饮食限制对二乙基亚硝胺诱导小鼠肝细胞癌的抑制作用及机制探究

发布时间：2018-03-12 06:28

本文选题：饮食限制　切入点：肝细胞癌　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：肝细胞癌(Hepatocellular carcinoma,HCC)是最常见的致死癌症之一,乙肝或丙肝病毒感染、黄曲霉毒素、酒精、肥胖、烟草等导致的慢性肝病是诱导HCC的主要原因。目前,针对肝癌病人的治疗手段十分有限,且效果欠佳,这给医疗业造成极大负荷、给肝癌患者及其家庭带来严重的精神痛苦及经济负担。研究表明,在不涉及营养不良的情况下,饮食限制(Dietary restriction,DR)能够成功抑制原发性及化学诱导的肿瘤发生发展,但目前对于DR的这种抗癌效果的研究不够全面且缺乏深入的机制探索。因此,建立合适的肝肿瘤模型,观察DR对肝肿瘤的干预效应,并进一步探究其分子机制,将为临床治疗策略及肝病预防提供可靠参考,具有重要的意义。为了研究DR对肝癌的干预效应并揭示其分子机制,我们建立了二乙基亚硝胺(diethylnitrosamine,DEN)诱导小鼠HCC模型。两周龄雄性C57BL/6小鼠被随机分配到control俎、DR组、DEN组、DEN+DR组,DEN组及DEN+DR组给予单次腹腔注射25 mg/kg DEN,control组及DR组给予单次注射相应体积的生理盐水(0.9%)。DEN处理一周后,DR及DEN+DR组分别以control组与DEN组作为参照,以逐渐递减方式给予DR干预(90%DR 一周,80%DR一周,70%DR至实验终点),control组及DEN组小鼠始终给予自由进食,所有小鼠自由进水。DEN处理30周后,处死小鼠,收集标本。我们发现:与DEN组相比,DEN+DR组小鼠肝肿瘤数目减少,肿瘤体积减小,病理进程延缓;DEN+DR组小鼠肝脏凋亡、自噬水平上升,增殖受到抑制,DNA损伤降低,炎症减轻。同时,我们发现DR能够抑制DEN对mTOR及NF-1κB通路的活化,并且上调与DR抗肿瘤效果密切相关通路Keap1-Nrf2。另外,体外实验证实了 DR抑制Hepa1-6细胞增殖并诱导其凋亡。通过RNA测序及基因差异表达分析,我们发现DR有效逆转DEN对小鼠肝脏转录组的改变。根据测序结果的提示,我们在DEN组小鼠肝脏发现Braf基因突变及ERK通路的活化,这种现象并未在DEN+DR组出现。在体外实验中,通过ERK激动剂TPA、抑制剂SCH772984,我们进一步证实ERK通路影响Hepa1-6细胞活力且在DR对Hepa1-6细胞的抑制作用中发挥重要角色。为了明确DR诱导的自噬对其抗肿瘤效果的作用,按照野生型小鼠DEN建模及DR干预的操作,我们利用Ulk1敲除致使DR诱导自噬发生障碍。结果表明在Ulk1敲除小鼠中DR仍能发挥抗肿瘤效应,但与在野生型小鼠中相比,这种抑制作用减弱。更为重要的是,我们发现Ulk1敲除本身能够有效抑制DEN诱导HCC,且Ull1敲除与DR的抗癌效应之间存在交互作用。综上,我们认为:DR能够有效抑制DEN诱导小鼠HCC,DR的这种抗肿瘤作用主要与其对增殖、凋亡的调控有关,但自噬并不是DR抗肿瘤的关键;Braf/MEK/ERK信号通路对DR抗肿瘤作用具有重要意义。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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