小细胞肺癌细胞系H446肿瘤干细胞能量代谢的初步研究
发布时间：2018-10-31 07:44
【摘要】：研究目的1.阐明小细胞肺癌干细胞的能量代谢状态。2.揭示小细胞肺癌干细胞的主要产能途径。3.探讨干预主要产能途径对小细胞肺癌干细胞干性的影响。研究方法1.课题组前期工作证明在小细胞肺癌细胞系H446中,u PAR+细胞群富集了干细胞样细胞。因此,本实验通过流式细胞术从H446中分选出u PAR+细胞作为肿瘤干细胞,u PAR-细胞作为非干癌细胞。2.比较u PAR+细胞与u PAR-细胞的能量代谢状态:(1)利用Cell Titer-Glo#174;发光法细胞活力检测试剂盒,检测u PAR+细胞与u PAR-细胞的ATP含量;(2)检测反映u PAR+细胞与u PAR-细胞氧化磷酸化水平的相关指标:a.利用海马生物能量测定仪XF24,检测细胞的氧消耗率和线粒体储备能力;b.采用活性氧检测试剂盒,检测细胞的活性氧水平;c.利用透射电镜技术,观察细胞的线粒体结构。(3)检测反映u PAR+细胞与u PAR-细胞糖酵解水平的相关指标:a.利用海马生物能量测定仪XF24,检测细胞的细胞外酸化率和糖酵解的储备能力;b.利用荧光标记的2-脱氧葡萄糖类似物2-NBDG,检测细胞的葡萄糖摄取能力;c.采用乳酸测定试剂盒,检测细胞的乳酸生成率。3.比较u PAR+细胞与u PAR-细胞的主要产能途径:(1)单独给予糖酵解抑制剂2-DG后,比较u PAR+细胞与u PAR-细胞的ATP含量变化。(2)单独给予氧化磷酸化抑制剂寡霉素后,比较u PAR+细胞与u PAR-细胞的ATP含量变化。(3)联合应用寡霉素和2-DG后,比较u PAR+细胞与u PAR-细胞的ATP含量变化。4.探究寡霉素对u PAR+细胞增殖及自我更新能力的影响:(1)通过MTT实验,比较寡霉素处理前后u PAR+细胞增殖能力的变化。(2)通过肿瘤球形成实验,比较寡霉素处理前后u PAR+细胞体外成球能力的变化。(3)通过裸鼠移植瘤实验,比较寡霉素处理前后u PAR+细胞体内致瘤能力的影响。结果1.小细胞肺癌干细胞与非干癌细胞能量代谢状态的比较(1)u PAR+细胞的ATP含量显著低于u PAR-细胞(P0.05)。(2)u PAR+细胞的基础氧消耗率和线粒体储备能力均低于u PAR-细胞(P0.05);进一步检测两群细胞的活性氧水平,结果显示u PAR+细胞的活性氧水平低于u PAR-细胞;但透射电镜的结果表明u PAR+细胞的线粒体结构与u PAR-细胞没有明显差异。(3)u PAR+细胞的细胞外酸化率和糖酵解储备能力均低于u PAR-细胞(P0.05);葡萄糖摄取率检测结果显示u PAR+细胞的葡萄糖摄取能力低于u PAR-细胞(P0.05);而且u PAR+细胞的乳酸生成率也显著低于u PAR-细胞(P0.05)。2.小细胞肺癌干细胞与非干癌细胞主要产能途径的比较(1)单独应用寡霉素后,u PAR+细胞ATP下降幅度大于u PAR-细胞(P0.05)。(2)单独应用2-DG后,u PAR-细胞ATP下降幅度较大(P0.05)。(3)联合应用2-DG和寡霉素后,u PAR+细胞的ATP含量高于u PAR-细胞(P0.05);在缺氧环境下,联合抑制后的u PAR+细胞ATP水平较常氧环境更高(P0.05),u PAR-细胞则无明显变化(P0.05)。3.抑制主要产能途径对小细胞肺癌干细胞增殖和自我更新能力的影响(1)MTT实验结果显示:2-DG能抑制u PAR-细胞的增殖却不能抑制u PAR+细胞的增殖,而寡霉素能够同时抑制u PAR-细胞与u PAR+细胞的增殖(P0.05);(2)肿瘤球形成实验结果显示:氧化磷酸化的抑制剂寡霉素可显著影响u PAR+细胞的成球率和成球体积(P0.05),而2-DG对其几乎没有影响;(3)动物移植瘤实验结果显示:寡霉素处理的u PAR+细胞致瘤率、致瘤体积和瘤体重量均低于未处理组(P0.05)。结论1.小细胞肺癌干细胞与非干癌细胞能量代谢状态不同,是一群代谢休眠的细胞。2.小细胞肺癌干细胞与非干癌细胞的产能途径不同,其主要依赖氧化磷酸化产能。而且,在缺氧条件下还可以通过线粒体底物水平磷酸化产生部分能量。3.抑制氧化磷酸化可以降低小细胞肺癌干细胞的干性。综上所述,小细胞肺癌干细胞是一群能量代谢状态相对不活跃的细胞,氧化磷酸化是其主要产能途径,临床上针对糖酵解的治疗策略只能杀伤非干癌细胞而对肿瘤干细胞无效。在缺氧条件下,小细胞肺癌干细胞可以通过线粒体底物水平磷酸化产生部分能量;同时,抑制氧化磷酸化可降低小细胞肺癌干细胞的干性。因此,该研究将对研发靶向癌干细胞代谢的相关治疗有重要价值。
[Abstract]:Study objective 1. state of energy metabolism of small cell lung cancer stem cells. To reveal the main productive way of small cell lung cancer stem cells. To investigate the effect of main capacity of intervention on stem cell drying in small cell lung cancer. Study Method 1. The preliminary work of the team demonstrated that in the small cell lung cancer cell line H446, the u PAR + cell population enriched the stem cell-like cells. Therefore, u PAR + cells were selected from H446 as tumor stem cells by flow cytometry, and u PAR-cells were used as non-dry cancer cells. The energy metabolism states of u PAR + cells and u PAR-cells were compared: (1) Cell viability test kit was used to detect the ATP content of u PAR + cells and u PAR-cells. (2) detecting the correlation index of the oxidative phosphorylation level of the u PAR + cell and the u PAR-cell: a. detecting the oxygen consumption rate and the mitochondrial reserve capacity of the cell by using the hippocampal biological energy tester XF24; b, adopting an active oxygen detection kit to detect the active oxygen level of the cell; c. The mitochondrial structure of cells was observed by transmission electron microscope (TEM). (3) detecting the correlation index reflecting the level of the u PAR + cell and the u PAR-cell sugar level: a. detecting the extracellular acidification rate of the cell and the storage capacity of the glycolysis by using the hippocampal biological energy determinator XF24; b. using the fluorescent label 2-deoxyglucose analog 2-NBDG, detecting the glucose uptake capacity of the cells; c. using a lactic acid measurement kit to detect the lactic acid generation rate of the cells. The main productivity approaches of u PAR + cells and u PAR-cells were compared: (1) After administration of glycolytic inhibitor 2-DG alone, the ATP content of u PAR + cells and u PAR-cells were compared. (2) The changes of the ATP content of u PAR + cells and u PAR-cells were compared after the oxidative phosphorylation inhibitor was administered alone. (3) After the combination of oligomycin and 2-DG, the change of ATP content in u PAR + cells and u PAR-cells was compared. The effect of oligomycin on the proliferation and self-renewal of u PAR + cells was studied: (1) The change of uPAR + cell proliferation was compared by MTT assay. (2) By the experiment of tumor ball formation, the change of spherical ability of u PAR + cells before and after treatment with oligomycin was compared. (3) To compare the effect of oligomycin on the tumorigenic ability of u PAR + cells before and after treatment by nude mouse transplantation tumor. Result 1. Compared with the state of energy metabolism of non-dry cancer cells, the ATP content of the small cell lung cancer stem cells and non-dry cancer cells was significantly lower than that of u PAR-cells (P0.05). (2) The basal oxygen consumption rate and mitochondrial reserve capacity of u PAR + cells were lower than that of u PAR-cells (P0.05). However, the results of transmission electron microscope showed that the mitochondrial structure of u PAR + cells was not significantly different than that of u PAR-cells. (3) The extracellular acidification rate and sugar-uptake capacity of u PAR + cells were lower than that of u PAR-cells (P0.05), and the glucose uptake ability of u PAR + cells was lower than that of u PAR-cells (P0.05). Moreover, the lactic acid production rate of u PAR + cells was significantly lower than that of u PAR-cells (P0.05). Compared with the main production capacity of small cell lung cancer stem cells and non-dry cancer cells (1), the decrease of ATP in u PAR + cells was greater than that of u PAR-cells (P0.05). (2) After 2-DG alone, the decrease of u PAR-cell ATP was larger (P0.05). (3) After combined application of 2-DG and oligomycin, the ATP content of u PAR + cells was higher than that of u PAR-cells (P0.05). Inhibition of main production capacity on proliferation and self-renewal of small cell lung cancer stem cells (1) MTT assay showed that 2-DG could inhibit the proliferation of u PAR-cells but could not inhibit the proliferation of u PAR + cells. The results of the experimental results showed that the inhibitor of oxidative phosphorylation could significantly affect the percentage and volume of u PAR + cells (P0.05), but the 2-DG had little effect on it. (3) The experimental results showed that the tumor rate, tumor volume and body weight of the u PAR + cells treated by oligomycin were lower than those in untreated group (P0.05). Conclusion 1. Small cell lung cancer stem cells differ from non-dry cell energy metabolism, a group of dormant cells. The production capacity of small cell lung cancer stem cells and non-dry cancer cells is different, which mainly depends on the oxidative phosphorylation capacity. Moreover, partial energy can also be generated by phosphorylation of the mitochondrial substrate at anoxic conditions. Inhibition of oxidative phosphorylation can reduce the dryness of small cell lung cancer stem cells. To sum up, small cell lung cancer stem cells are a group of cells with relatively inactive energy metabolism state, oxidative phosphorylation is its main production capacity, and the clinical treatment strategy for glycolysis can only kill non-dry cancer cells and is ineffective for tumor stem cells. in anoxic condition, small cell lung cancer stem cells can generate partial energy through horizontal phosphorylation of mitochondrial substrate; meanwhile, inhibition of oxidative phosphorylation can reduce dry of small cell lung cancer stem cells. Therefore, this study will have an important value in the development of related therapies for the metabolism of targeted cancer stem cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
本文编号：2301424
[Abstract]:Study objective 1. state of energy metabolism of small cell lung cancer stem cells. To reveal the main productive way of small cell lung cancer stem cells. To investigate the effect of main capacity of intervention on stem cell drying in small cell lung cancer. Study Method 1. The preliminary work of the team demonstrated that in the small cell lung cancer cell line H446, the u PAR + cell population enriched the stem cell-like cells. Therefore, u PAR + cells were selected from H446 as tumor stem cells by flow cytometry, and u PAR-cells were used as non-dry cancer cells. The energy metabolism states of u PAR + cells and u PAR-cells were compared: (1) Cell viability test kit was used to detect the ATP content of u PAR + cells and u PAR-cells. (2) detecting the correlation index of the oxidative phosphorylation level of the u PAR + cell and the u PAR-cell: a. detecting the oxygen consumption rate and the mitochondrial reserve capacity of the cell by using the hippocampal biological energy tester XF24; b, adopting an active oxygen detection kit to detect the active oxygen level of the cell; c. The mitochondrial structure of cells was observed by transmission electron microscope (TEM). (3) detecting the correlation index reflecting the level of the u PAR + cell and the u PAR-cell sugar level: a. detecting the extracellular acidification rate of the cell and the storage capacity of the glycolysis by using the hippocampal biological energy determinator XF24; b. using the fluorescent label 2-deoxyglucose analog 2-NBDG, detecting the glucose uptake capacity of the cells; c. using a lactic acid measurement kit to detect the lactic acid generation rate of the cells. The main productivity approaches of u PAR + cells and u PAR-cells were compared: (1) After administration of glycolytic inhibitor 2-DG alone, the ATP content of u PAR + cells and u PAR-cells were compared. (2) The changes of the ATP content of u PAR + cells and u PAR-cells were compared after the oxidative phosphorylation inhibitor was administered alone. (3) After the combination of oligomycin and 2-DG, the change of ATP content in u PAR + cells and u PAR-cells was compared. The effect of oligomycin on the proliferation and self-renewal of u PAR + cells was studied: (1) The change of uPAR + cell proliferation was compared by MTT assay. (2) By the experiment of tumor ball formation, the change of spherical ability of u PAR + cells before and after treatment with oligomycin was compared. (3) To compare the effect of oligomycin on the tumorigenic ability of u PAR + cells before and after treatment by nude mouse transplantation tumor. Result 1. Compared with the state of energy metabolism of non-dry cancer cells, the ATP content of the small cell lung cancer stem cells and non-dry cancer cells was significantly lower than that of u PAR-cells (P0.05). (2) The basal oxygen consumption rate and mitochondrial reserve capacity of u PAR + cells were lower than that of u PAR-cells (P0.05). However, the results of transmission electron microscope showed that the mitochondrial structure of u PAR + cells was not significantly different than that of u PAR-cells. (3) The extracellular acidification rate and sugar-uptake capacity of u PAR + cells were lower than that of u PAR-cells (P0.05), and the glucose uptake ability of u PAR + cells was lower than that of u PAR-cells (P0.05). Moreover, the lactic acid production rate of u PAR + cells was significantly lower than that of u PAR-cells (P0.05). Compared with the main production capacity of small cell lung cancer stem cells and non-dry cancer cells (1), the decrease of ATP in u PAR + cells was greater than that of u PAR-cells (P0.05). (2) After 2-DG alone, the decrease of u PAR-cell ATP was larger (P0.05). (3) After combined application of 2-DG and oligomycin, the ATP content of u PAR + cells was higher than that of u PAR-cells (P0.05). Inhibition of main production capacity on proliferation and self-renewal of small cell lung cancer stem cells (1) MTT assay showed that 2-DG could inhibit the proliferation of u PAR-cells but could not inhibit the proliferation of u PAR + cells. The results of the experimental results showed that the inhibitor of oxidative phosphorylation could significantly affect the percentage and volume of u PAR + cells (P0.05), but the 2-DG had little effect on it. (3) The experimental results showed that the tumor rate, tumor volume and body weight of the u PAR + cells treated by oligomycin were lower than those in untreated group (P0.05). Conclusion 1. Small cell lung cancer stem cells differ from non-dry cell energy metabolism, a group of dormant cells. The production capacity of small cell lung cancer stem cells and non-dry cancer cells is different, which mainly depends on the oxidative phosphorylation capacity. Moreover, partial energy can also be generated by phosphorylation of the mitochondrial substrate at anoxic conditions. Inhibition of oxidative phosphorylation can reduce the dryness of small cell lung cancer stem cells. To sum up, small cell lung cancer stem cells are a group of cells with relatively inactive energy metabolism state, oxidative phosphorylation is its main production capacity, and the clinical treatment strategy for glycolysis can only kill non-dry cancer cells and is ineffective for tumor stem cells. in anoxic condition, small cell lung cancer stem cells can generate partial energy through horizontal phosphorylation of mitochondrial substrate; meanwhile, inhibition of oxidative phosphorylation can reduce dry of small cell lung cancer stem cells. Therefore, this study will have an important value in the development of related therapies for the metabolism of targeted cancer stem cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
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