差異(P0.01)。結(jié)論成功篩選和鑒定具有高增殖能力的CD133+HepG2腫瘤干細胞,在Tro引起肝細胞毒性方面較HepG2細胞更為敏感性,顯示顯著細胞選擇毒性差異,為Tro靶向CD133+HepG2的細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 鹵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 鹵1.25 渭 mol/L after Tro treatment for 12 h and 24 h and 48 h for 72 h. 99.08 鹵1.90 渭 mol / L, 43.96 鹵0.71 渭 mol / L and 14.81 鹵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.
【學位授予單位】：廣東藥科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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