【摘要】：研究目的1.闡明小細(xì)胞肺癌干細(xì)胞的能量代謝狀態(tài)。2.揭示小細(xì)胞肺癌干細(xì)胞的主要產(chǎn)能途徑。3.探討干預(yù)主要產(chǎn)能途徑對小細(xì)胞肺癌干細(xì)胞干性的影響。研究方法1.課題組前期工作證明在小細(xì)胞肺癌細(xì)胞系H446中,u PAR+細(xì)胞群富集了干細(xì)胞樣細(xì)胞。因此,本實驗通過流式細(xì)胞術(shù)從H446中分選出u PAR+細(xì)胞作為腫瘤干細(xì)胞,u PAR-細(xì)胞作為非干癌細(xì)胞。2.比較u PAR+細(xì)胞與u PAR-細(xì)胞的能量代謝狀態(tài):(1)利用Cell Titer-Glo#174;發(fā)光法細(xì)胞活力檢測試劑盒,檢測u PAR+細(xì)胞與u PAR-細(xì)胞的ATP含量;(2)檢測反映u PAR+細(xì)胞與u PAR-細(xì)胞氧化磷酸化水平的相關(guān)指標(biāo):a.利用海馬生物能量測定儀XF24,檢測細(xì)胞的氧消耗率和線粒體儲備能力;b.采用活性氧檢測試劑盒,檢測細(xì)胞的活性氧水平;c.利用透射電鏡技術(shù),觀察細(xì)胞的線粒體結(jié)構(gòu)。(3)檢測反映u PAR+細(xì)胞與u PAR-細(xì)胞糖酵解水平的相關(guān)指標(biāo):a.利用海馬生物能量測定儀XF24,檢測細(xì)胞的細(xì)胞外酸化率和糖酵解的儲備能力;b.利用熒光標(biāo)記的2-脫氧葡萄糖類似物2-NBDG,檢測細(xì)胞的葡萄糖攝取能力;c.采用乳酸測定試劑盒,檢測細(xì)胞的乳酸生成率。3.比較u PAR+細(xì)胞與u PAR-細(xì)胞的主要產(chǎn)能途徑:(1)單獨(dú)給予糖酵解抑制劑2-DG后,比較u PAR+細(xì)胞與u PAR-細(xì)胞的ATP含量變化。(2)單獨(dú)給予氧化磷酸化抑制劑寡霉素后,比較u PAR+細(xì)胞與u PAR-細(xì)胞的ATP含量變化。(3)聯(lián)合應(yīng)用寡霉素和2-DG后,比較u PAR+細(xì)胞與u PAR-細(xì)胞的ATP含量變化。4.探究寡霉素對u PAR+細(xì)胞增殖及自我更新能力的影響:(1)通過MTT實驗,比較寡霉素處理前后u PAR+細(xì)胞增殖能力的變化。(2)通過腫瘤球形成實驗,比較寡霉素處理前后u PAR+細(xì)胞體外成球能力的變化。(3)通過裸鼠移植瘤實驗,比較寡霉素處理前后u PAR+細(xì)胞體內(nèi)致瘤能力的影響。結(jié)果1.小細(xì)胞肺癌干細(xì)胞與非干癌細(xì)胞能量代謝狀態(tài)的比較(1)u PAR+細(xì)胞的ATP含量顯著低于u PAR-細(xì)胞(P0.05)。(2)u PAR+細(xì)胞的基礎(chǔ)氧消耗率和線粒體儲備能力均低于u PAR-細(xì)胞(P0.05);進(jìn)一步檢測兩群細(xì)胞的活性氧水平,結(jié)果顯示u PAR+細(xì)胞的活性氧水平低于u PAR-細(xì)胞;但透射電鏡的結(jié)果表明u PAR+細(xì)胞的線粒體結(jié)構(gòu)與u PAR-細(xì)胞沒有明顯差異。(3)u PAR+細(xì)胞的細(xì)胞外酸化率和糖酵解儲備能力均低于u PAR-細(xì)胞(P0.05);葡萄糖攝取率檢測結(jié)果顯示u PAR+細(xì)胞的葡萄糖攝取能力低于u PAR-細(xì)胞(P0.05);而且u PAR+細(xì)胞的乳酸生成率也顯著低于u PAR-細(xì)胞(P0.05)。2.小細(xì)胞肺癌干細(xì)胞與非干癌細(xì)胞主要產(chǎn)能途徑的比較(1)單獨(dú)應(yīng)用寡霉素后,u PAR+細(xì)胞ATP下降幅度大于u PAR-細(xì)胞(P0.05)。(2)單獨(dú)應(yīng)用2-DG后,u PAR-細(xì)胞ATP下降幅度較大(P0.05)。(3)聯(lián)合應(yīng)用2-DG和寡霉素后,u PAR+細(xì)胞的ATP含量高于u PAR-細(xì)胞(P0.05);在缺氧環(huán)境下,聯(lián)合抑制后的u PAR+細(xì)胞ATP水平較常氧環(huán)境更高(P0.05),u PAR-細(xì)胞則無明顯變化(P0.05)。3.抑制主要產(chǎn)能途徑對小細(xì)胞肺癌干細(xì)胞增殖和自我更新能力的影響(1)MTT實驗結(jié)果顯示:2-DG能抑制u PAR-細(xì)胞的增殖卻不能抑制u PAR+細(xì)胞的增殖,而寡霉素能夠同時抑制u PAR-細(xì)胞與u PAR+細(xì)胞的增殖(P0.05);(2)腫瘤球形成實驗結(jié)果顯示:氧化磷酸化的抑制劑寡霉素可顯著影響u PAR+細(xì)胞的成球率和成球體積(P0.05),而2-DG對其幾乎沒有影響;(3)動物移植瘤實驗結(jié)果顯示:寡霉素處理的u PAR+細(xì)胞致瘤率、致瘤體積和瘤體重量均低于未處理組(P0.05)。結(jié)論1.小細(xì)胞肺癌干細(xì)胞與非干癌細(xì)胞能量代謝狀態(tài)不同,是一群代謝休眠的細(xì)胞。2.小細(xì)胞肺癌干細(xì)胞與非干癌細(xì)胞的產(chǎn)能途徑不同,其主要依賴氧化磷酸化產(chǎn)能。而且,在缺氧條件下還可以通過線粒體底物水平磷酸化產(chǎn)生部分能量。3.抑制氧化磷酸化可以降低小細(xì)胞肺癌干細(xì)胞的干性。綜上所述,小細(xì)胞肺癌干細(xì)胞是一群能量代謝狀態(tài)相對不活躍的細(xì)胞,氧化磷酸化是其主要產(chǎn)能途徑,臨床上針對糖酵解的治療策略只能殺傷非干癌細(xì)胞而對腫瘤干細(xì)胞無效。在缺氧條件下,小細(xì)胞肺癌干細(xì)胞可以通過線粒體底物水平磷酸化產(chǎn)生部分能量;同時,抑制氧化磷酸化可降低小細(xì)胞肺癌干細(xì)胞的干性。因此,該研究將對研發(fā)靶向癌干細(xì)胞代謝的相關(guān)治療有重要價值。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R734.2

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