本文選題：肝細胞核因子-1β + 肝癌細胞��； 參考：《第二軍醫(yī)大學》2017年碩士論文

【摘要】：研究目的及背景:世界衛(wèi)生組織(WHO)將原發(fā)性肝癌分為肝細胞癌(hepatocullular carcinoma,HCC)、肝內膽管細胞癌(intrahepatic cholangiocarcinoma,ICC)和兼具前兩者病理學特征的混合型肝細胞-膽管細胞癌(combined hepatocellular-cholangiocarinoma,CHC)。由于腫瘤異質性,不同病理類型的肝癌之間的預后存在很大的差異,ICC的預后要明顯比HCC差。許多研究表明腫瘤異質性與腫瘤干細胞(cancer stem cells,CSCs)相關。CSCs是存在于腫瘤組織中的一類細胞,它們具有干細胞的特征,即具有無限的自我增殖和分化潛能,從而維持腫瘤細胞活力。而肝癌中CSCs可能是導致肝癌復發(fā)轉移的原因之一。有研究表明,部分肝細胞癌同時也表達干細胞表型,如CK7和CK19等,并且這部分肝細胞癌的預后較CK7、CK19陰性的肝細胞癌的預后更差,治療后的復發(fā)率更高。肝細胞核因子-1β(HNF-1β)是肝細胞核因子家族的成員,其參與調節(jié)脂質,碳水化合物和蛋白質的代謝,并且在調節(jié)肝臟發(fā)育和肝細胞分化中起著重要的作用。而原發(fā)性肝癌的發(fā)生發(fā)展又和腫瘤干細胞密切相關。我們前期研究發(fā)現,HNF-1β表達水平與原發(fā)性肝癌的不同病理類型相關,ICC腫瘤組織中的HNF-1β表達高于HCC。因此,我們推測,HNF-1β高表達可能促使HCC干性能力增強,從而提高HCC的惡性程度,影響HCC預后。本課題回顧性地分析肝細胞癌患者腫瘤中HNF-1β的表達和預后的關系,并通過建立HNF-1β過表達的肝癌細胞系來觀察肝癌細胞干性的變化,最后探討HNF-1β對于肝癌細胞干性特征的影響機制。研究方法:(1)收集臨床資料:回顧性地收集90例肝細胞癌患者的蠟塊標本和臨床資料,該90例患者均行手術治療,術后隨訪以上患者的復發(fā)及生存情況。利用免疫組織化學方法檢測以上患者腫瘤組織中的HNF-1β表達水平,由兩位病理醫(yī)師分別獨立進行主觀評價HNF-1β表達水平高低,將90例患者分為HNF-1β高表達組和HNF-1β低表達組,用統(tǒng)計學方法分析患者癌組織中HNF-1β表達水平與患者預后的相關性。(2)在人類肝癌細胞系中穩(wěn)定轉染HNF-1β,使其過表達HNF-1β,利用Western-Blotting、RT-PCR和細胞免疫熒光方法檢測肝癌細胞和過表達HNF-1β的肝癌細胞中HNF-1β和干系標志物(CK7、CK19、SOX9和CD133)的表達水平差異。利用細胞克隆形成實驗觀察肝癌細胞和過表達HNF-1β的肝癌細胞在HNF-1β表達水平差異的情況下細胞的克隆形成能力。(3)利用transwell侵襲實驗觀察HNF-1β對肝癌細胞侵襲能力的影響。利用Western-Blotting、RT-PCR和細胞免疫熒光方法檢測HNF-1β對肝癌細胞上皮間質轉化(EMT)的影響。(4)進一驗證HNF-1β對體內腫瘤形成能力的影響,將肝癌細胞和過表達HNF-1β的肝癌細胞分別種植于裸鼠皮下,觀察不同HNF-1β表達水平的肝癌細胞的成瘤能力,利用免疫組織化學方法觀察腫瘤組織中干性標志物(CK7、CK19、SOX9和CD133)的表達水平差異。(5)進一步研究HNF-1β增強肝癌細胞干性特征的作用機制。利用Western-Blotting、RT-PCR方法檢測HNF-1β與Notch信號通路相關基因的表達。研究結果:(1)HNF-1β表達水平較高的HCC患者的無病生存期較HNF-1β低表達組更短,HNF-1β高表達提示更差的預后。(2)HNF-1β過表達的肝癌細胞的干性標志物(CK7、CK19、SOX9和CD133)的表達水平較對照組明顯增高。這表示HNF-1β陽性細胞可能保留一些干/祖細胞樣特征,肝癌的惡性程度和HNF-1β之間的相關性可能是由于肝臟祖細胞標志物的上調和腫瘤細胞的干性增強。(3)Transwell侵襲實驗發(fā)現HNF-1β過表達的肝癌細胞侵襲轉移能力較對照組明顯升高。(4)HNF-1β可以促進肝癌細胞的裸鼠皮下成瘤能力,并且免疫組織化學染色發(fā)現HNF-1β過表達形成的腫瘤組織中,干性標志物(CK7,CK19,SOX9和CD133)表達較對照組高。(5)HNF-1β過表達可以增強肝癌細胞中Notch信號通路相關分子(Notch1和Hes1)的表達水平。這說明HNF-1β可以通過Notch通路增強肝癌細胞的干性。結論:我們的研究結果表明,HNF-1β可以通過激活Notch信號通路從而調控肝癌細胞的干性特征,并且HNF-1β還可以促使肝癌細胞上皮間質轉化,進而增強肝癌細胞的自我更新以及侵襲轉移能力,促使肝癌患者預后更差。
[Abstract]:Objective and background: the WHO (WHO) divides primary liver cancer into hepatocullular carcinoma (HCC), intrahepatic cholangiocarcinoma (intrahepatic cholangiocarcinoma, ICC) and mixed hepatocyte cholangiocarcinoma (combined hepatocellular-cholangiocarinoma, CHC) with the pathological features of the previous two. There is a great difference between the tumor heterogeneity and the prognosis of different pathological types of liver cancer. The prognosis of ICC is obviously worse than that of HCC. Many studies show that the tumor heterogeneity and the cancer stem cells (CSCs) related.CSCs are a kind of cells in the tumor tissue, and they have the characteristics of stem cells, that is, it has unlimited self proliferation. And the potential of differentiation to maintain the viability of tumor cells, and CSCs may be one of the reasons for the recurrence of HCC. Some studies have shown that some hepatocellular carcinoma also expresses stem cell phenotype, such as CK7 and CK19, and the prognosis of this part of HCC is worse than that of CK7, CK19 negative liver cell carcinoma, and the recurrence rate after treatment. The liver nuclear factor -1 beta (HNF-1 beta) is a member of the liver nuclear factor family, which is involved in regulating the metabolism of lipids, carbohydrates and proteins, and plays an important role in regulating the development of liver and differentiation of liver cells. The development of primary liver cancer is closely related to the stem cells of the tumor. Our previous study found that HNF The expression level of -1 beta is related to the different pathological types of primary liver cancer. The expression of HNF-1 beta in ICC tumor tissues is higher than that of HCC.. We speculate that the high expression of HNF-1 beta may promote the enhancement of HCC dry ability, thus improving the malignant degree of HCC and affecting the prognosis of HCC. This topic is a retrospective analysis of the expression and pre expression of HNF-1 beta in the tumor of hepatocellular carcinoma patients. After the establishment of HNF-1 beta overexpressed hepatocellular carcinoma cell lines to observe the changes in liver cancer stem cells, the mechanism of the effect of HNF-1 beta on the dry characteristics of hepatoma cells was discussed. (1) the clinical data were collected: the specimens and clinical data of 90 patients with hepatocellular carcinoma were collected retrospectively, and all of the 90 patients underwent surgery. The HNF-1 beta expression level in the tumor tissues of the above patients was detected by immunohistochemical method. The level of HNF-1 beta expression was evaluated by two pathologists independently, and the 90 patients were divided into HNF-1 beta high expression group and HNF-1 beta low expression group, and the statistical method was used. The correlation between the expression level of HNF-1 beta in the cancer tissues and the prognosis of the patients was analyzed. (2) the stable transfection of HNF-1 beta in human hepatoma cell lines was used to overexpress HNF-1 beta, and Western-Blotting, RT-PCR and cell immunofluorescence were used to detect the HNF-1 beta and main markers of the liver cancer cells and the liver cancer cells overexpressing HNF-1 beta (CK7, CK19, SOX9 and CD). 133) difference of expression level. Cell clone formation ability of hepatoma cells and overexpressed HNF-1 beta hepatoma cells with the difference of HNF-1 beta expression level was observed by cell clone formation. (3) the effect of HNF-1 beta on the invasion ability of hepatoma cells was observed by Transwell invasion experiment. Western-Blotting, RT-PCR and cell immunity were used. The effect of HNF-1 beta on the epithelial mesenchymal transformation (EMT) of hepatoma cells was detected by the immunofluorescence method. (4) the effect of HNF-1 beta on the ability of tumor formation in the body was verified, the hepatoma cells and the hepatoma cells overexpressing HNF-1 beta were implanted subcutaneously in nude mice, and the tumor formation ability of the hepatoma cells with different HNF-1 beta levels was observed and the immuno histochemistry was used. Methods the expression levels of the dry markers (CK7, CK19, SOX9 and CD133) in the tumor tissues were observed. (5) further study the mechanism of HNF-1 beta enhancement of the dry characteristics of hepatoma cells. The expression of HNF-1 beta and Notch signaling pathway related genes was detected by Western-Blotting and RT-PCR. The results were: (1) HCC suffering from higher HNF-1 beta expression level. The patient's disease-free survival is shorter than that of HNF-1 beta low expression group, and the high expression of HNF-1 beta suggests a worse prognosis. (2) the expression level of HNF-1 beta overexpressed liver cancer cells (CK7, CK19, SOX9 and CD133) is significantly higher than that of the control group. This indicates that HNF-1 beta positive cells may retain some dry / progenitor like characteristics, the malignancy and HNF of liver cancer. The correlation between -1 beta may be due to the up-regulation of liver progenitor cells and the enhancement of tumor cells. (3) Transwell invasion experiment found that the invasion and metastasis of HNF-1 beta overexpressed hepatoma cells were significantly higher than those of the control group. (4) HNF-1 beta could promote the subcutaneous tumor formation of hepatoma cells and immunohistochemical staining. The expression of HNF-1 beta (CK7, CK19, SOX9 and CD133) was higher than that of the control group. (5) the expression of HNF-1 beta over expression could enhance the expression level of Notch signaling pathway related molecules (Notch1 and Hes1) in hepatoma cells. This suggests that HNF-1 beta can enhance the stem nature of hepatoma cells through the Notch pathway. Conclusion: our research The results show that HNF-1 beta can regulate the dry characteristics of liver cancer cells by activating the Notch signaling pathway, and HNF-1 beta can also promote the transformation of the epithelial mesenchymal cells of the hepatoma cells, and then enhance the self renewal and invasion and metastasis of hepatoma cells, and make the prognosis of the liver cancer patients worse.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

【參考文獻】

相關期刊論文 前1條

1 Soomin Ahn;Jiyeon Hyeon;Cheol-Keun Park;;Notch1 and Notch4 are markers for poor prognosis of hepatocellular carcinoma[J];Hepatobiliary & Pancreatic Diseases International;2013年03期

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本文編號：1897337