發(fā)布時間：2018-01-31 13:43

  本文關(guān)鍵詞： 肝細胞癌 ARK5 TGF-β1 Akt　出處：《鄭州大學》2016年博士論文　論文類型：學位論文

【摘要】：肝細胞癌(Hepatocellular carcinoma,HCC)為世界范圍內(nèi)最常見的惡性腫瘤之一,有研究報道HBV和HCV感染為HCC最主要的病因,在歐美等其他一些發(fā)達國家或地區(qū),酒精性肝硬化以及與肥胖相關(guān)的非酒精性脂肪肝亦是造成罹患HCC的主要原因。惡性腫瘤的發(fā)生和發(fā)展通常是由體內(nèi)外多種因素共同作用的多階段參與的復雜過程。HCC的發(fā)生過程是由體內(nèi)外多種因素以及多條信號通路共同作用的結(jié)果,盡管目前臨床已廣泛使用常規(guī)放療以及化療等技術(shù)有效控制了HCC的發(fā)生和發(fā)展,但大部分患者在接受上述療法之后的預后情況依然不容樂觀。靶向治療是目前新發(fā)現(xiàn)的、能夠有效治療HCC的新途徑,已成為基礎(chǔ)腫瘤醫(yī)學界研究的熱點和難點內(nèi)容。廣大的醫(yī)學工作者們正在試圖尋找能夠成為臨床上有效治療HCC的新靶點,以期為有效控制HCC和改善患者預后奠定基礎(chǔ)。ARK5(AMPK-related protein kinase 5)的異常表達與臨床上多種惡性腫瘤的發(fā)生和發(fā)展存在密切聯(lián)系,還與惡性腫瘤的侵襲和轉(zhuǎn)移相關(guān)。目前研究已證實ARK5基因在人類的多種惡性腫瘤組織中存在高表達,特別是在一些侵襲和轉(zhuǎn)移性較高的惡性腫瘤中,但其在HCC中表達研究相對較少。目前對于Akt(Protein Kinase B)在HCC發(fā)生和發(fā)展過程中如何調(diào)控ARK5與HCC腫瘤的增殖、凋亡以及侵襲過程中的相關(guān)機制的研究依然較少,TGF-β1(transforming growth factor-beta 1)信號通路出現(xiàn)異常是惡性腫瘤發(fā)生、發(fā)展、浸潤、轉(zhuǎn)移等過程的重要因素,TGF-β1信號通路出現(xiàn)異常是惡性腫瘤發(fā)生、發(fā)展、浸潤、轉(zhuǎn)移等過程的重要因素。本研究擬觀察ark5在hcc患者腫瘤組織中的表達及意義,分析ark5表達的變化對hcc腫瘤細胞增殖、凋亡、侵襲的影響,探究tgf-β1和akt信號活化對hcc腫瘤細胞中ark5表達的調(diào)控作用及其對hcc腫瘤細胞生物學行為的影響,旨在為了解hcc的發(fā)生和發(fā)展機制并為hcc的臨床診斷及靶向治療提供可靠依據(jù)。目的本研究擬觀察ark5在肝細胞癌(hepatocellularcarcinoma,hcc)患者腫瘤組織中的表達及意義,分析ark5表達的變化對hcc腫瘤細胞增殖、凋亡、侵襲的影響,探究tgf-β1和akt信號活化對hcc腫瘤細胞中ark5表達的調(diào)控作用及其對hcc腫瘤細胞生物學行為的影響,旨在為了解hcc的發(fā)生和發(fā)展機制并為hcc的臨床診斷及靶向治療提供可靠依據(jù)。方法(1)運用熒光定量pcr對20例hcc組織以及相應(yīng)癌旁正常組織樣品中ark5mrna的表達情況進行測定;運用westernblot法檢測hcc癌組織及其相應(yīng)癌旁正常組織中ark5蛋白的表達;運用免疫組織化學法檢測樣品中ark5蛋白的表達;分析ark5蛋白的表達與hcc患者臨床病理參數(shù)以及患者生存率的相關(guān)性。(2)將sirna-ark5轉(zhuǎn)染人肝癌細胞ammc-7721,以此降低細胞中ark5的表達;熒光定量pcr和westernblot法檢測轉(zhuǎn)染后ammc-7721細胞中ark5mrna和相應(yīng)蛋白的表達;運用mtt法檢測轉(zhuǎn)染后細胞的增殖活性;annexinv-fitc/pi雙染法檢測轉(zhuǎn)染對細胞凋亡的影響;用transweii小室法檢測轉(zhuǎn)染后細胞的侵襲能力;細胞劃痕實驗檢測轉(zhuǎn)染后對細胞遷移能力的影響;檢測轉(zhuǎn)染后細胞中caspase-3活性。(3)將tgf-β1抑制劑ly364947作用于人肝癌細胞ammc-7721,以此降低細胞中tgf-β1的表達,brdu細胞增殖實驗檢測細胞的增殖能力;用transweii小室法檢測細胞的侵襲能力;細胞劃痕實驗檢測細胞的遷移能力;westernblot法檢測細胞中tgf-β1、akt、ark5蛋白的表達。結(jié)果(1)ARK5 mRNA及其相應(yīng)蛋白在HCC癌組織中的表達顯著高于其在相應(yīng)癌旁正常肝臟組織中的表達;ARK5高表達與HCC腫瘤大小、組織分化程度、腫瘤分期顯著相關(guān)(P0.05);而與患者年齡、肝硬化、HBsAg、血清AFP無關(guān)(P0.05);ARK5蛋白表達能夠作為HCC的一個獨立預后判斷因素。(2)轉(zhuǎn)染siRNA-ARK5后AMMC-7721細胞中ARK5 mRNA和相應(yīng)蛋白的表達顯著降低(P0.05);siRNA-ARK5組AMMC-7721細胞的增殖活性顯著低于siRNA-NC組和空白組,且siRNA-ARK5組細胞生長抑制率在48 h為最高,與siRNA-NC組相比差異顯著(P0.01);轉(zhuǎn)染48 h后,siRNA-ARK5組細胞的凋亡率明顯增高,與空白組相比差異顯著(P0.01);siRNA-ARK5組細胞的侵襲能力明顯降低,與與空白組相比差異顯著(P0.05);與空白組相比,siRNA-ARK5組細胞的遷移能力明顯降低(P0.01);siRNA-ARK5組細胞的caspase-3活性較空白組明顯增高(P0.05)(3)TGF-β1抑制劑Ly364947作用于AMMC-7721細胞后,TGF-β1 inhibitor組細胞的增殖活性較對照組明顯降低(P0.01);TGF-β1 inhibitor組細胞的侵襲能力較對照組明顯降低(P0.05);TGF-β1 inhibitor組細胞的遷移能力較對照組明顯降低(P0.01);TGF-β1被抑制后,細胞中的Akt和ARK5蛋白表達量較對照組顯著下降(P0.05)。結(jié)論HCC組織中ARK5的表達與腫瘤大小、組織分化程度、腫瘤分期之間存在密切聯(lián)系,這可能是與TGF-β1刺激Akt/ARK5信號轉(zhuǎn)導通路促進了AMMC-7721細胞的增殖、侵襲和轉(zhuǎn)移相關(guān)。
[Abstract]:Hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) is one of the most common malignant tumors in the world, studies have reported that HBV and HCV HCC infection was the major cause in Europe and some other developed countries or regions, alcoholic liver cirrhosis associated with obesity and nonalcoholic fatty liver disease is mainly caused by the risk of HCC the occurrence and development of malignant tumor is usually occurred during.HCC complex multistep process interaction in vivo by various factors involved in the body is composed of many factors and the interaction of multi pathway results, although at present has been widely used in clinical routine radiotherapy and chemotherapy technology to effectively control the occurrence and development of HCC the prognosis of most patients, but after receiving the therapy is still not optimistic. Targeted therapy is a new discovery, a new way to treat HCC effectively, has become As a hot and difficult content based medical oncology research. The majority of medical workers who are attempting to become a new target for clinical treatment of HCC, in order to lay the foundation for the prognosis of patients with.ARK5 and improve the effective control of HCC (AMPK-related protein kinase 5) abnormal expression is closely related with the occurrence and development of clinical a variety of malignant tumors, and malignant tumor invasion and metastasis. It has been confirmed that ARK5 is highly expressed in a variety of malignant tumors in human tissues, especially in the invasion and metastasis of highly malignant tumors, but its expression in HCC research is relatively small. The Akt (Protein Kinase B) in HCC and how to regulate ARK5 and HCC in the development of the tumor proliferation, apoptosis and invasion mechanism of the process is still less, beta 1 (transforming growth FA TGF- Ctor-beta 1) signaling pathway abnormalities are malignant tumor occurrence, development, invasion, metastasis and other important factors in the process of TGF-, beta 1 signaling pathway abnormalities are the malignant tumor occurrence, development, invasion, metastasis and other important factors in the process. This study was to investigate the expression and significance of ARK5 in patients with HCC tumor tissues, analysis the expression of ARK5 on proliferation and apoptosis of HCC tumor cells, invasion effect, explore tgf- beta 1 and Akt signal activation on the regulation of the expression of ARK5 HCC in tumor cells and its effect on the biological behavior of HCC cells to tumor, clinical diagnosis and target for understanding the mechanism of occurrence and development of HCC and HCC to provide a reliable basis for the purpose of this study is to observe the treatment. ARK5 in hepatocellular carcinoma (hepatocellularcarcinoma, HCC) expression and significance of tumor patients, analysis of the expression of ARK5 on proliferation and apoptosis of HCC tumor cells, invasion. Ring, explore tgf- beta 1 and Akt signal activation on the regulation of the expression of ARK5 HCC in tumor cells and its effect on the biological behavior of HCC cells to tumor, clinical diagnosis and target for understanding the mechanism of occurrence and development of HCC and HCC to provide a reliable basis for treatment. Methods (1) using fluorescence quantitative PCR in 20 cases HCC and ark5mrna expression in adjacent normal tissue samples were measured; ARK5 protein expression detected by using the Westernblot method of HCC tissues and adjacent normal tissues; ARK5 protein expression detected by immunohistochemistry method in the analysis; the expression of ARK5 and HCC with clinicopathological parameters and survival rate the correlation. (2) sirna-ark5 transfected human hepatoma cell line ammc-7721, in order to reduce the expression of ARK5 in the cells; ammc-7721 cells detected by fluorescence quantitative PCR and Westernblot method ark5mrna And the corresponding protein expression; MTT was used to measure the proliferation of transfected cells; annexinv-fitc/pi double staining method to detect the transfection effect on cell apoptosis; invasion detection of transfected cells by transweii chamber assay; cell scratch assay after transfection can influence on cell migration force; detection of Caspase-3 in the transfected cells (activity. 3) tgf- beta 1 inhibitor ly364947 on human hepatocellular carcinoma cell line ammc-7721, in order to reduce the expression of tgf- beta 1 cells, BrdU cell proliferation assay to detect cell proliferation; cell detected by transweii chamber invasion; migration of cell scratch assay; cell Westernblot detection of tgf- beta 1, Akt, the expression of ARK5 protein. Results (1) the expression of ARK5 and related protein mRNA in HCC carcinoma tissues was significantly higher than the expression in adjacent normal liver tissues; the high expression of ARK5 and HCC Tumor size, histological grade, tumor stage was significantly correlated (P0.05); and the patient's age, liver cirrhosis, serum HBsAg, independent of AFP (P0.05); the expression of ARK5 protein can be used as a HCC independent prognosis factors. (2) the expression of ARK5 AMMC-7721 cells in mRNA and protein after transfection of siRNA-ARK5 significantly decreased (P0.05); group siRNA-ARK5 AMMC-7721 cell proliferation activity was significantly lower than that of siRNA-NC group and blank group, siRNA-ARK5 group and the cell growth inhibition rate at 48 h was the highest, compared with the siRNA-NC group (P0.01); 48 h after transfection, the apoptosis rate of cells in siRNA-ARK5 group increased significantly compared with the control group had significant difference (P0.01) group siRNA-ARK5; cell invasion ability decreased significantly, with significant difference compared with the control group (P0.05); compared with the control group, the migration ability of siRNA-ARK5 cells was significantly reduced (P0.01); the activity of Caspase-3 cells in siRNA-ARK5 group Significantly higher than the control group (P0.05) (3) TGF- beta 1 inhibitor Ly364947 on AMMC-7721 cell, TGF- beta 1 group inhibitor cell proliferation activity was significantly lower than the control group (P0.01); group TGF- beta 1 inhibitor cell invasion ability was significantly lower than the control group (P0.05); the migration ability of TGF- beta 1 inhibitor the cell group was significantly lower than the control group (P0.01); TGF- beta 1 is inhibited, the expression of Akt and ARK5 protein were significantly decreased compared to the control group (P0.05). The expression of ARK5 and tumor size. Conclusion HCC tissues, tissue differentiation, there is a close relationship between tumor stage, it is possible to promote the proliferation of AMMC-7721 cells and TGF- beta 1 stimulation of Akt/ARK5 signal transduction pathway, invasion and metastasis.

【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7

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5 嚴曉娣;shRNA干預IGF-I受體活化對肝癌細胞增殖抑制及其分子機制研究[D];南通大學;2014年

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8 李鳳霞;Myrothecine A通過miR-221調(diào)控肝癌細胞增殖及樹突狀細胞成熟機制的研究[D];揚州大學;2015年

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10 武承鳳;MIF通過ERK信號通路對肝癌細胞增殖的實驗研究[D];甘肅中醫(yī)藥大學;2016年

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