【摘要】：背景膽管癌組織學(xué)起源于膽道被覆上皮細(xì)胞,與肝細(xì)胞癌分別居于肝膽系統(tǒng)最常見惡性腫瘤的前兩位。相比于肝細(xì)胞癌,有較多研究指出,膽管癌具有較明顯的原發(fā)多藥耐藥性,對順鉑等化療藥物普遍不敏感。對于兩種不同組織來源的腫瘤細(xì)胞——肝細(xì)胞癌細(xì)胞和膽管癌細(xì)胞——可能因代謝差異而導(dǎo)致自身抗氧化能力不同,并因此對順鉑等抗腫瘤藥物表現(xiàn)出不同的敏感性。由于糖代謝與細(xì)胞抗氧化能力的密切關(guān)系,而順鉑在誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡時會增加細(xì)胞內(nèi)ROS水平,尤其是線粒體ROS,因此,推測膽管癌對順鉑耐藥可能與其抗氧化能力增強(qiáng)有關(guān)。糖代謝為細(xì)胞提供了合成生物大分子所需的原料和各種生命活動所需的能量。磷酸戊糖途徑作為糖代謝的重要分支之一,不僅能生成核苷酸的原料——磷酸核糖,還能產(chǎn)生大量NADPH。后者是細(xì)胞內(nèi)重要的還原性底物,能夠維持GSH的還原性,參與細(xì)胞內(nèi)氧化還原平衡的調(diào)節(jié)。自噬是維持細(xì)胞代謝功能的必要條件,也可以被線粒體功能紊亂和氧化應(yīng)激誘導(dǎo)激活。異常功能的蛋白和細(xì)胞器可以通過分子伴侶介導(dǎo)自噬或巨自噬降解,而自噬的降解產(chǎn)物可以參與代謝過程,重新用于生成生物大分子或提供能量。作為經(jīng)典的自噬抑制劑CQ,能夠抑制肺癌、結(jié)腸癌等腫瘤發(fā)展,具有治療腫瘤的潛在可能。研究指出,自噬被抑制后細(xì)胞內(nèi)ROS水平顯著升高,由于自噬與代謝關(guān)系密切,推測自噬通過與代謝途徑的相互作用發(fā)揮維持細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)的作用。與之相反的,通過抑制自噬是否能夠抑制細(xì)胞代謝活性,進(jìn)而降低其抗氧化能力,增加腫瘤細(xì)胞對化療藥物敏感性,成為我們所關(guān)注的重點(diǎn)。目的本實(shí)驗(yàn)選擇肝臟不同組織來源的腫瘤,肝細(xì)胞癌Hep G2細(xì)胞和膽管細(xì)胞癌QBC939細(xì)胞,通過對比二者順鉑耐藥性差異與代謝、抗氧化能力,探討自噬抑制劑CQ通過抑制細(xì)胞PPP途徑、促進(jìn)細(xì)胞內(nèi)ROS產(chǎn)生等多個途徑增加細(xì)胞內(nèi)mt ROS水平,促進(jìn)順鉑誘導(dǎo)QBC939細(xì)胞死亡,進(jìn)而為通過抑制細(xì)胞自噬-溶酶體途徑降低細(xì)胞糖代謝活性,而增加腫瘤細(xì)胞對順鉑的敏感性機(jī)制的探索提供新的途徑。方法(1)細(xì)胞活力和細(xì)胞凋亡率變化:利用MTT法分別檢測藥物作用后細(xì)胞活力變化;利用Annexin V/PI染色后流式細(xì)胞術(shù)檢測藥物作用后細(xì)胞凋亡率變化。(2)細(xì)胞內(nèi)ROS水平和線粒體膜電勢檢測:應(yīng)用熒光探針DCFH-DA對細(xì)胞進(jìn)行染色后在倒置熒光顯微鏡下觀察總體ROS水平變化;利用熒光探針Mito SOX對細(xì)胞進(jìn)行染色后在倒置熒光顯微鏡下觀察線粒體ROS水平變化;利用JC-1對細(xì)胞染色后通過流式細(xì)胞術(shù)檢測細(xì)胞內(nèi)熒光強(qiáng)度變化。(3)標(biāo)準(zhǔn)化培養(yǎng)基葡萄糖消耗及乳酸釋放的檢測:利用葡萄糖檢測試劑盒和乳酸試劑盒分別檢測培養(yǎng)基原液及培養(yǎng)細(xì)胞后所含葡萄糖及乳酸的量,通過計算二者差值得出細(xì)胞對培養(yǎng)基葡萄糖消耗和乳酸釋放的量。利用Bradford法檢測培養(yǎng)基內(nèi)總細(xì)胞蛋白量后,將所測得的葡萄糖變化值及乳酸變化值與總蛋白數(shù)作比,得出標(biāo)準(zhǔn)化后的葡萄糖消耗量及乳酸釋放量。(4)G6PDH活性檢測:收集細(xì)胞并裂解后,裂解液利用G6PDH活性檢測試劑盒處理并通過比色法檢測G6PDH活性變化。(5)GSH/GSSG比值、NADPH/NADP比值及羥自由基含量檢測:收集細(xì)胞并裂解,裂解液分別利用GSH/GSSG檢測試劑盒、NADPH/NADP檢測試劑盒和羥自由基檢測試劑盒處理并通過比色法檢測GSH、GSSG、NADPH、NADP和細(xì)胞內(nèi)羥自由基的含量,通過分別作比,得出GSH/GSSG比值、NADPH/NADP比值和相對自由基。(6)自噬相關(guān)蛋白檢測:收集細(xì)胞并裂解,裂解液利用免疫印跡法檢測細(xì)胞內(nèi)自噬相關(guān)蛋白LC3和p62的水平變化。結(jié)果(1)與肝細(xì)胞癌Hep G2相比,膽管細(xì)胞癌QBC939在CDDP處理后表現(xiàn)出細(xì)胞活力變化較小、凋亡發(fā)生率較低和細(xì)胞內(nèi)線粒體ROS水平變化較小的特點(diǎn)。(2)與Hep G2相比,QBC939糖代謝方面表現(xiàn)出葡萄糖攝取及消耗能力明顯增強(qiáng)、乳酸釋放增多的特點(diǎn),磷酸戊糖途徑關(guān)鍵酶G6PDH的表達(dá)及活性、NADPH/NADP比值和GSH/GSSG比值也明顯增高。(3)2-DG和DHEA均能使細(xì)胞內(nèi)ROS水平升高,與Hep G2相比,QBC939對DHEA的敏感性較低,在DHEA作用下QBC939細(xì)胞內(nèi)總ROS水平和線粒體ROS水平均升高程度較小。(4)CQ與3-MA作用下Hep G2與QBC939細(xì)胞活力被顯著抑制,細(xì)胞內(nèi)的LC3與p62水平也明顯升高,其中QBC939細(xì)胞對CQ較為敏感。與Hep G2相比,在CQ作用下QBC939細(xì)胞內(nèi)總ROS和線粒體ROS升高均較明顯,同時對CDDP誘導(dǎo)凋亡敏感性也顯著升高。(5)與Hep G2細(xì)胞相比,CQ處理后QBC939細(xì)胞表現(xiàn)出G6PDH活性、GSH/GSSG比值和NADPH/NADP比值均顯著降低,線粒體膜電勢也降低較明顯,而細(xì)胞內(nèi)羥自由基含量顯著增加。結(jié)論(1)肝細(xì)胞癌細(xì)胞Hep G2和膽管癌細(xì)胞QBC939之間存在代謝模式差異。(2)磷酸戊糖途徑活性增強(qiáng)可能是QBC939細(xì)胞對順鉑敏感性低的機(jī)制之一。(3)自噬抑制劑氯喹通過抑制細(xì)胞磷酸戊糖途徑活性、降低抗氧化能力,可能是其增加QBC939細(xì)胞順鉑敏感性的機(jī)制之一。(4)氯喹通過抑制損傷線粒體清除和促進(jìn)了芬頓反應(yīng)發(fā)生,升高了細(xì)胞內(nèi)ROS水平,促進(jìn)了細(xì)胞對順鉑敏感性的增加。綜上,我們以膽管細(xì)胞癌QBC939和肝細(xì)胞癌Hep G2為研究對象,通過對比研究細(xì)胞糖代謝相關(guān)抗氧化能力與順鉑耐藥之間的關(guān)系,并發(fā)現(xiàn)自噬抑制劑氯喹通過抑制細(xì)胞磷酸戊糖途徑等多種方式升高了細(xì)胞內(nèi)ROS水平,增加了細(xì)胞對順鉑的敏感性。提示抑制自噬-溶酶體途徑可能會成為抑制膽管癌細(xì)胞的新思路。
[Abstract]:BACKGROUND Histologically, cholangiocarcinoma originates from coated epithelial cells of the biliary tract and is the first two most common malignant tumors of the hepatobiliary system, respectively. Compared with hepatocellular carcinoma, many studies have shown that cholangiocarcinoma has obvious primary multidrug resistance and is generally insensitive to cisplatin and other therapeutic drugs. Tumor cells, hepatocellular carcinoma cells and cholangiocarcinoma cells, may have different antioxidant abilities due to metabolic differences, and therefore exhibit different sensitivity to antitumor drugs such as cisplatin. Endochondrial ROS levels, especially mitochondrial ROS, suggest that resistance to cisplatin in cholangiocarcinoma may be associated with increased antioxidant capacity. Glucose metabolism provides cells with the raw materials needed to synthesize biological macromolecules and the energy needed for various life activities. Pentose phosphate pathway, as an important branch of glucose metabolism, can not only produce nucleotide progenitors. Phosphorus ribose also produces large amounts of NADPH. The latter is an important reductive substrate in cells, which maintains GSH reducibility and participates in the regulation of redox balance in cells. Autophagy is a necessary condition for maintaining cell metabolic function, and can also be activated by mitochondrial dysfunction and oxidative stress. Organelles can mediate autophagy or macrophagy degradation through molecular chaperones, and the degradation products of autophagy can participate in metabolic processes, and can be reused to produce biological macromolecules or provide energy. As a classical autophagy inhibitor, CQ can inhibit the development of lung cancer, colon cancer and other tumors, and has the potential to treat tumors. ROS levels were significantly elevated after treatment. Because of the close relationship between autophagy and metabolism, it is speculated that autophagy plays a role in maintaining homeostasis of cellular environment by interacting with metabolic pathways. Objective To compare the cisplatin resistance, metabolism and antioxidant capacity of hepatocellular carcinoma Hep G2 cells and cholangiocarcinoma QBC939 cells from different tissues of the liver, and to explore the effect of autophagy inhibitor CQ on the production of ROS by inhibiting PPP pathway. Methods (1) Changes of cell viability and apoptosis rate: MTT assay was used to detect the drug respectively. Cell viability was measured by Annexin V/PI staining and apoptosis rate was detected by flow cytometry. (2) ROS level and mitochondrial membrane potential were measured by fluorescent probe DCFH-DA staining, and ROS level was observed by inverted fluorescence microscope. Mitochondrial ROS level was observed under inverted fluorescence microscope after staining, and fluorescence intensity was detected by flow cytometry after JC-1 staining. (3) Detection of glucose consumption and lactic acid release in standardized medium: glucose detection kit and lactic acid kit were used to detect the original medium respectively. The amount of glucose and lactic acid in the culture medium was calculated by calculating the difference between them, and the amount of glucose consumption and lactic acid release in the culture medium was calculated. Consumption and lactic acid release. (4) G6PDH activity detection: after the cells were collected and lysed, the lysate was treated with G6PDH activity detection kit and the activity of G6PDH was detected by colorimetry. (5) GSH / GSSG ratio, NADPH / NADP ratio and hydroxyl radical content detection: cells were collected and lysed, and the lysate was detected by GSH / GSSG detection kit, NADPH / NADP respectively. The contents of GSH, GSSG, NADPH, NADP and intracellular hydroxyl radicals were detected by colorimetric method. The ratios of GSH/GSSG, NADPH/NADP and relative free radicals were obtained by comparing them. 6. Autophagy-associated protein detection: cells were collected and lysed, lysate was detected by immunoblotting. Results (1) Compared with hepatocellular carcinoma Hep G2, cholangiocarcinoma QBC939 showed less changes in cell viability, lower apoptosis rate and less changes in mitochondrial ROS level after CDDP treatment. (2) Compared with Hep G2, QBC939 showed glucose uptake and consumption in glucose metabolism. The expression and activity of G6PDH, NADPH/NADP ratio and GSH/GSSG ratio were also significantly increased. (3) Both 2-DG and DHEA increased the intracellular ROS level. Compared with Hep G2, QBC939 was less sensitive to DHEA, and the total ROS level and mitochondrial R in QBC939 cells were increased by DHEA. The activity of Hep G2 and QBC939 cells was significantly inhibited by CQ and 3-MA, and the levels of LC3 and p62 were also significantly increased. QBC939 cells were more sensitive to CQ. Compared with Hep G2, the total ROS and mitochondrial ROS in QBC939 cells were significantly increased by CQ and 3-MA, and the sensitivity to CDDP-induced apoptosis was also obvious. (5) Compared with Hep G2 cells, QBC939 cells treated with CQ showed G6PDH activity, GSH / GSSG ratio and NADPH / NADP ratio decreased significantly, mitochondrial membrane potential decreased significantly, and intracellular hydroxyl free radical content increased significantly. Conclusion (1) There was a metabolic pattern difference between hepatocellular carcinoma cells Hep G2 and cholangiocarcinoma cells QBC939. Increased activity of pentose phosphate pathway may be one of the mechanisms underlying the low sensitivity of QBC939 cells to cisplatin. (3) Chloroquine, an autophagy inhibitor, inhibits the activity of pentose phosphate pathway and decreases the antioxidant capacity of QBC939 cells, which may be one of the mechanisms by which it increases the sensitivity of QBC939 cells to cisplatin. (4) Chloroquine enhances the clearance of damaged mitochondria In summary, we studied the relationship between antioxidant capacity related to glucose metabolism and cisplatin resistance in cholangiocarcinoma QBC939 and hepatocellular carcinoma Hep G2, and found that chloroquine, an autophagy inhibitor, inhibited cell phosphoric acid by inhibiting cell phosphoric acid. Inhibition of autophagy-lysosome pathway may be a new way to inhibit cholangiocarcinoma cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.8

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