本文選題：肝細(xì)胞癌 + UHRF1��； 參考：《南方醫(yī)科大學(xué)》2016年碩士論文

【摘要】：背景與目的原發(fā)性肝癌(Primary carcinoma of the liver)是目前世界上最常見(jiàn)的惡性腫瘤之一,最近一份調(diào)查報(bào)告表明：我國(guó)原發(fā)性肝癌年發(fā)病率為26.39/10萬(wàn),占惡性腫瘤發(fā)病率第4位；死亡率為23.93/10萬(wàn),占惡性腫瘤死亡率第2位。原發(fā)性肝癌按組織學(xué)分型可分為肝細(xì)胞型、膽管細(xì)胞型和混合型,其中以肝細(xì)胞型最為多見(jiàn),約占原發(fā)性肝癌的90%,又稱肝細(xì)胞癌(Hepatocellular carcinoma, HCC)。在我國(guó),肝細(xì)胞癌發(fā)生的主要病因?yàn)橐倚透窝撞《靖腥?我們前期的研究顯示,85%以上的肝細(xì)胞癌患者同時(shí)合并有乙型肝炎病毒感染。近年來(lái),由于各種腫瘤標(biāo)志物的臨床應(yīng)用和影像學(xué)技術(shù)的進(jìn)步,特別是甲胎蛋白(alpha fetoprotein, AFP)和超聲顯像用于肝癌高危人群的監(jiān)測(cè),使肝癌能夠在無(wú)明顯癥狀或體征的亞臨床期被診斷,加上外科手術(shù)技術(shù)的成熟,以及各種局部治療等非手術(shù)治療方法的發(fā)展,肝癌患者的總體預(yù)后較過(guò)去有了明顯改善,但仍不盡如人意。肝癌具有高復(fù)發(fā)和高轉(zhuǎn)移的生物學(xué)特征,有資料顯示肝癌患者在接受根治性切除手術(shù)后五年內(nèi)的復(fù)發(fā)率高達(dá)50-70%。如此高的術(shù)后復(fù)發(fā)率可能是由于患者手術(shù)前己合并存在目前醫(yī)療手段尚不能檢測(cè)到的微轉(zhuǎn)移灶。有研究發(fā)現(xiàn),近一半接受肝癌切除術(shù)的患者在術(shù)前已合并有肝內(nèi)微血管侵犯,進(jìn)一步分析提示肝癌術(shù)前合并微血管侵犯是影響術(shù)后患者無(wú)瘤生存時(shí)間的獨(dú)立危險(xiǎn)因素。肝癌在早期缺乏典型癥狀,而患者一旦出現(xiàn)明顯臨床表現(xiàn)時(shí),肝癌往往已經(jīng)進(jìn)展為晚期。因此,早發(fā)現(xiàn)、早診斷、早治療是肝癌防治的關(guān)鍵。目前,研究者一致認(rèn)為腫瘤是多因素、多步驟、多基因、多突變的結(jié)果,肝癌亦不例外。隨著分子生物學(xué)等技術(shù)的進(jìn)步,分子治療也應(yīng)運(yùn)而生；近年來(lái),科學(xué)家們發(fā)現(xiàn)了許多與肝癌患者預(yù)后相關(guān)的分子標(biāo)志物,這些研究成果讓我們對(duì)肝癌的發(fā)病機(jī)制及其治療有了新的認(rèn)識(shí),為肝癌患者的預(yù)后評(píng)估及管理提供了新的思路和方法。然而,目前我們?nèi)晕茨苷业揭环N能夠有效監(jiān)測(cè)肝癌微轉(zhuǎn)移灶的指標(biāo)或系統(tǒng)。為此,我們有必要繼續(xù)努力去發(fā)現(xiàn)與肝癌轉(zhuǎn)移及復(fù)發(fā)相關(guān)的生物標(biāo)志物。含植物同源結(jié)構(gòu)和環(huán)指域泛素樣蛋白1(ubiquitin-like with PHD and ring finger domains 1, UHRF1),又稱為Np95或ICBP90,是新近發(fā)現(xiàn)的一種與腫瘤發(fā)生發(fā)展有關(guān)的核蛋白基因,定位于染色體19p13.3。研究表明,UHRF1通過(guò)與DNA甲基轉(zhuǎn)移酶(DNA methyltransferase 1, DNMT1)以及組蛋白去乙�；�(histone deacetylase 1, HDAC1)等相互作用,參與基因表達(dá)調(diào)控及染色質(zhì)修飾(如調(diào)節(jié)DNA甲基化,抑制抑癌基因的表達(dá)等)。同時(shí),研究者還發(fā)現(xiàn)UHRF1在多種腫瘤組織中均存在過(guò)表達(dá)現(xiàn)象,且與腫瘤患者的預(yù)后呈負(fù)相關(guān)。此外,UHRF1的表達(dá)被敲低后,腫瘤細(xì)胞的增殖及侵襲能力等明顯下降。這提示UHRF1有可能成為一個(gè)全新的阻止腫瘤細(xì)胞遷移和入侵的藥物靶點(diǎn)。然而截至目前,UHRF1在肝細(xì)胞癌中尚未有一個(gè)較全面且深入的研究。本課題通過(guò)熒光定量PCR技術(shù)和免疫組織化學(xué)染色了解UHRF1在肝細(xì)胞癌組織中的表達(dá)情況及其與肝細(xì)胞癌生物學(xué)特征的關(guān)系,并探討其在肝細(xì)胞癌發(fā)生、發(fā)展中可能的作用。此外,為進(jìn)一步研究UHRF1在肝細(xì)胞癌中的作用,我們采用RNA干擾技術(shù)特異性敲低肝癌細(xì)胞株中UHRF1的表達(dá),并通過(guò)體內(nèi)和體外兩個(gè)水平研究UHRF1在肝細(xì)胞癌發(fā)生、發(fā)展中的作用,為將來(lái)肝細(xì)胞癌患者的預(yù)后評(píng)估及個(gè)體化治療等提供新的理論依據(jù)。第一章UHRF1 mRNA在肝細(xì)胞癌中的表達(dá)目的探索UHRF1 mRNA在肝細(xì)胞癌組織中的表達(dá)情況及其與肝細(xì)胞癌生物學(xué)特征和肝細(xì)胞癌患者預(yù)后的關(guān)系。方法1.實(shí)驗(yàn)組織標(biāo)本的獲�。航M織標(biāo)本來(lái)自于2010年11月至2014年11月期間在南方醫(yī)科大學(xué)南方醫(yī)院肝膽外科接受根治性手術(shù)治療的80例經(jīng)病理證實(shí)的肝細(xì)胞癌患者,所有患者術(shù)前血清學(xué)檢查均提示有乙型肝炎病毒感染；2.實(shí)時(shí)熒光定量PCR檢測(cè)UHRF1 mRNA在肝細(xì)胞癌組織及對(duì)應(yīng)非癌組織中的表達(dá)情況；3.采用統(tǒng)計(jì)學(xué)方法分析UHRF1 mRNA在肝細(xì)胞癌患者癌組織中的表達(dá)水平及其與臨床病理參數(shù)和預(yù)后的關(guān)系。結(jié)果1.在80例肝細(xì)胞癌患者中,54例患者癌組織中UHRF1 mRNA的表達(dá)水平高于非癌組織,高表達(dá)率約為67.5%(P0.05)；2.經(jīng)統(tǒng)計(jì)分析,肝細(xì)胞癌組織中UHRF1 mRNA的表達(dá)水平與肝細(xì)胞癌患者腫瘤的大小、TNM分期、病理分化程度、微癌栓、轉(zhuǎn)移、術(shù)后復(fù)發(fā)及術(shù)后無(wú)瘤生存時(shí)間等病理參數(shù)相關(guān)(P0.05)；3. Kaplan-Meier生存分析表明：高表達(dá)UHRF1 mRNA的肝細(xì)胞癌患者術(shù)后無(wú)瘤生存時(shí)間顯著縮短(P=0.002)。結(jié)論1.UHRF1 mRNA在肝細(xì)胞癌組織中的表達(dá)顯著高于非癌組織；2. UHRF1 mRNA在肝細(xì)胞癌組織中的表達(dá)水平與肝細(xì)胞癌患者腫瘤的大小、TNM分期、微癌栓、術(shù)前轉(zhuǎn)移、術(shù)后復(fù)發(fā)及術(shù)后無(wú)瘤生存時(shí)間等病理參數(shù)相關(guān)；3. UHRF1 mRNA高表達(dá)的肝細(xì)胞癌患者術(shù)后無(wú)瘤生存時(shí)間較低表達(dá)的患者顯著縮短；4.肝細(xì)胞癌患者癌組織中UHRF1 mRNA高表達(dá)提示預(yù)后不良。第二章UHRF1蛋白在肝細(xì)胞癌中的表達(dá)目的探索UHRF1蛋白在肝細(xì)胞癌組織中的表達(dá)水平及其與肝細(xì)胞癌生物學(xué)特征和肝細(xì)胞癌患者預(yù)后的關(guān)系。方法1.實(shí)驗(yàn)組織標(biāo)本的獲�。航M織標(biāo)本來(lái)自于2010年11月至2014年11月期間在南方醫(yī)科大學(xué)南方醫(yī)院肝膽外科接受根治性手術(shù)治療的102例經(jīng)病理證實(shí)的肝細(xì)胞癌患者,所有患者術(shù)前血清學(xué)檢查均提示有乙型肝炎病毒感染；2.免疫組織化學(xué)染色檢測(cè)’UHRF1蛋白在肝細(xì)胞癌組織及非癌組織中的表達(dá)情況;3.采用統(tǒng)計(jì)學(xué)方法分析肝細(xì)胞癌患者癌組織中UHRF1蛋白陽(yáng)性表達(dá)與臨床病理參數(shù)及預(yù)后的關(guān)系。結(jié)果1.在102例肝細(xì)胞癌患者中,癌組織中UHRF1蛋白陽(yáng)性表達(dá)率為57.8%；而在非癌組織中為32.7%,進(jìn)一步分析表明,肝細(xì)胞癌組織中UHRF1蛋白的陽(yáng)性表達(dá)率顯著高于非癌組織(P0.05)。2.經(jīng)統(tǒng)計(jì)分析,肝細(xì)胞癌組織中UHRF1蛋白陽(yáng)性表達(dá)與肝細(xì)胞癌患者的腫瘤大小、病理分化程度、微癌栓、TNM分期、術(shù)前轉(zhuǎn)移、術(shù)后復(fù)發(fā)及術(shù)后無(wú)瘤生存時(shí)間等病理參數(shù)相關(guān)(P0.05)；3. Kaplan-Meier生存分析表明：UHRF1蛋白陽(yáng)性表達(dá)組肝細(xì)胞癌患者的術(shù)后無(wú)瘤生存時(shí)間顯著縮短(P0.05)結(jié)論1. UHRF1蛋白在肝細(xì)胞癌組織中的陽(yáng)性表達(dá)率顯著高于非癌組織；2. UHRF1蛋白陽(yáng)性表達(dá)與肝細(xì)胞癌患者腫瘤的大小、病理分化程度、微癌栓、TNM分期、術(shù)前轉(zhuǎn)移、術(shù)后復(fù)發(fā)及術(shù)后無(wú)瘤生存時(shí)間等病理參數(shù)相關(guān)；3. UHRF1蛋白陽(yáng)性表達(dá)的肝細(xì)胞癌患者術(shù)后無(wú)瘤生存時(shí)間較陰性表達(dá)的患者顯著縮短；4.肝細(xì)胞癌患者癌組織UHRF1蛋白陽(yáng)性表達(dá)可能提示預(yù)后不良。第三章UHRF1在肝細(xì)胞癌中作用的體外研究目的研究UHRF1表達(dá)敲低后肝癌細(xì)胞的增殖、遷移及侵襲能力以及細(xì)胞周期和凋亡等的變化：同時(shí)探討mTOR抑制劑AZD2014對(duì)肝癌細(xì)胞中UHRF1表達(dá)的影響。方法1.實(shí)時(shí)熒光定量PCR檢測(cè)UHRF1 mRNA在不同肝癌細(xì)胞株及L02中的表達(dá)情況；2. siRNA特異性敲低肝癌細(xì)胞HepG2及HCCLM3中UHRF1的表達(dá)；3.CCK-8法檢測(cè)UHRF1表達(dá)敲低后肝癌細(xì)胞增殖能力的變化；4.流式細(xì)胞術(shù)和Western Blot檢鋇UHRF1表達(dá)敲低后肝癌細(xì)胞中細(xì)胞周期及細(xì)胞周期相關(guān)蛋白表達(dá)的變化；5.流式細(xì)胞術(shù)檢測(cè)UHRF1表達(dá)敲低后肝癌細(xì)胞中細(xì)胞凋亡的變化;6. Transwell法檢測(cè)UHRF1表達(dá)敲低后肝癌細(xì)胞遷移及侵襲能力的變化,Western Blot檢測(cè)肝癌細(xì)胞中EMT相關(guān)蛋白表達(dá)的變化；7.實(shí)時(shí)熒光定量PCR及Western Blot檢測(cè)]mTOR抑制劑AZD2014對(duì)肝癌細(xì)胞HCCLM3中UHRF1表達(dá)的影響。結(jié)果1. UHRF1 mRNA在肝癌細(xì)胞株中的表達(dá)水平較L02高,且UHRF1 mRNA在HepG2、HCCLM3及Hep3B中的表達(dá)顯著高于L02(P0.05)；2.實(shí)時(shí)熒光定量PCR及Western Blot結(jié)果顯示我們選取的的兩條siRNA可顯著敲低HepG2和HCCLM3細(xì)胞中UHRF1的表達(dá)；3. UHRF1表達(dá)敲低后可以顯著抑制HepG2和HCCLM3細(xì)胞的增殖能力(P0.05);4.UHRF1表達(dá)敲低后,HepG2和HCCLM3細(xì)胞中的G2/M期細(xì)胞比例顯著升高(P0.05),G1期細(xì)胞比例明顯降低(P0.05),S期細(xì)胞比例無(wú)明顯變化；Western Blot檢測(cè)結(jié)果顯示：UHRF1表達(dá)敲低后,cyclinB1表達(dá)上調(diào),cyclinD1表達(dá)下調(diào)；5. Annexin V-FITC/PI流式檢測(cè)顯示,UHRF1表達(dá)敲低后,HepG2和HCCLM3細(xì)胞中各組間出現(xiàn)凋亡的細(xì)胞數(shù)無(wú)明顯區(qū)別；6. Transwell遷移及侵襲實(shí)驗(yàn)發(fā)現(xiàn),UHRF1表達(dá)敲低后,HepG2和HCCLM3細(xì)胞穿過(guò)Transwell小室的細(xì)胞數(shù)較對(duì)照組均顯著減少(P0.05)；Western Blot結(jié)果顯示：UHRF1表達(dá)敲低后,HepG2和HCCLM3細(xì)胞中上皮表型標(biāo)志物E-cadherin表達(dá)上調(diào),而間質(zhì)表型標(biāo)志物N-cadherin、β-cateni、Vimenti、Slug和Snail表達(dá)下調(diào)；7.實(shí)時(shí)熒光定量PCR及Western Blot結(jié)果顯示,mTOR抑制劑AZD2014可以降低HCCLM3細(xì)胞中UHRF1的表達(dá)水平。結(jié)論1.敲低肝癌細(xì)胞HepG2和HCCLM3中UHRF1的表達(dá)可顯著抑制細(xì)胞的增殖、遷移和侵襲能力,誘導(dǎo)細(xì)胞出現(xiàn)G2/M期阻滯,抑制EMT進(jìn)程；2. UHRF1可能是mTOR抑制劑AZD2014發(fā)揮抗腫瘤作用的藥物靶點(diǎn)。第四章UHRF1在肝細(xì)胞癌中作用的體內(nèi)研究目的研究UHRF1表達(dá)敲低后對(duì)肝癌細(xì)胞在體內(nèi)的生物學(xué)功能的影響,并進(jìn)一步探討UHRF1在肝細(xì)胞癌發(fā)生、發(fā)展中的作用。方法1.采用腺病毒介導(dǎo)的shRNA穩(wěn)定敲低肝癌細(xì)胞HCCLM3中UHRF1的表達(dá)；2.將穩(wěn)定轉(zhuǎn)染sh-NC和sh-UHRF1的HCCLM3細(xì)胞接種至裸鼠皮下,建立荷瘤模型；3.采用蘇木素-伊紅及免疫組織化學(xué)染色對(duì)裸鼠皮下腫瘤進(jìn)行進(jìn)一步檢測(cè)。結(jié)果1.熒光倒置顯微鏡下觀察及流式細(xì)胞術(shù)檢測(cè)均提示腺病毒介導(dǎo)的sh-NC和sh-UHRF1轉(zhuǎn)染細(xì)胞后具有理想的轉(zhuǎn)染效率,熒光定量PCR提示：腺病毒介導(dǎo)的sh-UHRF1可顯著敲低HCCLM3中UHRF1的表達(dá)；2.裸鼠成瘤試驗(yàn)結(jié)果顯示：裸鼠皮下均可見(jiàn)腫瘤形成,但sh-UHRF1組裸鼠皮下形成的腫瘤體積明顯小于sh-NC組,皮下形成的腫瘤重量也顯著小于sh-NC組(P0.05)；腫瘤生長(zhǎng)曲線提示sh-UHRF1組裸鼠的腫瘤生長(zhǎng)顯著受到抑制(P0.05)；熒光定量PCR結(jié)果顯示sh-UHRF1組瘤塊中UHRF1 mRNA的表達(dá)量也顯著低于sh-NC組(P0.05);3.蘇木素-伊紅染色顯示：sh-NC組和sh-UHRF1組裸鼠皮下形成的腫瘤均可見(jiàn)明顯的異型細(xì)胞；免疫組織化學(xué)染色結(jié)果提示：sh-UHRF1組裸鼠皮下腫瘤中UHRF1、Ki-67、N-cadherin和Vimentin的表達(dá)較sh-NC組降低,E-cadherin的表達(dá)升高。結(jié)論UHRF1表達(dá)敲低后可顯著抑制腫瘤的生長(zhǎng)和EMT進(jìn)程。
[Abstract]:Background and objective primary liver cancer (Primary carcinoma of the liver) is one of the most common malignant tumors in the world. The latest report shows that the annual incidence of primary liver cancer in China is 26.39/10 million, accounting for fourth of the malignant tumor incidence, the mortality rate is 23.93/10 million, and the mortality rate of malignant tumor is second. The histological type can be divided into hepatocyte type, bile duct cell type and mixed type, among which hepatocyte type is the most common, about 90% of primary liver cancer, also known as Hepatocellular carcinoma (HCC). In China, the main disease of hepatocellular carcinoma is hepatitis B virus infection. Our previous study showed that more than 85% of liver cells were thin. In recent years, the clinical application of various tumor markers and advances in imaging techniques, especially the alpha fetoprotein (AFP) and ultrasound imaging, have been used to monitor the high risk population of liver cancer in recent years, so that the liver cancer can be diagnosed in subclinical stage without obvious symptoms or signs, plus The development of surgical techniques, as well as the development of non-surgical methods, such as local treatment, has improved the overall prognosis of the patients with liver cancer than in the past, but it is still unsatisfactory. The biological characteristics of high recurrence and high metastasis of the liver cancer show that the recurrence rate of the liver cancer patients within five years after the radical resection is high. The recurrence rate of such a high 50-70%. may be due to the presence of the micrometastases that are not yet detected by the medical treatment before the operation. The independent risk factors for the free survival time of the patients. There is a lack of typical symptoms in the early stage of the liver cancer, and the liver cancer is often advanced in the case of obvious clinical manifestation. Therefore, early detection, early diagnosis and early treatment are the key to the prevention and treatment of liver cancer. As a result, liver cancer is no exception. Molecular therapy has also emerged as molecular biology advances. In recent years, scientists have discovered a number of molecular markers related to the prognosis of patients with liver cancer. These results have given us a new understanding of the pathogenesis and treatment of liver cancer, and for the evaluation of the prognosis of the liver cancer patients and Management provides new ideas and methods. However, we are still unable to find an indicator or system that can effectively monitor liver cancer micrometastases. To this end, it is necessary to continue our efforts to identify biomarkers associated with metastasis and recurrence of liver cancer. Containing plant homologous structure and ubiquitin-like with PHD a Nd ring finger domains 1, UHRF1), also known as Np95 or ICBP90, is a newly discovered nuclear protein gene associated with the development of tumor, and is located in chromosome 19p13.3. studies indicating that UHRF1 passes through the DNA methyltransferase (DNA methyltransferase 1) and histone deacetylase (1). It is involved in the regulation of gene expression and chromatin modification (such as regulating DNA methylation and inhibiting the expression of tumor suppressor genes, etc.). At the same time, the researchers also found that UHRF1 has overexpression in various tumor tissues and has a negative correlation with the prognosis of tumor patients. In addition, the proliferation and invasion ability of the tumor cells after the knockout of UHRF1 This suggests that UHRF1 may be a new drug target to prevent tumor cell migration and invasion. However, up to now, UHRF1 has not been in a more comprehensive and in-depth study in hepatocellular carcinoma. This topic is to understand the expression of UHRF1 in hepatocellular carcinoma by fluorescence quantitative PCR and immunohistochemical staining. In addition, in order to further study the role of UHRF1 in hepatocellular carcinoma, we used RNA interference to specifically knock down the expression of UHRF1 in the hepatocellular carcinoma cell lines, and study the UHRF1 in the liver by two levels in vivo and in vitro. The role of cell carcinogenesis and development to provide a new theoretical basis for the prognosis assessment and individualized treatment of patients with hepatocellular carcinoma in the future. Chapter 1 UHRF1 mRNA expression in hepatocellular carcinoma to explore the expression of UHRF1 mRNA in hepatocellular carcinoma and its relationship with the biological characteristics of hepatocellular carcinoma and the prognosis of hepatocellular carcinoma patients Methods 1. experimental tissue specimens were obtained from 80 cases of hepatocellular carcinoma confirmed by pathology at the Department of hepatobiliary surgery of the Southern Hospital of Southern Medical University from November 2010 to November 2014. All the patients were diagnosed with hepatitis B virus infection before the preoperative serological examination; 2. real time fluorescence determination. The expression of UHRF1 mRNA in the tissues of hepatocellular carcinoma and corresponding non cancer tissues was measured by PCR. 3. the expression of UHRF1 mRNA in the cancer tissues of patients with hepatocellular carcinoma and its relationship with the clinicopathological parameters and prognosis were analyzed by statistical method. Results 1. the expression of UHRF1 mRNA in 80 cases of hepatocellular carcinoma and 54 cases of cancer tissues were expressed. The high expression rate was about 67.5% (P0.05). 2. by statistical analysis, the expression level of UHRF1 mRNA in HCC tissues was related to the tumor size, TNM staging, pathological differentiation, microcarcinoma thrombus, metastasis, postoperative recurrence and postoperative tumor free survival (P0.05), and 3. Kaplan-Meier survival points. The analysis showed that the tumor free survival time of HCC patients with high expression of UHRF1 mRNA was significantly shorter (P=0.002). Conclusion the expression of 1.UHRF1 mRNA in HCC tissues was significantly higher than that of non cancer tissues; the expression level of 2. UHRF1 mRNA in HCC tissues was associated with the size of tumor, TNM staging, microcarcinoma thrombus, and preoperative metastasis in hepatocellular carcinoma. The postoperative recurrence and postoperative tumor free survival time were related to pathological parameters. 3. UHRF1 mRNA high expression of hepatocellular carcinoma patients were significantly shorter than those with low expression of tumor free time. 4. UHRF1 mRNA expression in the carcinoma tissue of patients with hepatocellular carcinoma showed poor prognosis. The expression of second chapter UHRF1 egg white in hepatocellular carcinoma was UHRF The expression level of 1 protein in hepatocellular carcinoma and its relationship with the biological characteristics of hepatocellular carcinoma and the prognosis of hepatocellular carcinoma. Method 1. experimental tissue specimens: tissue specimens from 102 cases of radical surgery in Department of hepatobiliary surgery, Southern Hospital of Southern Medical University from November 2010 to November 2014. In all patients with hepatocellular carcinoma, all patients showed hepatitis B virus infection before operation; 2. immunohistochemical staining was used to detect the expression of UHRF1 protein in the tissues of hepatocellular carcinoma and non cancer tissues; 3. the positive expression of UHRF1 protein in the cancer tissues of patients with hepatocellular carcinoma and clinical disease were analyzed by statistical method. Results 1. of the 102 patients with hepatocellular carcinoma, the positive expression rate of UHRF1 protein was 57.8% and 32.7% in non cancer tissue. The further analysis showed that the positive rate of UHRF1 protein in the hepatocellular carcinoma tissue was significantly higher than that of non cancer tissue (P0.05).2., and the UHRF1 egg in the hepatocellular carcinoma tissue was UHRF1 The white positive expression was related to the tumor size, the degree of pathological differentiation, the microcarcinoma thrombus, TNM staging, preoperative metastasis, postoperative recurrence and postoperative tumor free survival time (P0.05). 3. Kaplan-Meier survival analysis showed that the tumor free survival time of the patients with UHRF1 protein positive expression of HCC was significantly shortened (P0.05 Conclusion the positive expression rate of 1. UHRF1 protein in hepatocellular carcinoma was significantly higher than that of non cancer tissue, and the positive expression of 2. UHRF1 protein was related to the size of tumor, the degree of pathological differentiation, the microcarcinoma thrombus, TNM staging, preoperative metastasis, postoperative recurrence and postoperative tumor free survival. The positive expression of 3. UHRF1 protein was positive. The tumor free survival time of the patients with hepatocellular carcinoma was significantly shorter than those with negative expression; 4. the positive expression of UHRF1 protein in the carcinoma tissue of the patients with hepatocellular carcinoma may indicate poor prognosis. The purpose of the third chapter in the study of hepatocellular carcinoma in vitro is to study the proliferation, migration and invasion ability of hepatoma cells and the cells after UHRF1 knockout. Changes in cycle and apoptosis, and the effect of mTOR inhibitor AZD2014 on the expression of UHRF1 in hepatoma cells. Method 1. real-time quantitative PCR PCR was used to detect the expression of UHRF1 mRNA in different hepatocellular carcinoma cell lines and L02; the expression of UHRF1 in the HepG2 and HCCLM3 in the 2. siRNA specific knockout of hepatoma cells; The changes in the proliferation ability of HCC cells, the changes in cell cycle and cell cycle related protein expression in hepatoma cells after 4. flow cytometry and Western Blot examination of barium UHRF1, and 5. flow cytometry to detect the changes of apoptosis in the hepatoma cells after the UHRF1 expression was knocked low; and the 6. Transwell method was used to detect the liver cancer after the UHRF1 knockout. Changes in cell migration and invasion ability, Western Blot detection of EMT related protein expression in hepatoma cells; 7. real time fluorescence quantitative PCR and Western Blot to detect the effect of]mTOR inhibitor AZD2014 on the expression of UHRF1 in liver cancer cell HCCLM3. Results the expression level of 1. UHRF1 mRNA in liver cancer cell lines is higher. The expression in CLM3 and Hep3B was significantly higher than that of L02 (P0.05); 2. real-time fluorescent quantitative PCR and Western Blot results showed that our selected two siRNA could significantly knock down the UHRF1 expression in the HepG2 and HCCLM3 cells, and the 3. UHRF1 decreased after knockout. The proportion of G2/M cells in CCLM3 cells increased significantly (P0.05), the proportion of G1 cells decreased significantly (P0.05), and there was no significant change in the proportion of cells in the S phase. The Western Blot detection results showed that the cyclinB1 expression was up and the cyclinD1 expression was down after the low expression of UHRF1 expression. There was no significant difference in the number of apoptotic cells in the cells. 6. Transwell migration and invasion experiments found that after UHRF1 knockout, the number of HepG2 and HCCLM3 cells passing through the Transwell cells decreased significantly (P0.05). The Western Blot results showed that the epithelial phenotype in HepG2 and HCCLM3 cells after the UHRF1 table was low. The expression of E-cadherin was up and the expression of interstitial phenotypes N-cadherin, beta -cateni, Vimenti, Slug and Snail were down regulated. 7. real time fluorescent quantitative PCR and Western Blot results showed that mTOR inhibitor AZD2014 could reduce the expression level in HCCLM3 cells. Conclusion 1. knockout low liver cancer cells and the expression of Western are significantly inhibited. The proliferation, migration and invasion of cells, inducing G2/M phase block and inhibiting EMT process; 2. UHRF1 may be a drug target for anti-tumor effect of mTOR inhibitor AZD2014. In vivo study on the role of UHRF1 in hepatocellular carcinoma in vivo And further explore the role of UHRF1 in the development of hepatocellular carcinoma (HCC). Method 1. the expression of UHRF1 in HCCLM3 cells was stabilized by adenovirus mediated shRNA; 2. the HCCLM3 cells transfected with sh-NC and sh-UHRF1 were inoculated subcutaneously into nude mice, and the tumor bearing model was established; 3. using hematoxylin eosin and immunohistochemical staining. Further detection of subcutaneous tumor in nude mice. Results 1. fluorescence inverted microscope observation and flow cytometry showed that adenovirus mediated sh-NC and sh-UHRF1 transfected cells had ideal transfection efficiency. Fluorescence quantitative PCR suggested that adenovirus mediated sh-UHRF1 could significantly reduce the expression of UHRF1 in HCCLM3, and 2. nude mice were tumorigenic test. The results showed that tumor formation was observed in the subcutaneous tissue of nude mice, but the volume of tumor subcutaneously formed in nude mice in group sh-UHRF1 was significantly smaller than that in group sh-NC.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.7

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8 張金山;要靈活運(yùn)用影像學(xué)提供的方法和手段[N];中國(guó)高新技術(shù)產(chǎn)業(yè)導(dǎo)報(bào);2001年

9 李杰;不能手術(shù)切除肝細(xì)胞癌的治療[N];科技日?qǐng)?bào);2006年

10 ;修復(fù)肝細(xì)胞 改善肝功能[N];人民日?qǐng)?bào)海外版;2006年

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