本文選題：TIGAR + 凋亡��； 參考：《蘇州大學》2015年博士論文

【摘要】：目的:觀察化療藥表阿霉素對肝癌細胞p53誘導的糖酵解和凋亡調節(jié)子(TIGAR)蛋白表達的影響以及TIGAR在肝癌細胞對表阿霉素化療耐受中的作用;探討TIGAR影響肝癌細胞對表阿霉素耐藥的初步機制。方法:Western blot法檢測表阿霉素對肝癌細胞TIGAR蛋白表達的影響;采用小干擾RNA技術干擾肝癌細胞TIGAR表達后通過MTT及克隆形成實驗分別檢測干擾TIGAR表達聯(lián)合表阿霉素治療對肝癌細胞生存的短期和長期影響;Annexin V-FITC/PI雙染流式細胞術檢測干擾TIGAR表達聯(lián)合表阿霉素治療對肝癌細胞凋亡的影響;采用慢病毒構建的TIGAR-sh RNA感染Hep G2肝癌細胞,經(jīng)嘌呤霉素篩選,western blot及細胞免疫熒光法檢測干擾效率,得到穩(wěn)轉株后通過裸鼠成瘤實驗檢測干擾TIGAR表達對肝癌腫瘤形成、生長及其對表阿霉素化療療效的影響;2’,7’-二氯二氫熒光素雙醋酸鹽(DCFH2-DA)標記流式細胞術、GSH檢測試劑盒及NADPH檢測試劑盒分別檢測細胞內活性氧(ROS)水平、GSH/GSSG比值及NADPH水平;Western blot檢測細胞內凋亡相關蛋白水平;細胞免疫熒光法、western blot及電鏡技術檢測干擾TIGAR表達對肝癌細胞自噬的影響;彗星實驗檢測細胞DNA損傷情況;采用細胞核質分離技術分離細胞核后western blot分別檢測胞漿及胞核中TIGAR的表達;運用細胞免疫熒光及western blot技術檢測DNA損傷修復中相關蛋白ATM、Cdk5等的表達。結果:為了研究表阿霉素對肝癌細胞TIGAR表達的影響,我們采用不同濃度的表阿霉素作用肝癌Hep G2細胞不同時間后,western blot檢測TIGAR表達,結果顯示,低濃度短時間的表阿霉素作用可誘導Hep G2肝癌細胞TIGAR表達,高濃度長時間的表阿霉素作用后TIGAR表達下調。且表阿霉素誘導的TIGAR表達是依賴p53變化的。為了進一步探討表阿霉素誘導的肝癌細胞TIGAR高表達在肝癌細胞對表阿霉素療效中的作用,我們采用小干擾RNA干擾肝癌Hep G2細胞TIGAR表達后通過MTT、克隆形成實驗及流式等體外實驗檢測發(fā)現(xiàn),干擾TIGAR表達聯(lián)合表阿霉素作用后肝癌Hep G2細胞的存活明顯下降,細胞凋亡顯著增加;此外,通過體內實驗即裸鼠成瘤實驗檢測干擾TIGAR表達對肝癌細胞成瘤的影響以及移植瘤對表阿霉素療效的影響,結果顯示,干擾TIGAR表達抑制肝癌的形成和生長,同時也增加了其對表阿霉素的化療敏感性。研究還發(fā)現(xiàn),不僅對于肝癌細胞,表阿霉素也增加了肺癌A549細胞的TIGAR表達且干擾TIGAR表達增加肺癌A549細胞對表阿霉素的化療敏感性。腫瘤的化療耐受與多種機制相關,其中ROS適應性反應、細胞自噬及細胞DNA損傷修復在腫瘤化療耐藥的形成中起著重要作用,TIGAR作為p53下游基因,通過抑制細胞糖酵解及增加磷酸戊糖通路產(chǎn)生NADPH降低細胞內ROS水平,從而降低ROS引起的細胞凋亡、ROS介導的細胞自噬激活及ROS誘導的DNA損傷修復,此外,磷酸戊糖通路的激活增加了DNA合成原料核苷酸的生成,有利于受損DNA的修復。因此,為了進一步探討干擾TIGAR表達在肝癌細胞對表阿霉素化療增敏中的機制,我們從細胞凋亡、細胞自噬及細胞DNA損傷修復三方面來進行研究。第一,干擾TIGAR表達顯著增加表阿霉素誘導的肝癌細胞凋亡,并與ROS水平增加、Caspase3激活相關。采用NADPH或NAC清除細胞ROS水平或采用Z-VAD-FMK抑制Caspase3后可部分減輕細胞凋亡。提示,干擾TIGAR表達增加肝癌細胞對表阿霉素化療敏感性與干擾TIGAR表達增加細胞凋亡有關。第二,干擾TIGAR表達顯著增加表阿霉素誘導的肝癌細胞自噬激活,表現(xiàn)為LC3I向LC3 II轉化增加、自噬體增多及自噬底物p62蛋白水平的下調。干擾TIGAR表達增加表阿霉素誘導的肝癌細胞自噬激活通過增加細胞ROS水平及抑制m TOR通路來實現(xiàn),NADPH清除ROS后可部分抑制干擾TIGAR表達聯(lián)合表阿霉素作用激活的自噬。采用3-MA或干擾Atg5抑制細胞自噬后進一步增加了干擾TIGAR表達聯(lián)合表阿霉素作用所致的肝癌細胞凋亡。提示,干擾TIGAR表達增加表阿霉素誘導的肝癌細胞自噬激活對細胞的生存起保護作用,能夠抵抗表阿霉素的毒性作用,在此基礎上抑制細胞自噬能得到額外的抗腫瘤效果。即干擾TIGAR誘導的細胞自噬激活降低肝癌細胞對表阿霉素的化療敏感性。第三,TIGAR增加磷酸戊糖通路產(chǎn)生NADPH減少細胞ROS從而減少DNA損傷,此外,產(chǎn)生的戊糖作為DNA合成原料有利于受損DNA的修復。表阿霉素除了誘導肝癌細胞TIGAR表達外還促進其入核,調控腫瘤細胞DNA損傷修復。干擾TIGAR表達顯著增加表阿霉素誘導的肝癌細胞DNA損傷,當給予磷酸戊糖途徑產(chǎn)生的戊糖、NADPH或ROS清除劑NAC后可部分緩解DNA損傷。提示干擾TIGAR表達除了通過抑制磷酸戊糖通路增加表阿霉素誘導的肝癌細胞DNA損傷外,還有其他機制的存在。進一步實驗研究顯示,TIGAR通過調節(jié)Cdk5來磷酸化DNA損傷修復關鍵蛋白ATM進而調控細胞DNA損傷修復。綜上,TIGAR通過磷酸戊糖途徑及Cdk5-AMT信號通路來調控表阿霉素對肝癌細胞DNA的損傷與修復進而影響肝癌細胞對表阿霉素的化療敏感性。結論:表阿霉素誘導肝癌細胞TIGAR表達;干擾TIGAR表達增加肝癌細胞對表阿霉素的化療敏感性,其機制涉及細胞凋亡增加、自噬水平激活以及細胞DNA的損傷與修復。TIGAR表達在肝癌細胞對表阿霉素耐受中的作用提示TIGAR成為抗腫瘤治療作用靶點的可能,為臨床抗腫瘤治療尤其是對表阿霉素耐受的肝癌治療提供理論基礎及依據(jù)。
[Abstract]:Objective: To observe the effect of epirubicin on the expression of glycolysis and apoptosis regulator (TIGAR) induced by p53 in hepatoma cells and the role of TIGAR in the tolerance of liver cancer cells to adriamycin chemotherapy, and to explore the mechanism of TIGAR influence on the resistance of liver cancer cells to adriamycin. Methods: Western blot assay was used to detect epirubicin to liver cancer. The effect of the expression of TIGAR protein in cells; using small interference RNA technique to interfere with the expression of TIGAR in hepatoma cells, the short-term and long-term effects of TIGAR expression combined with epirubicin on the survival of hepatoma cells were detected by MTT and clonogenic experiments, and Annexin V-FITC/PI double dye flow cytometry interfered with TIGAR expression combined with epirubicin The effect of treatment on the apoptosis of hepatoma cells; TIGAR-sh RNA infected by lentivirus, Hep G2 hepatoma cells, the screening of purinomycin, Western blot and cell immunofluorescence method to detect the interference efficiency. After the stable transgenic mice were obtained, the interference of TIGAR expression to the formation of the cancer of the liver cancer, the growth and the chemotherapy of doxorubicin on the liver cancer were detected. Effect: 2 ', 7' - two chlorine two fluorescein diacetate (DCFH2-DA) labeled flow cytometry, GSH detection kit and NADPH detection kit to detect intracellular reactive oxygen species (ROS) level, GSH/GSSG ratio and NADPH level, Western blot detection of intracellular apoptotic phase protein level, cell immunofluorescence, Western blot and electron microscopy The effect of interfering TIGAR expression on autophagy of hepatoma cells was detected; Comet assay was used to detect DNA damage in cells; Western blot was used to detect the expression of TIGAR in cytoplasm and nucleus after cell nuclear separation technique, and cell immunofluorescence and Western blot were used to detect the associated protein ATM, Cdk5, etc. in DNA damage repair. Results: in order to study the effect of epirubicin on the expression of TIGAR in hepatoma cells, we used Western blot to detect TIGAR expression after different concentrations of epirubicin on the liver cancer Hep G2 cells. The results showed that the low concentration of epirubicin could induce the TIGAR expression of Hep G2 hepatoma cells, and the high concentration and long time table could be used. The expression of TIGAR was down regulated by doxorubicin, and the expression of epirubicin induced TIGAR was dependent on p53. In order to further explore the effect of TIGAR high expression of epirubicin induced hepatoma cells in the therapeutic effect of hepatoma cells on epuxorubicin, we used small interference RNA to interfere with the expression of TIGAR in Hep G2 cells to form solid clones through MTT. In vitro test and flow test, it was found that the survival of Hep G2 cells in HCC significantly decreased and apoptosis increased significantly after TIGAR expression combined with epirubicin. In addition, the effects of interfering TIGAR expression on the tumor formation of hepatoma cells and the effect of xenograft on adriamycin effect were detected by in vivo experiments. The results show that interfering TIGAR expression inhibits the formation and growth of liver cancer and also increases its sensitivity to epuxorubicin. The study also found that epirubicin also increased the TIGAR expression of lung cancer A549 cells and interfered with the expression of TIGAR in lung cancer cells and increased the chemosensitivity of A549 cells to epirubicin. Tolerance is related to a variety of mechanisms, in which ROS adaptive response, cell autophagy and cell DNA damage repair play an important role in the formation of chemotherapeutic drug resistance. TIGAR, as a downstream p53 gene, reduces cell apoptosis induced by ROS by inhibiting cell glycolysis and increasing NADPH in pentose phosphoric acid pathway to reduce the level of intracellular ROS induced by ROS, ROS Mediated cell autophagy activation and ROS induced DNA damage repair. In addition, the activation of pentose phosphate pathway increases the formation of DNA synthesis nucleotides and is beneficial to the repair of damaged DNA. Therefore, in order to further explore the mechanism of interference TIGAR expression in the chemosensitivity of hepatoma cells to adriamycin chemotherapy, we are from cell apoptosis and autophagy. Three aspects of cell DNA damage repair were studied. First, interfering TIGAR expression significantly increased the apoptosis of HCC induced by epirubicin, and was related to the increase of ROS level and Caspase3 activation. NADPH or NAC was used to remove the cell ROS level or to reduce the apoptosis after the use of Z-VAD-FMK to inhibit Caspase3. It suggested that the expression of TIGAR increased in TIGAR. The chemosensitivity of hepatoma cells to epuxorubicin was associated with increased cell apoptosis by interfering TIGAR expression. Second, interfering TIGAR expression significantly increased autophagy induced by epirubicin induced hepatoma cells, showing an increase in LC3I to LC3 II, the increase of autophagosome and the downregulation of p62 protein levels of autophagic substrates. Interference with TIGAR expression increased epirubicin induced induction. Autophagy of hepatoma cells is activated by increasing cell ROS level and inhibiting the m TOR pathway. NADPH scavenging ROS can partially inhibit the interference of TIGAR expression and autophagy activated by the action of epirubicin. 3-MA or interference Atg5 inhibits autophagy of the cells to further increase the interference of the TIGAR table to the action of the combined adriamycin action. It is suggested that the interference of TIGAR expression to increase the autophagy induced by epirubicin induced activation of autophagy can protect the survival of the cells, and can resist the toxicity of epirubicin. On this basis, the inhibition of autophagy by cell autophagy can result in an additional anti-tumor effect. That is, interfering with autophagy induced by TIGAR to reduce hepatoma cells to opiophos Third, TIGAR increase the pentose phosphate pathway to produce NADPH to reduce cell ROS and reduce DNA damage. In addition, the pentose produced as a DNA synthetic material is beneficial to the repair of damaged DNA. Epirubicin also promotes its nucleation in addition to inducing the TIGAR expression of hepatoma cells and regulates the repair of DNA damage in tumor cells. Significantly increased DNA damage induced by epirubicin, when pentose pentose produced by pentose phosphate pathway was given, NADPH or ROS scavenger NAC could partially alleviate DNA damage. It suggests that there are other mechanisms in the presence of interference TIGAR expression in addition to increasing the DNA damage induced by epirubicin pathway by inhibiting the pentose phosphate pathway. The experimental studies have shown that TIGAR regulates the DNA damage and repair key protein ATM by regulating the phosphorylation of Cdk5 to regulate the repair of cell DNA damage. To sum up, TIGAR regulates the damage and repair of epirubicin on the liver cancer cell DNA by pentose phosphate pathway and Cdk5-AMT signaling pathway and then affects the chemosensitivity of hepatoma cells to adriamycin. Epirubicin induced the expression of TIGAR in hepatoma cells; interference with TIGAR expression increases the chemosensitivity of hepatoma cells to adriamycin, and its mechanism involves increased apoptosis, activation of autophagy, DNA damage and the role of.TIGAR expression in the tolerance of liver cancer cells to adriamycin tolerance, suggesting that TIGAR becomes an antitumor therapeutic target. It may provide a theoretical basis and basis for clinical antitumor therapy, especially for the treatment of epirubicin tolerant liver cancer.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7

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