【摘要】：研究背景和目的原發(fā)性肝癌(primary liver cancer)是世界上第五位最常見的惡性腫瘤,其預(yù)后差、病死率極高。在2008年,大約有75萬新發(fā)現(xiàn)的病例,并有近70萬病例死于肝癌。而到了2012年,新發(fā)現(xiàn)的病例數(shù)已經(jīng)增加到了78萬多人。肝細(xì)胞癌(hepatocellular carcinoma,HCC)是原發(fā)性肝癌中最常見的組織類型,占到全部肝癌的85%-90%。HCC起源于構(gòu)成肝臟實(shí)質(zhì)細(xì)胞的肝細(xì)胞,其發(fā)病情況在世界范圍內(nèi)分布很不均勻,超過80%的病例發(fā)生在非洲和東亞地區(qū)。而在所有HCC病例中,有50%來自于中國。恰恰相反,南北美洲及歐洲的HCC發(fā)病率很低。有許多因素造成了HCC分布的不均一性,其中環(huán)境因素也是其中很重要的一個(gè)方面。社會(huì)心理因素是一類十分重要的環(huán)境因素,而應(yīng)激作為對(duì)機(jī)體生理病理影響最大的一類社會(huì)心理因素,其對(duì)腫瘤的影響也日益受到人們的關(guān)注。近期有許多證據(jù)支持慢性應(yīng)激能夠影響腫瘤生長和進(jìn)展這一假說。研究表明,交感神經(jīng)遞質(zhì),如兒茶酚胺類物質(zhì)或神經(jīng)肽類,既能影響腫瘤細(xì)胞生長,又能引起腫瘤血管的生成。因此,腎上腺素系統(tǒng)的激活對(duì)不同類型腫瘤的生長均有促進(jìn)作用,并參與調(diào)解了應(yīng)激對(duì)腫瘤進(jìn)展的促進(jìn)作用。腎上腺素系統(tǒng)在應(yīng)激過程中釋放的兒茶酚胺類物質(zhì)作為配體,是通過結(jié)合并激活細(xì)胞表面的腎上腺素能受體來發(fā)揮作用的。因此,研究應(yīng)激對(duì)腫瘤調(diào)控的本質(zhì)就是研究其激活的表達(dá)在腫瘤細(xì)胞表面上的腎上腺素能受體對(duì)腫瘤細(xì)胞具有哪些調(diào)控作用。本研究擬首先通過建立經(jīng)典的二乙基亞硝胺(DEN)誘導(dǎo)肝細(xì)胞癌模型,來觀察兒茶酚胺類物質(zhì)能否在這個(gè)模型中促進(jìn)肝細(xì)胞癌的發(fā)生及發(fā)展。同時(shí),我們要進(jìn)一步檢測肝癌組織和細(xì)胞中各類腎上腺素能受體的表達(dá)水平,來確定哪種受體的激活能夠介導(dǎo)兒茶酚胺類物質(zhì)對(duì)肝細(xì)胞癌生長的調(diào)控。接下來,我們將重點(diǎn)研究該受體的激活通過怎樣的方式促進(jìn)了腫瘤細(xì)胞的生長。這不僅能夠闡明應(yīng)激是否對(duì)肝細(xì)胞癌的發(fā)生和發(fā)展產(chǎn)生影響,更能為臨床控制肝細(xì)胞癌進(jìn)展及改善患者預(yù)后提供新的方法,相關(guān)的研究目前在國內(nèi)外均未見報(bào)道。實(shí)驗(yàn)方法1、建立小鼠二乙基亞硝胺(DEN)誘導(dǎo)肝細(xì)胞癌模型,通過給予小鼠腹腔注射腎上腺素或同時(shí)阻滯β2型腎上腺素能受體(ADRB2)來觀察對(duì)小鼠肝細(xì)胞癌發(fā)生情況及腫瘤生長情況的影響；2、使用ADRB2的特異性抑制劑ICI118,551,并通過CCK8細(xì)胞增殖實(shí)驗(yàn)、克隆形成實(shí)驗(yàn)、流式細(xì)胞技術(shù)檢測凋亡細(xì)胞比例等實(shí)驗(yàn)方法,體外檢測阻滯ADRB2信號(hào)通路對(duì)肝癌細(xì)胞系增殖及存活能力的影響；3、使用電子透射電鏡觀察ICI118,551對(duì)細(xì)胞自噬的誘導(dǎo),并通過Western Blot技術(shù)及GFP-LC3質(zhì)粒轉(zhuǎn)染的方法等檢測ICI118,551對(duì)細(xì)胞自噬的調(diào)節(jié)：4、構(gòu)建干擾ADRB2慢病毒所需的質(zhì)粒、包裝純化慢病毒。通過ADRB2干擾細(xì)胞系進(jìn)一步證實(shí)ADRB2信號(hào)通路對(duì)自噬的調(diào)控；5、構(gòu)建體外誘導(dǎo)自噬的模型,并觀察ADRB2特異性激動(dòng)劑福莫特羅(FOR)對(duì)自噬的調(diào)節(jié)；6、利用Western Blot技術(shù)、免疫共沉淀技術(shù)(IP)等方法研究ADRB2信號(hào)通路對(duì)自噬調(diào)控的機(jī)制；7、通過裸鼠皮下荷瘤模型檢測ADRB2信號(hào)通路對(duì)腫瘤細(xì)胞體內(nèi)增殖能力的影響及p受體阻滯劑抑制腫瘤生長的效果；8、利用索拉菲尼進(jìn)行自噬誘導(dǎo),通過Western Blot技術(shù)及GFP-LC3質(zhì)粒轉(zhuǎn)染的方法檢測ADRB2信號(hào)通路對(duì)索拉菲尼誘導(dǎo)的自噬的調(diào)節(jié),并通過耐藥克隆形成實(shí)驗(yàn)、CCK8細(xì)胞增殖實(shí)驗(yàn)及裸鼠皮下荷瘤模型檢測ADRB2信號(hào)通路對(duì)索拉菲尼抗腫瘤效果的影響。結(jié)果1、腎上腺素能夠促進(jìn)DEN誘導(dǎo)的肝細(xì)胞癌的發(fā)生及進(jìn)展,并且這種促進(jìn)作用是通過激活A(yù)DRB2實(shí)現(xiàn)的；2、阻滯ADRB2信號(hào)通路能夠在體外抑制肝癌細(xì)胞系的增殖和存活；3、ADRB2信號(hào)通路對(duì)自噬具有負(fù)向調(diào)控的作用,并通過激活A(yù)kt及調(diào)節(jié)Beclinl參與的自噬復(fù)合物來發(fā)揮作用；4、ADRB2信號(hào)通路激活后能夠抑制HIFIA的自噬性降解,從而促進(jìn)葡萄糖代謝；5、裸鼠皮下荷瘤實(shí)驗(yàn)證實(shí)ADRB2信號(hào)通路激活能夠促進(jìn)皮下腫瘤的生長,β受體阻滯劑普萘洛爾能夠在一定程度上抑制腫瘤的生長；6、ADRB2信號(hào)通路能夠抑制索拉菲尼誘導(dǎo)的細(xì)胞自噬,并且阻滯ADRB2信號(hào)通路后能夠促進(jìn)索拉菲尼的抗腫瘤效果；7、肝癌組織中ADRB2的表達(dá)聯(lián)合HIFIA的表達(dá)能夠很好地預(yù)測肝癌患者的預(yù)后。結(jié)論本研究從不同的動(dòng)物實(shí)驗(yàn)?zāi)Ｐ秃腕w外培養(yǎng)的肝癌細(xì)胞系這兩個(gè)方面,首次證明了在應(yīng)激條件下能夠被激活的ADRB2信號(hào)通路對(duì)肝癌發(fā)生及發(fā)展的影響。通過實(shí)驗(yàn),我們發(fā)現(xiàn),一方面,ADRB2信號(hào)通路激活后能夠增強(qiáng)肝癌細(xì)胞的體內(nèi)成瘤性和體外的增殖能力；另一方面,ADRB2信號(hào)通路的激活還能夠影響索拉菲尼的藥物敏感性使機(jī)體對(duì)索拉菲尼更加耐受。而這些作用均是通過對(duì)細(xì)胞自噬的負(fù)向調(diào)控進(jìn)而增加HIF1A穩(wěn)定性實(shí)現(xiàn)的。我們的研究首次報(bào)道了ADRB2信號(hào)通路與細(xì)胞自噬之間的關(guān)系,以及通過自噬對(duì)肝癌細(xì)胞增殖的影響。這為更好地理解應(yīng)激等外在環(huán)境因素對(duì)肝癌進(jìn)展的影響提供了線索和依據(jù),同時(shí)也為臨床探索新的治療肝癌的方法指明了一個(gè)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7

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