ADRB2信号通路调控肝癌发生及发展的机制研究

发布时间：2018-07-31 05:22

【摘要】：研究背景和目的原发性肝癌(primary liver cancer)是世界上第五位最常见的恶性肿瘤,其预后差、病死率极高。在2008年,大约有75万新发现的病例,并有近70万病例死于肝癌。而到了2012年,新发现的病例数已经增加到了78万多人。肝细胞癌(hepatocellular carcinoma,HCC)是原发性肝癌中最常见的组织类型,占到全部肝癌的85%-90%。HCC起源于构成肝脏实质细胞的肝细胞,其发病情况在世界范围内分布很不均匀,超过80%的病例发生在非洲和东亚地区。而在所有HCC病例中,有50%来自于中国。恰恰相反,南北美洲及欧洲的HCC发病率很低。有许多因素造成了HCC分布的不均一性,其中环境因素也是其中很重要的一个方面。社会心理因素是一类十分重要的环境因素,而应激作为对机体生理病理影响最大的一类社会心理因素,其对肿瘤的影响也日益受到人们的关注。近期有许多证据支持慢性应激能够影响肿瘤生长和进展这一假说。研究表明,交感神经递质,如儿茶酚胺类物质或神经肽类,既能影响肿瘤细胞生长,又能引起肿瘤血管的生成。因此,肾上腺素系统的激活对不同类型肿瘤的生长均有促进作用,并参与调解了应激对肿瘤进展的促进作用。肾上腺素系统在应激过程中释放的儿茶酚胺类物质作为配体,是通过结合并激活细胞表面的肾上腺素能受体来发挥作用的。因此,研究应激对肿瘤调控的本质就是研究其激活的表达在肿瘤细胞表面上的肾上腺素能受体对肿瘤细胞具有哪些调控作用。本研究拟首先通过建立经典的二乙基亚硝胺(DEN)诱导肝细胞癌模型,来观察儿茶酚胺类物质能否在这个模型中促进肝细胞癌的发生及发展。同时,我们要进一步检测肝癌组织和细胞中各类肾上腺素能受体的表达水平,来确定哪种受体的激活能够介导儿茶酚胺类物质对肝细胞癌生长的调控。接下来,我们将重点研究该受体的激活通过怎样的方式促进了肿瘤细胞的生长。这不仅能够阐明应激是否对肝细胞癌的发生和发展产生影响,更能为临床控制肝细胞癌进展及改善患者预后提供新的方法,相关的研究目前在国内外均未见报道。实验方法1、建立小鼠二乙基亚硝胺(DEN)诱导肝细胞癌模型,通过给予小鼠腹腔注射肾上腺素或同时阻滞β2型肾上腺素能受体(ADRB2)来观察对小鼠肝细胞癌发生情况及肿瘤生长情况的影响；2、使用ADRB2的特异性抑制剂ICI118,551,并通过CCK8细胞增殖实验、克隆形成实验、流式细胞技术检测凋亡细胞比例等实验方法,体外检测阻滞ADRB2信号通路对肝癌细胞系增殖及存活能力的影响；3、使用电子透射电镜观察ICI118,551对细胞自噬的诱导,并通过Western Blot技术及GFP-LC3质粒转染的方法等检测ICI118,551对细胞自噬的调节：4、构建干扰ADRB2慢病毒所需的质粒、包装纯化慢病毒。通过ADRB2干扰细胞系进一步证实ADRB2信号通路对自噬的调控；5、构建体外诱导自噬的模型,并观察ADRB2特异性激动剂福莫特罗(FOR)对自噬的调节；6、利用Western Blot技术、免疫共沉淀技术(IP)等方法研究ADRB2信号通路对自噬调控的机制；7、通过裸鼠皮下荷瘤模型检测ADRB2信号通路对肿瘤细胞体内增殖能力的影响及p受体阻滞剂抑制肿瘤生长的效果；8、利用索拉菲尼进行自噬诱导,通过Western Blot技术及GFP-LC3质粒转染的方法检测ADRB2信号通路对索拉菲尼诱导的自噬的调节,并通过耐药克隆形成实验、CCK8细胞增殖实验及裸鼠皮下荷瘤模型检测ADRB2信号通路对索拉菲尼抗肿瘤效果的影响。结果1、肾上腺素能够促进DEN诱导的肝细胞癌的发生及进展,并且这种促进作用是通过激活ADRB2实现的；2、阻滞ADRB2信号通路能够在体外抑制肝癌细胞系的增殖和存活；3、ADRB2信号通路对自噬具有负向调控的作用,并通过激活Akt及调节Beclinl参与的自噬复合物来发挥作用；4、ADRB2信号通路激活后能够抑制HIFIA的自噬性降解,从而促进葡萄糖代谢；5、裸鼠皮下荷瘤实验证实ADRB2信号通路激活能够促进皮下肿瘤的生长,β受体阻滞剂普萘洛尔能够在一定程度上抑制肿瘤的生长；6、ADRB2信号通路能够抑制索拉菲尼诱导的细胞自噬,并且阻滞ADRB2信号通路后能够促进索拉菲尼的抗肿瘤效果；7、肝癌组织中ADRB2的表达联合HIFIA的表达能够很好地预测肝癌患者的预后。结论本研究从不同的动物实验模型和体外培养的肝癌细胞系这两个方面,首次证明了在应激条件下能够被激活的ADRB2信号通路对肝癌发生及发展的影响。通过实验,我们发现,一方面,ADRB2信号通路激活后能够增强肝癌细胞的体内成瘤性和体外的增殖能力；另一方面,ADRB2信号通路的激活还能够影响索拉菲尼的药物敏感性使机体对索拉菲尼更加耐受。而这些作用均是通过对细胞自噬的负向调控进而增加HIF1A稳定性实现的。我们的研究首次报道了ADRB2信号通路与细胞自噬之间的关系,以及通过自噬对肝癌细胞增殖的影响。这为更好地理解应激等外在环境因素对肝癌进展的影响提供了线索和依据,同时也为临床探索新的治疗肝癌的方法指明了一个新的方向。
[Abstract]:Background and objective primary liver cancer (primary liver cancer) is the fifth most common malignant tumor in the world, with poor prognosis and high mortality. In 2008, about 75 thousands of newly discovered cases and nearly 700 thousand cases died of liver cancer. In 2012, the number of newly discovered cases had increased to more than 780 thousand. (hepatoce). Llular carcinoma, HCC) is the most common type of tissue in primary liver cancer, and the 85%-90%.HCC of all liver cancer originates from liver cells that form the parenchymal cells of the liver. The incidence of the disease is unevenly distributed worldwide. More than 80% of the cases occur in Africa and East Asia. In all HCC cases, 50% come from China. On the contrary, the incidence of HCC in North America and Europe is very low. There are many factors that cause the heterogeneity of HCC distribution, and environmental factors are also one of the most important aspects. Psychosocial factors are a very important environmental factor, and stress is a kind of social psychological factor which is the biggest response to the body's physiological and pathological changes. There is a lot of evidence to support the hypothesis that chronic stress can affect the growth and progression of cancer. Research shows that sympathetic neurotransmitters, such as catecholamines or neuropeptides, can affect both tumor cell growth and tumor angiogenesis. Therefore, the activation of adrenaline system The growth of different types of tumor has a promoting effect and participates in mediating the effect of stress on the progression of tumor. The catecholamines released by the adrenergic system act as a ligand to bind and activate the adrenergic receptors on the surface of the cell. This study is to investigate the effects of the adrenergic receptor on the tumor cells on the tumor cells to regulate the tumor cells. This study is to investigate whether catecholamines can promote the development and development of hepatocellular carcinoma in this model by establishing a classic model of two ethyl nitrosamine (DEN) induced hepatocellular carcinoma. At the same time, we should further detect the expression level of all kinds of adrenergic receptors in liver cancer tissues and cells to determine which receptor activation can mediate the regulation of catecholamines on the growth of hepatocellular carcinoma. It is only possible to clarify whether stress affects the occurrence and development of hepatocellular carcinoma, and can provide a new method for clinical control of the progression of hepatocellular carcinoma and improving the prognosis of patients. The related research has not been reported at home and abroad. Method 1, the model of hepatocarcinoma induced by two ethyl nitrosamines (DEN) in mice was established by giving mice abdominal cavity. Injecting adrenaline or blocking beta 2 adrenergic receptor (ADRB2) to observe the occurrence of hepatocellular carcinoma and the growth of tumor in mice. 2, using the specific inhibitor ICI118551 of ADRB2, and through the proliferation test of CCK8 cells, cloning and forming experiment, flow cytometry to detect the proportion of apoptotic cells, and so on. The effect of ADRB2 signaling pathway on the proliferation and survival of hepatoma cell lines was detected by external detection. 3, the induction of autophagy by ICI118551 was observed by electronic transmission electron microscopy, and the regulation of autophagy by ICI118551 was detected by Western Blot and GFP-LC3 plasmid transfection: 4, the plasmids needed to interfere with the ADRB2 lentivirus were constructed. Package and purify lentivirus. Through ADRB2 interference cell lines further confirm the regulation of autophagy by ADRB2 signaling pathway; 5, construct a model of autophagy induced in vitro, and observe the regulation of autophagy by ADRB2 specific agonist, formoterol (FOR); 6, use Western Blot technology, immunoprecipitation (IP) and other methods to study the ADRB2 signaling pathway. The mechanism of autophagy regulation; 7, the effect of ADRB2 signaling pathway on the proliferation of tumor cells and the effect of P receptor blocker on tumor growth were detected by subcutaneous tumor bearing model in nude mice. 8, autophagy induced by Sola Feeney was used to detect the ADRB2 signaling pathway by Western Blot technique and GFP-LC3 plasmid transfer. Regulation of induction of autophagy, and through the formation of drug resistant clones, CCK8 cell proliferation test and nude mouse subcutaneous tumor bearing model to detect the effect of ADRB2 signaling pathway on the antitumor effect of sorafeni. Results 1, adrenaline can promote the development and progression of DEN induced hepatocellular carcinoma, and this promotion is achieved by activating ADRB2 2, blocking ADRB2 signaling pathway can inhibit the proliferation and survival of liver cancer cell lines in vitro; 3, ADRB2 signaling pathway has a negative regulation of autophagy, and plays a role by activating Akt and regulating the autophagic complex involved in Beclinl; 4, ADRB2 signaling pathway can inhibit the autophagy degradation of HIFIA and thus promote the grape 5, the subcutaneous tumor test in nude mice confirmed that the activation of ADRB2 signaling pathway could promote the growth of subcutaneous tumors. The beta blocker propranolol could inhibit the growth of the tumor to a certain extent; 6, the ADRB2 signaling pathway could inhibit the autophagy induced by Sola Feeney, and could promote Sola Feeney after blocking the ADRB2 signaling pathway. 7, the expression of ADRB2 in liver cancer tissue and expression of HIFIA can predict the prognosis of liver cancer patients well. Conclusion this study is the first evidence that the ADRB2 signaling pathway can be activated in stress conditions for the occurrence and development of liver cancer in the two aspects of different animal models and in vitro cultured hepatoma cell lines. On the one hand, we found that, on the one hand, the activation of the ADRB2 signaling pathway can enhance the tumorigenicity of the liver cancer cells and the ability to proliferate in vitro; on the other hand, the activation of the ADRB2 signaling pathway also affects the drug sensitivity of sorafeni, which makes the body more tolerant to sorafeni. The negative regulation of cytosolic autophagy increases the stability of HIF1A. Our study for the first time reports the relationship between ADRB2 signaling pathway and autophagy and the effect of autophagy on the proliferation of hepatoma cells. This provides a clue and basis for better understanding of the effects of stress on the progression of liver cancer. The bed explores new ways to treat liver cancer, indicating a new direction.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

【相似文献】