【摘要】：肝癌是全球第五大腫瘤疾病,具有極高的發(fā)病率及致死率。目前雖然在臨床上主要采取化學(xué)療法、切除法、肝移植、免疫療法等多種方法治療肝癌,但是在發(fā)達(dá)國(guó)家,肝癌疾病的生存率僅為3-5%,這促使人們不得不繼續(xù)尋找新的治療肝癌疾病的方法。雙環(huán)醇(4,4’-二甲氧基-5,6,5’,6’-雙(亞甲二氧基)-2-羥甲基-2’-甲氧羰基聯(lián)苯)為抗肝炎化學(xué)合成新藥,在臨床上已被廣泛應(yīng)用于治療HBV感染。此外,一些研究證明雙環(huán)醇還具有提高肝功能、保護(hù)肝臟免受化學(xué)物毒害的作用；同時(shí)也有研究發(fā)現(xiàn)雙環(huán)醇可以誘導(dǎo)肝癌細(xì)胞中熱休克蛋白HSP27/70的表達(dá),從而抑制細(xì)胞線粒體途徑的凋亡。但是,對(duì)雙環(huán)醇的抗腫瘤效果以及雙環(huán)醇在腫瘤細(xì)胞周期與自噬方面功能的研究仍少有涉及,而相關(guān)文獻(xiàn)中對(duì)雙環(huán)醇作用后的癌細(xì)胞進(jìn)行高通量篩選差異表達(dá)基因等初步研究表明雙環(huán)醇具有作為抗癌藥物的潛能。因此本研究重點(diǎn)探索了雙環(huán)醇對(duì)HepG2細(xì)胞的毒性作用,并且在體外和體內(nèi)環(huán)境探究了雙環(huán)醇對(duì)肝癌細(xì)胞作用的分子機(jī)制。結(jié)果如下：1.雙環(huán)醇顯著抑制HepG2細(xì)胞增殖,而對(duì)正常肝細(xì)胞LO2的毒性很低。MTT實(shí)驗(yàn)表明,雙環(huán)醇對(duì)HepG2的IC50值為L(zhǎng)O2細(xì)胞的三分之一。而通過(guò)流式細(xì)胞術(shù)研究發(fā)現(xiàn),雙環(huán)醇對(duì)HepG2細(xì)胞活性的抑制作用主要源于其能夠抑制細(xì)胞周期以及誘導(dǎo)細(xì)胞產(chǎn)生自噬,而在雙環(huán)醇作用下的HepG2的凋亡水平?jīng)]有發(fā)生變化。在細(xì)胞周期方面,雙環(huán)醇能夠誘導(dǎo)HepG2細(xì)胞發(fā)生時(shí)間和濃度梯度依賴的G1期細(xì)胞阻滯。在細(xì)胞自噬方面,經(jīng)過(guò)雙環(huán)醇處理后,細(xì)胞內(nèi)自噬標(biāo)志蛋白LC3Ⅰ向LC3 Ⅱ轉(zhuǎn)化。另外,通過(guò)熒光顯微鏡對(duì)GFP-RFP-LC3轉(zhuǎn)染細(xì)胞的觀察和透射電鏡直接觀察顯示,細(xì)胞內(nèi)自噬小體和自噬溶酶體的數(shù)量在雙環(huán)醇誘導(dǎo)處理后顯著增加。這些結(jié)果說(shuō)明雙環(huán)醇通過(guò)抑制細(xì)胞周期和誘導(dǎo)細(xì)胞自噬,來(lái)抑制HepG2的細(xì)胞活性。2.雙環(huán)醇同時(shí)抑制PI3K/AKT通路和Ras/Raf/MEK/ERK通路。通過(guò)western blotting方法,證明雙環(huán)醇影響了G1期調(diào)控相關(guān)蛋白的活性。雙環(huán)醇能夠誘導(dǎo)p21CIP1和p27KIP1表達(dá),抑制CDK2-cyclinE和CDK4-cyclinD復(fù)合體水平,將Rb蛋白去磷酸化導(dǎo)致細(xì)胞周期阻滯于G1期,同時(shí)本課題還發(fā)現(xiàn)雙環(huán)醇通過(guò)抑制mTOR蛋白活性誘導(dǎo)了細(xì)胞自噬。通過(guò)進(jìn)一步研究周期與自噬相關(guān)上游信號(hào)通路,發(fā)現(xiàn)雙環(huán)醇顯著抑制了AKT蛋白的磷酸化,下調(diào)了PI3K/AKT通路。轉(zhuǎn)染AKTcDNA使雙環(huán)醇對(duì)腫瘤細(xì)胞的周期抑制和自噬誘導(dǎo)效果降低,而轉(zhuǎn)染AKT siRNA或加入化學(xué)抑制劑LY294002分別使雙環(huán)醇的抑瘤作用加強(qiáng)。這些結(jié)果表明AKT在雙環(huán)醇抑制腫瘤細(xì)胞活性過(guò)程中發(fā)揮重要的作用。另外,Ras/Raf/MEK/ERK通路也被雙環(huán)醇所抑制。轉(zhuǎn)染ERK siRNA或加入化學(xué)抑制劑PD98059也分別使雙環(huán)醇周期抑制及自噬誘導(dǎo)效果增強(qiáng)。以上結(jié)果說(shuō)明雙環(huán)醇的細(xì)胞毒性效果與PI3K/AKT通路和Ras/Raf/MEK/ERK通路有密切關(guān)系。3.通過(guò)裸鼠移植瘤模型研究雙環(huán)醇在體內(nèi)環(huán)境的作用。利用腋下移植方法將HepG2細(xì)胞移植于裸鼠,通過(guò)口服給藥雙環(huán)醇與索拉非尼。結(jié)果表明,雙環(huán)醇減小了小鼠體內(nèi)的腫瘤體積并且降低了腫瘤的重量,同時(shí)對(duì)小鼠不產(chǎn)生毒性。HE染色和TUNEL染色方法表明在腫瘤組織中雙環(huán)醇引起了腫瘤細(xì)胞一定程度的死亡,而在肝組織中沒(méi)有發(fā)現(xiàn)類似現(xiàn)象。對(duì)腫瘤組織進(jìn)行western blotting和免疫組化實(shí)驗(yàn),發(fā)現(xiàn)雙環(huán)醇降低了腫瘤組織內(nèi)的AKT和ERK磷酸化水平,抑制了蛋白活性。綜上所述,雙環(huán)醇對(duì)于腫瘤細(xì)胞的抑制增殖作用來(lái)源于細(xì)胞周期抑制與細(xì)胞自噬。在體外和體內(nèi)環(huán)境中,雙環(huán)醇的毒性與其對(duì)PI3K/AKT通路和Ras/Raf/MEK/ERK通路的抑制作用有關(guān)。這些結(jié)果使人們對(duì)雙環(huán)醇的藥物效果有了更深的理解,為發(fā)展新的抗腫瘤藥物奠定了一定的理論基礎(chǔ)。
[Abstract]:Hepatocellular carcinoma is the fifth largest tumor in the world and has a very high morbidity and mortality. Although a variety of methods such as chemotherapy, excision, liver transplantation, and immunotherapy have been used to treat the liver cancer, the survival rate of the liver cancer is only 3-5% in the developed countries, which has prompted the people to continue to find new methods of treating the liver cancer. Bicyclol (4,4 '-dimethoxy-5,6,5',6 '-bis (methylenedioxy) -2-hydroxymethyl-2'-methoxyphenyl) is a new drug for anti-hepatitis chemical synthesis, and has been widely used in the treatment of HBV infection. In addition, some studies have shown that the bicyclol also has the function of improving the liver function, It is also found that the double-ring alcohol can induce the expression of the heat shock protein HSP27/70 in the liver cancer cell, thereby inhibiting the apoptosis of the mitochondrial pathway of the cell. The anti-tumor effect of the double-ring alcohol and the study of the function of the double-ring alcohol in the cell cycle and autophagy of the tumor are still rare, The preliminary study on the high-throughput screening of differentially expressed genes in the cancer cells after the double-ring alcohol in the relevant literature shows that the double-ring alcohol has the potential as an anti-cancer drug, and therefore, the research focuses on the toxic effect of the bicyclol on the HepG2 cells, And the molecular mechanism of the double-ring alcohol on the action of the liver cancer cells is explored in the in vitro and in vivo environment, and the results are as follows:1. the double-ring alcohol can obviously inhibit the proliferation of the HepG2 cells, and the toxicity to the normal liver cells LO2 is very low. The IC50 value of bicyclol on HepG2 cells is one-third of the LO2 cell, and by flow cytometry it is found that the inhibitory effect of bicyclol on the activity of HepG2 cells is mainly due to its ability to inhibit cell cycle and to induce autophagy of the cells, and the level of apoptosis of HepG2 cells under the action of the bicyclol is not changed. In the aspect of the cell cycle, the double-ring alcohol can induce the G1 phase cell block which is dependent on the time and the concentration gradient of the HepG2 cells, In addition, the observation of GFP-RFP-LC3 transfected cells and the direct observation of transmission electron microscope (TEM) of GFP-RFP-LC3 were observed by fluorescence microscope. The number of autophagy bodies and autophagy lysosomes in the cells increased significantly after the double-ring alcohol induction treatment. These results indicated that the double-ring alcohol inhibited the cell activity of HepG2 by inhibiting the cell cycle and the autophagy of the cells.2. The bicyclol inhibited the PI3K/ AKT pathway and the Ras/ Raf/ MEK/ ERK pathway at the same time. The double-ring alcohol can induce the expression of p21CIP1 and p27KIP1, inhibit the level of CDK2-cyclinE and CDK4-cyclinD complex, and dephosphorylation of Rb protein results in cell cycle arrest in G1 phase. At the same time, it was found that the double-ring alcohol induced autophagy of the cell by inhibiting the activity of mTOR protein. By further studying the upstream signal path of the cycle and autophagy, it was found that the double-ring alcohol significantly inhibited the phosphorylation of the AKT protein. The PI3K/ AKT pathway was downregulated. The effect of Bicyclol on the cycle inhibition and autophagy of the tumor cells was reduced by the transfection of AKTcDNA. The results show that AKT plays an important role in the inhibition of tumor cell activity. The results indicated that the cytotoxicity of the double-ring alcohol was closely related to the PI3K/ AKT pathway and the Ras/ Raf/ MEK/ ERK pathway. The effect of the alcohol in the in vivo environment is that the HepG2 cells are transplanted to a nude mouse by an underarm transplantation method, and the bicyclol and sorafenib are administered by oral administration. The results show that the bicyclol reduces the tumor volume in the mice and reduces the weight of the tumor, The method of HE staining and TUNEL staining showed that the double-ring alcohol in the tumor tissue caused a certain degree of death of the tumor cells, and no similar phenomenon was found in the liver tissue. Western blotting and immunohistochemistry were performed on the tumor tissues. In conclusion, the inhibition and proliferation of the bicyclol on the tumor cells is derived from cell cycle inhibition and autophagy. In vitro and in vivo environment, The toxicity of the double-ring alcohol is related to the inhibition of the PI3K/ AKT pathway and the Ras/ Raf/ MEK/ ERK pathway.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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6 祁文s，

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