本文選題：有氧糖酵解 + PKM2��； 參考：《北京協(xié)和醫(yī)學院》2017年博士論文

【摘要】：背景:肝細胞肝癌(Hepatocellular carcinoma,HCC)是肝臟最常見的惡性腫瘤之一。多數(shù)肝癌患者初診時已經(jīng)處于病程的中晚期,多靶點酪氨酸激酶抑制劑索拉非尼(Sorafenib,NexavarR)是治療不可手術(shù)的進展期HCC的一線靶向用藥。但是由于肝癌復雜的生物學特性,索拉非尼臨床療效異質(zhì)性較大。因此探討索拉非尼耐藥的機制對于改善進展期肝癌患者的預后顯得極為重要。有氧糖酵解是腫瘤細胞糖代謝的主要方式,糖酵解中的關(guān)鍵酶如丙酮酸激酶(Pyruvate kinase M2,PKM2)和乳酸脫氫酶(Lactate dehydrogenase,LDH)對于腫瘤的發(fā)生發(fā)展有重要作用,糖酵解的關(guān)鍵酶PKM2和LDHA等與索拉非尼耐藥的相關(guān)性有待進一步深入研究。Extracellular signal-regulated kinase(ERK)5 通路也稱為 BMK1 通路,是Mitogen-activated protein kinase(MAPK)家族中新近發(fā)現(xiàn)并加以研究的分子通路,近年來研究發(fā)現(xiàn)ERK5的表達水平和多種惡性腫瘤的發(fā)生發(fā)展具有一定關(guān)系。既往研究提示ERK1/2可調(diào)節(jié)PKM2基因入核而影響細胞惡性生物行為。作為索拉非尼主要作用靶點之一,ERK1/2基因表達變化在索拉非尼耐藥中具有重要作用。而同作為MAPK家族的ERK5對PKM2是否有調(diào)節(jié)作用,及其與索拉非尼耐藥的關(guān)系還亟待進一步深入研究。材料和方法:1.回顧性分析119例行索拉非尼治療的晚期乙肝相關(guān)肝癌患者臨床資料。根據(jù)治療前血清乳酸脫氫酶水平將患者分為2組。分析血清乳酸脫氫酶水平的預后預測價值及與其他臨床病理因素的關(guān)系;2.應用免疫組織化學方法檢測肝癌腫瘤組織、癌旁組織中LDHA的蛋白表達水平。3.構(gòu)建PKM2基因過表達及敲減的慢病毒,并經(jīng)篩選構(gòu)建穩(wěn)定敲減及過表達PKM2基因的肝癌細胞株。4.分別觀察下調(diào)及上調(diào)PKM2基因表達對索拉非尼引起的細胞增殖、凋亡、遷移、葡萄糖消耗率、乳酸產(chǎn)率等的影響,并使用RT-PCR及Western Blot檢測LDHA的mRNA及蛋白質(zhì)表達變化。5.觀察ERK5特異性抑制劑XMD8-92對索拉非尼引起的肝癌細胞增殖、凋亡、遷移、葡萄糖消耗率、乳酸產(chǎn)率等的影響。并使用RT-PCR及Western Blot檢測PKM2及LDHA的mRNA及蛋白質(zhì)表達變化。6.觀察上調(diào)PKM2基因?qū)MD8-92及XMD8-92聯(lián)合索拉非尼處理的肝癌細胞的增殖、凋亡、遷移、葡萄糖消耗率、乳酸產(chǎn)率等的影響,并使用RT-PCR及Western Blot檢測LDHA的mRNA及蛋白質(zhì)表達變化。結(jié)果:1.患者分組的界值定為221U/L。中位隨訪時長15(范圍:3-73)個月,91個患者達到隨訪終點。多因素分析發(fā)現(xiàn)治療前乳酸脫氫酶水平是影響總生存時間和無進展生存時間的獨立危險因素。對于治療前乳酸脫氫酶水平高于221U/L的患者,治療后3個月乳酸脫氫酶升高者預后更差。治療前LDH偏高與低白蛋白、大血管癌栓、高Child-Pugh分級、高T分級、高AFP水平和高總膽紅素有關(guān)。2.接受索拉非尼治療的肝癌組織中LDHA表達顯著高于癌旁組織。3.成功構(gòu)建了 PKM2的敲減及過表達慢病毒,并成功轉(zhuǎn)染Huh7細胞得到穩(wěn)轉(zhuǎn)株。4.下調(diào)及上調(diào)PKM2基因表達可分別增強和減弱索拉非尼引起的肝癌細胞抑制增殖、促進凋亡、抑制遷移、減少葡萄糖消耗率、減少乳酸產(chǎn)率等的影響。下調(diào)及上調(diào)PKM2基因表達可分別下調(diào)或上調(diào)LDHA的mRNA及蛋白質(zhì)表達變化。5.ERK5特異性抑制劑XMD8-92可增強索拉非尼引起的對肝癌細胞抑制增殖、促進凋亡、抑制遷移、減少葡萄糖消耗率、減少乳酸產(chǎn)率等的影響,且呈劑量依賴效應。XMD8-92可降低肝癌細胞核中PKM2表達,以及細胞中LDHA的表達,且XMD8-92與索拉非尼聯(lián)合用藥較索拉非尼單藥處理對上述基因的表達抑制效果更為顯著,且呈劑量依賴效應。6.上調(diào)PKM2基因可減弱XMD8-92及XMD8-92聯(lián)合索拉非尼處理的引起的肝癌細胞增殖、促進凋亡、抑制遷移、減少葡萄糖消耗率、減少乳酸產(chǎn)率等的影響,且PKM2過表達后XMD8-92組及XMD8-92聯(lián)合索拉非尼組細胞中LDHA升高。結(jié)論:PKM2介導的糖酵解通路改變可能是引起索拉非尼耐藥機制之一。血清乳酸脫氫酶水平是接受索拉非尼治療的晚期肝細胞肝癌患者的預后預測因素。PKM2介導的糖酵解通路可被ERK5特異性抑制劑XMD8-92調(diào)節(jié)。
[Abstract]:Background: hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) is one of the most common malignant tumor of the liver. The majority of patients with hepatocellular carcinoma diagnosed at an advanced stage of disease, multitargeted tyrosine kinase inhibitor Sola Fini (Sorafenib, NexavarR) is a target for the treatment of unresectable advanced HCC to medication. But due to the biological characteristics of hepatocellular carcinoma the Sola Fini complex, clinical heterogeneity. Therefore to explore the mechanism of sorafenib resistant to improve the prognosis of patients with advanced HCC is extremely important. Aerobic glycolysis is the main way of tumor cells in sugar metabolism, key enzyme in glycolysis such as pyruvate kinase (Pyruvate kinase, M2, PKM2) and lactic acid dehydrogenase (Lactate dehydrogenase, LDH) plays an important role in the occurrence and development of tumor related key glycolytic enzymes PKM2 and LDHA and Sola Fini further resistance Study of.Extracellular signal-regulated kinase (ERK) 5 pathway known as BMK1 pathway, Mitogen-activated protein kinase (MAPK) is a newly discovered family and molecular pathways to study, recent studies have found that the expression of ERK5 and tumor development has certain relationship. Previous studies suggested that ERK1/2 can regulate the PKM2 gene into the nucleus effect of biological behavior of malignant cells. As one of the main targets of Sola Fini, ERK1/2 gene expression plays an important role in the sorafenib resistance. But the same as the MAPK family of ERK5 whether the regulation of PKM2, and its relationship with Sola Fini resistance also need further research. Materials and methods: a retrospective analysis of 119 cases of Sora 1. sorafenib in the treatment of advanced HBV related HCC patients. According to the clinical data before treatment, serum lactate dehydrogenase levels in patients Divided into 2 groups. Analysis of the relationship between the prognostic value of serum lactate dehydrogenase level and other clinical pathological factors; detection of HCC tumor tissue 2. by immunohistochemical method LDHA in cancer tissue protein expression level of.3. gene over expression construct PKM2 and lentiviral knockdown, and screened stable knockdown and construction expression of PKM2 gene in hepatocellular carcinoma cell line.4. were observed by Sola Fini and upregulation of PKM2 gene expression on induced cell proliferation, apoptosis, migration, glucose consumption rate, lactic acid yield, and the use of RT-PCR and Western Blot to detect the expression of LDHA mRNA and.5. protein changes observed in cancer cells proliferation, ERK5 specific inhibitor of XMD8-92 Sola Fini induced apoptosis, migration, glucose consumption rate, the yield of lactic acid. The effect of the expression of mRNA and protein and using RT-PCR and Blot detection of Western PKM2 and LDHA .6. observed upregulation of PKM2 gene on proliferation of hepatocellular carcinoma cells XMD8-92, and combined treatment of Sola Fini XMD8-92 apoptosis, migration, glucose consumption rate, lactic acid yield, and protein expression of mRNA RT-PCR and Western Blot changes and the detection of LDHA. Results: 1. patients were grouped as 221U/L. value median follow-up duration 15 (range: 3-73 months), 91 patients at follow-up. Multivariate analysis showed that the end point before the treatment of lactate dehydrogenase levels are independent risk factors for overall survival and progression free survival time. Before treatment for lactate dehydrogenase level is higher than that of 221U/L patients, 3 months after the treatment of lactate dehydrogenase increased poorer prognosis LDH. Before the treatment with the high low albumin, vascular tumor thrombus, high grade Child-Pugh, high grade T, LDHA high expression level of AFP and high total bilirubin.2. received the Sola Fini treatment in hepatocellular carcinoma Compared with paracancerous tissues.3. was successfully constructed PKM2 knockdown and overexpression of lentivirus and transfected into Huh7 cells successfully expressed stable strain downregulation of.4. and upregulation of PKM2 gene can increase and decrease caused by Sola Fini hepatocellular carcinoma cells inhibit proliferation and promote apoptosis, inhibit migration, reduced glucose consumption rate, decrease the yield of lactic acid wait. Downregulation and upregulation of PKM2 gene were down regulated or up-regulated expression of LDHA mRNA and protein changes of.5.ERK5 specific inhibitor XMD8-92 can enhance sorafenib caused on hepatocellular carcinoma cell proliferation inhibition, apoptosis, inhibition of migration, reduced glucose consumption rate, reduce the effect of lactic acid yield, dose-dependent.XMD8-92 reduce the expression of PKM2 in hepatocellular carcinoma cells, and the expression of LDHA in the cells, and the XMD8-92 and sorafenib in combination with sorafenib monotherapy treatment on the gene expression. As the inhibitory effect is more significant, dose-dependent upregulation of.6. PKM2 gene can decrease XMD8-92 and induced the proliferation of hepatoma cells, combined with Sola Fini XMD8-92 promote apoptosis, inhibit migration, reduced glucose consumption rate, reduce the effect of lactic acid yield, and PKM2 LDHA increased in XMD8-92 group and XMD8-92 combined with sorafenib group cells expression. Conclusion: PKM2 mediated glycolytic pathway changes may be caused by one of the resistance mechanisms of Sola Fini. Serum lactate dehydrogenase level is sorafenib therapy in patients with advanced hepatocellular carcinoma and the prognostic factors of.PKM2 mediated glycolytic pathway by ERK5 specific inhibitor XMD8-92 regulation.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7

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