【摘要】：肝細(xì)胞癌是一種常見的原發(fā)性惡性腫瘤。乙型肝炎病毒是造成肝細(xì)胞癌發(fā)生的一個(gè)主要原因,但是以往的研究都是從間接的方式去闡述其致癌的機(jī)理,其致癌方式并沒有被清楚地說明。我們從文獻(xiàn)得知癌癥干細(xì)胞作為一種癌組織產(chǎn)生的源頭,能直接促進(jìn)癌癥的發(fā)生,因此我們想從癌癥干細(xì)胞方面研究乙型肝炎病毒介導(dǎo)的肝細(xì)胞癌形成原因。為了研究肝癌細(xì)胞與普通肝細(xì)胞之間的區(qū)別,我們檢測(cè)肝癌細(xì)胞系HepG2與正常肝細(xì)胞系L02的干細(xì)胞相關(guān)因子(Klf4、c-Myc、Nanog、Oct4和Sox2)的mRNA表達(dá)水平以及肝癌干細(xì)胞表面標(biāo)志物(CD90、CD117和CD133)的表達(dá)水平。我們發(fā)現(xiàn)HepG2細(xì)胞系的干細(xì)胞相關(guān)因子的mRNA表達(dá)水平和肝癌干細(xì)胞表面標(biāo)志物的表達(dá)水平都比L02細(xì)胞系高。這個(gè)發(fā)現(xiàn)告訴了我們肝細(xì)胞向肝癌細(xì)胞轉(zhuǎn)化的過程與細(xì)胞的去分化和癌干細(xì)胞表面標(biāo)志物表達(dá)緊密相關(guān)。緊接著我們對(duì)肝癌細(xì)胞系HepG2以及其轉(zhuǎn)入乙型肝炎病毒基因組的細(xì)胞系HepG2.2.15進(jìn)行研究,我們發(fā)現(xiàn)在HepG2.2.15細(xì)胞系的干細(xì)胞相關(guān)因子mRNA表達(dá)水平上明顯比HepG2細(xì)胞系高,癌干細(xì)胞表面標(biāo)志物的表達(dá)水平也顯著比HepG2高。隨后我們?cè)诩?xì)胞生長實(shí)驗(yàn)上發(fā)現(xiàn)HepG2.2.15細(xì)胞系的增殖能力和遷移能力比HepG2細(xì)胞系更強(qiáng)。以上的實(shí)驗(yàn)結(jié)果證實(shí)HepG2.2.15細(xì)胞系在乙型肝炎病毒基因組的影響下,其癌干細(xì)胞化的水平比HepG2細(xì)胞系高。在以往的文獻(xiàn)報(bào)道當(dāng)中,我們發(fā)現(xiàn)乙型肝炎病毒導(dǎo)致肝細(xì)胞癌的主要因素可能是病毒生命周期中產(chǎn)生的HBx蛋白和preS/S蛋白部分,我們?cè)谙嚓P(guān)文獻(xiàn)中也發(fā)現(xiàn)HBx、preS1和preS2與肝癌的發(fā)生發(fā)展可能相關(guān)。因此我們?cè)O(shè)計(jì)了一些實(shí)驗(yàn)檢測(cè)相關(guān)蛋白HBx、preS1和preS2對(duì)于肝細(xì)胞轉(zhuǎn)化成癌干細(xì)胞的能力。我們?cè)诟渭?xì)胞系L02細(xì)胞和肝癌細(xì)胞系HepG2細(xì)胞中分別穩(wěn)定表達(dá)了 HBx、preS1和preS2基因。在我們檢測(cè)各個(gè)構(gòu)建好的細(xì)胞系的癌干細(xì)胞相關(guān)指標(biāo)之前,我們驚奇地發(fā)現(xiàn)穩(wěn)定表達(dá)preS1的L02細(xì)胞系的生長情況和細(xì)胞形態(tài)發(fā)生了巨大的改變,隨后我們深入檢測(cè)preS1相關(guān)細(xì)胞系的癌干細(xì)胞水平。我們發(fā)現(xiàn)了 preS1能強(qiáng)烈地促進(jìn)相應(yīng)細(xì)胞系的癌干細(xì)胞化,使其干細(xì)胞相關(guān)因子mRNA表達(dá)水平顯著提高,癌干細(xì)胞標(biāo)志物的表達(dá)水平也部分提高了。隨后通過一系列細(xì)胞生長實(shí)驗(yàn)確定了 preS1基因能促進(jìn)肝細(xì)胞系L02的自我更新和增殖能力,揭示乙肝病毒的PreS1蛋白是導(dǎo)致肝癌干細(xì)胞發(fā)生發(fā)展的重要因素。
[Abstract]:Hepatocellular carcinoma is a common primary malignant tumor. Hepatitis B virus is one of the main causes of hepatocellular carcinoma, but previous studies have explained the mechanism of its carcinogenesis in an indirect way, and its carcinogenesis has not been clearly explained. We know from the literature that cancer stem cells as a source of cancer tissue can directly promote the occurrence of cancer, so we want to study the pathogenesis of Hepatitis B virus mediated hepatocellular carcinoma from the aspect of cancer stem cells. In order to study the difference between hepatoma cells and normal hepatocytes, we detected the mRNA expression level of stem cell related factors (Klf4C- Myct4 and Sox2) in HepG2 and L02, and the expression of CD90 CD117 and CD133 on the surface of hepatoma stem cells. We found that the mRNA expression level of stem cell related factors and the expression level of hepatoma stem cell surface marker in HepG2 cell line were higher than those in L02 cell line. This discovery tells us that the process of transforming hepatocytes into hepatoma cells is closely related to cell dedifferentiation and expression of tumor stem cell surface markers. Then we studied the hepatoma cell line HepG2 and the cell line HepG2.2.15 which was transferred to the genome of hepatitis B virus. We found that the mRNA expression level of stem cell related factors in HepG2.2.15 cell line was significantly higher than that in HepG2 cell line. The expression of tumor stem cell surface markers was also significantly higher than that of HepG2. Then we found that the proliferation and migration ability of HepG2.2.15 cell line was stronger than that of HepG2 cell line. The above results confirmed that the level of cancer stem cell transformation of HepG2.2.15 cell line was higher than that of HepG2 cell line under the influence of hepatitis B virus genome. In previous literature, we have found that the main factors of hepatitis B virus leading to hepatocellular carcinoma may be the HBx protein and preS/S protein part of the virus life cycle. We also found that HBX preS1 and preS2 may be associated with the occurrence and development of liver cancer. Therefore, we designed some experiments to detect the ability of HBxP preS1 and preS2 to transform hepatocytes into cancer stem cells. We stably expressed HBX preS1 and preS2 genes in L02 and HepG2 cells, respectively. We were surprised to find that the growth and morphology of the L02 cell line, which expressed preS1 stably, had changed dramatically before we examined the relevant markers of cancer stem cells in each constructed cell line. We then examined the level of cancer stem cells in preS1-associated cell lines. We found that preS1 could strongly promote the carcinomatosis of the corresponding cell lines and increase the mRNA expression level of stem cell related factors and the expression level of cancer stem cell markers. Then a series of cell growth experiments confirmed that preS1 gene can promote the self-renewal and proliferation of liver cell line L02. It is revealed that the PreS1 protein of hepatitis B virus is an important factor leading to the development of liver cancer stem cells.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Mustafa Sunbul;;Hepatitis B virus genotypes:Global distribution and clinical importance[J];World Journal of Gastroenterology;2014年18期

，

本文編號(hào)：2192304