本文選題：飲食限制　切入點：肝細胞癌　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：肝細胞癌(Hepatocellular carcinoma,HCC)是最常見的致死癌癥之一,乙肝或丙肝病毒感染、黃曲霉毒素、酒精、肥胖、煙草等導(dǎo)致的慢性肝病是誘導(dǎo)HCC的主要原因。目前,針對肝癌病人的治療手段十分有限,且效果欠佳,這給醫(yī)療業(yè)造成極大負荷、給肝癌患者及其家庭帶來嚴重的精神痛苦及經(jīng)濟負擔(dān)。研究表明,在不涉及營養(yǎng)不良的情況下,飲食限制(Dietary restriction,DR)能夠成功抑制原發(fā)性及化學(xué)誘導(dǎo)的腫瘤發(fā)生發(fā)展,但目前對于DR的這種抗癌效果的研究不夠全面且缺乏深入的機制探索。因此,建立合適的肝腫瘤模型,觀察DR對肝腫瘤的干預(yù)效應(yīng),并進一步探究其分子機制,將為臨床治療策略及肝病預(yù)防提供可靠參考,具有重要的意義。為了研究DR對肝癌的干預(yù)效應(yīng)并揭示其分子機制,我們建立了二乙基亞硝胺(diethylnitrosamine,DEN)誘導(dǎo)小鼠HCC模型。兩周齡雄性C57BL/6小鼠被隨機分配到control俎、DR組、DEN組、DEN+DR組,DEN組及DEN+DR組給予單次腹腔注射25 mg/kg DEN,control組及DR組給予單次注射相應(yīng)體積的生理鹽水(0.9%)。DEN處理一周后,DR及DEN+DR組分別以control組與DEN組作為參照,以逐漸遞減方式給予DR干預(yù)(90%DR 一周,80%DR一周,70%DR至實驗終點),control組及DEN組小鼠始終給予自由進食,所有小鼠自由進水。DEN處理30周后,處死小鼠,收集標本。我們發(fā)現(xiàn):與DEN組相比,DEN+DR組小鼠肝腫瘤數(shù)目減少,腫瘤體積減小,病理進程延緩;DEN+DR組小鼠肝臟凋亡、自噬水平上升,增殖受到抑制,DNA損傷降低,炎癥減輕。同時,我們發(fā)現(xiàn)DR能夠抑制DEN對mTOR及NF-1κB通路的活化,并且上調(diào)與DR抗腫瘤效果密切相關(guān)通路Keap1-Nrf2。另外,體外實驗證實了 DR抑制Hepa1-6細胞增殖并誘導(dǎo)其凋亡。通過RNA測序及基因差異表達分析,我們發(fā)現(xiàn)DR有效逆轉(zhuǎn)DEN對小鼠肝臟轉(zhuǎn)錄組的改變。根據(jù)測序結(jié)果的提示,我們在DEN組小鼠肝臟發(fā)現(xiàn)Braf基因突變及ERK通路的活化,這種現(xiàn)象并未在DEN+DR組出現(xiàn)。在體外實驗中,通過ERK激動劑TPA、抑制劑SCH772984,我們進一步證實ERK通路影響Hepa1-6細胞活力且在DR對Hepa1-6細胞的抑制作用中發(fā)揮重要角色。為了明確DR誘導(dǎo)的自噬對其抗腫瘤效果的作用,按照野生型小鼠DEN建模及DR干預(yù)的操作,我們利用Ulk1敲除致使DR誘導(dǎo)自噬發(fā)生障礙。結(jié)果表明在Ulk1敲除小鼠中DR仍能發(fā)揮抗腫瘤效應(yīng),但與在野生型小鼠中相比,這種抑制作用減弱。更為重要的是,我們發(fā)現(xiàn)Ulk1敲除本身能夠有效抑制DEN誘導(dǎo)HCC,且Ull1敲除與DR的抗癌效應(yīng)之間存在交互作用。綜上,我們認為:DR能夠有效抑制DEN誘導(dǎo)小鼠HCC,DR的這種抗腫瘤作用主要與其對增殖、凋亡的調(diào)控有關(guān),但自噬并不是DR抗腫瘤的關(guān)鍵;Braf/MEK/ERK信號通路對DR抗腫瘤作用具有重要意義。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common fatal cancers. Chronic liver disease caused by hepatitis B or hepatitis C virus infection, aflatoxin, alcohol, obesity, tobacco and so on is the main cause of inducing HCC. The treatment of liver cancer patients is very limited, and the effect is not good, which results in a heavy burden on the medical industry, serious mental pain and economic burden on the liver cancer patients and their families. Research shows that when malnutrition is not involved, Dietary restriction DR1 can successfully inhibit the development of primary and chemically induced tumors, but the current research on the anticancer effect of Dr is not comprehensive enough and lack of deep research on the mechanism. Therefore, an appropriate liver tumor model is established. It is of great significance to observe the intervention effect of Dr on liver tumor and further explore its molecular mechanism, which will provide reliable reference for clinical treatment strategy and prevention of liver disease, in order to study the interventional effect of Dr on liver cancer and reveal its molecular mechanism. HCC model was established in mice induced by diethylnitrosamine (DEN). Two-week-old male C57BL / 6 mice were randomly assigned to control group, den group, den group, DEN Dr group, and DEN Dr group. 25 mg/kg DENcontrol group and Dr group were given a single intraperitoneal injection of 25 mg/kg DENcontrol and Dr groups. The corresponding volume of normal saline 0.9g 路den was treated for one week, and the control group and the DEN group were used as the reference group, respectively, for the Dr and DEN Dr groups. The mice in the control group and DEN group were given free food, and all the mice were killed after 30 weeks of free water treatment. We found that compared with the DEN group, the number of liver tumors and tumor volume in the den Dr group decreased, the pathological process delayed the apoptosis of liver, the autophagy level increased, and the proliferation was inhibited in the den Dr group. At the same time, we found that Dr could inhibit the activation of mTOR and NF-1 魏 B pathway by DEN, and up-regulate Keap1-Nrf2associated with the anti-tumor effect of Dr. Dr inhibited the proliferation and induced apoptosis of Hepa1-6 cells in vitro. By RNA sequencing and gene differential expression analysis, we found Dr could effectively reverse the transcriptional changes of mouse liver by DEN. We found the mutation of Braf gene and activation of ERK pathway in the liver of DEN group, which was not found in DEN Dr group. Through ERK agonist TPA, inhibitor SCH772984, we further demonstrated that the ERK pathway affects the viability of Hepa1-6 cells and plays an important role in the inhibitory effect of Dr on Hepa1-6 cells. According to DEN modeling and Dr intervention in wild-type mice, Dr knockout was used to induce Dr induced autophagy. The results showed that Dr could still play an antitumor effect in Ulk1 knockout mice, but compared with that in wild type mice. More importantly, we found that Ulk1 knockout itself can effectively inhibit HCC- induced by DEN, and there is an interaction between Ull1 knockout and the anticancer effect of Dr. We think that the anti-tumor effect of DEN induced HCCG-DR is mainly related to the regulation of proliferation and apoptosis, but autophagy is not the key to Dr anti-tumor.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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