本文選題：肝細(xì)胞核因子-1β + 肝癌細(xì)胞��； 參考：《第二軍醫(yī)大學(xué)》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(xì)胞癌(hepatocullular carcinoma,HCC)、肝內(nèi)膽管細(xì)胞癌(intrahepatic cholangiocarcinoma,ICC)和兼具前兩者病理學(xué)特征的混合型肝細(xì)胞-膽管細(xì)胞癌(combined hepatocellular-cholangiocarinoma,CHC)。由于腫瘤異質(zhì)性,不同病理類型的肝癌之間的預(yù)后存在很大的差異,ICC的預(yù)后要明顯比HCC差。許多研究表明腫瘤異質(zhì)性與腫瘤干細(xì)胞(cancer stem cells,CSCs)相關(guān)。CSCs是存在于腫瘤組織中的一類細(xì)胞,它們具有干細(xì)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(xì)胞活力。而肝癌中CSCs可能是導(dǎo)致肝癌復(fù)發(fā)轉(zhuǎn)移的原因之一。有研究表明,部分肝細(xì)胞癌同時也表達(dá)干細(xì)胞表型,如CK7和CK19等,并且這部分肝細(xì)胞癌的預(yù)后較CK7、CK19陰性的肝細(xì)胞癌的預(yù)后更差,治療后的復(fù)發(fā)率更高。肝細(xì)胞核因子-1β(HNF-1β)是肝細(xì)胞核因子家族的成員,其參與調(diào)節(jié)脂質(zhì),碳水化合物和蛋白質(zhì)的代謝,并且在調(diào)節(jié)肝臟發(fā)育和肝細(xì)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(xì)胞密切相關(guān)。我們前期研究發(fā)現(xiàn),HNF-1β表達(dá)水平與原發(fā)性肝癌的不同病理類型相關(guān),ICC腫瘤組織中的HNF-1β表達(dá)高于HCC。因此,我們推測,HNF-1β高表達(dá)可能促使HCC干性能力增強(qiáng),從而提高HCC的惡性程度,影響HCC預(yù)后。本課題回顧性地分析肝細(xì)胞癌患者腫瘤中HNF-1β的表達(dá)和預(yù)后的關(guān)系,并通過建立HNF-1β過表達(dá)的肝癌細(xì)胞系來觀察肝癌細(xì)胞干性的變化,最后探討HNF-1β對于肝癌細(xì)胞干性特征的影響機(jī)制。研究方法:(1)收集臨床資料:回顧性地收集90例肝細(xì)胞癌患者的蠟塊標(biāo)本和臨床資料,該90例患者均行手術(shù)治療,術(shù)后隨訪以上患者的復(fù)發(fā)及生存情況。利用免疫組織化學(xué)方法檢測以上患者腫瘤組織中的HNF-1β表達(dá)水平,由兩位病理醫(yī)師分別獨(dú)立進(jìn)行主觀評價HNF-1β表達(dá)水平高低,將90例患者分為HNF-1β高表達(dá)組和HNF-1β低表達(dá)組,用統(tǒng)計學(xué)方法分析患者癌組織中HNF-1β表達(dá)水平與患者預(yù)后的相關(guān)性。(2)在人類肝癌細(xì)胞系中穩(wěn)定轉(zhuǎn)染HNF-1β,使其過表達(dá)HNF-1β,利用Western-Blotting、RT-PCR和細(xì)胞免疫熒光方法檢測肝癌細(xì)胞和過表達(dá)HNF-1β的肝癌細(xì)胞中HNF-1β和干系標(biāo)志物(CK7、CK19、SOX9和CD133)的表達(dá)水平差異。利用細(xì)胞克隆形成實(shí)驗觀察肝癌細(xì)胞和過表達(dá)HNF-1β的肝癌細(xì)胞在HNF-1β表達(dá)水平差異的情況下細(xì)胞的克隆形成能力。(3)利用transwell侵襲實(shí)驗觀察HNF-1β對肝癌細(xì)胞侵襲能力的影響。利用Western-Blotting、RT-PCR和細(xì)胞免疫熒光方法檢測HNF-1β對肝癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化(EMT)的影響。(4)進(jìn)一驗證HNF-1β對體內(nèi)腫瘤形成能力的影響,將肝癌細(xì)胞和過表達(dá)HNF-1β的肝癌細(xì)胞分別種植于裸鼠皮下,觀察不同HNF-1β表達(dá)水平的肝癌細(xì)胞的成瘤能力,利用免疫組織化學(xué)方法觀察腫瘤組織中干性標(biāo)志物(CK7、CK19、SOX9和CD133)的表達(dá)水平差異。(5)進(jìn)一步研究HNF-1β增強(qiáng)肝癌細(xì)胞干性特征的作用機(jī)制。利用Western-Blotting、RT-PCR方法檢測HNF-1β與Notch信號通路相關(guān)基因的表達(dá)。研究結(jié)果:(1)HNF-1β表達(dá)水平較高的HCC患者的無病生存期較HNF-1β低表達(dá)組更短,HNF-1β高表達(dá)提示更差的預(yù)后。(2)HNF-1β過表達(dá)的肝癌細(xì)胞的干性標(biāo)志物(CK7、CK19、SOX9和CD133)的表達(dá)水平較對照組明顯增高。這表示HNF-1β陽性細(xì)胞可能保留一些干/祖細(xì)胞樣特征,肝癌的惡性程度和HNF-1β之間的相關(guān)性可能是由于肝臟祖細(xì)胞標(biāo)志物的上調(diào)和腫瘤細(xì)胞的干性增強(qiáng)。(3)Transwell侵襲實(shí)驗發(fā)現(xiàn)HNF-1β過表達(dá)的肝癌細(xì)胞侵襲轉(zhuǎn)移能力較對照組明顯升高。(4)HNF-1β可以促進(jìn)肝癌細(xì)胞的裸鼠皮下成瘤能力,并且免疫組織化學(xué)染色發(fā)現(xiàn)HNF-1β過表達(dá)形成的腫瘤組織中,干性標(biāo)志物(CK7,CK19,SOX9和CD133)表達(dá)較對照組高。(5)HNF-1β過表達(dá)可以增強(qiáng)肝癌細(xì)胞中Notch信號通路相關(guān)分子(Notch1和Hes1)的表達(dá)水平。這說明HNF-1β可以通過Notch通路增強(qiáng)肝癌細(xì)胞的干性。結(jié)論:我們的研究結(jié)果表明,HNF-1β可以通過激活Notch信號通路從而調(diào)控肝癌細(xì)胞的干性特征,并且HNF-1β還可以促使肝癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化,進(jìn)而增強(qiáng)肝癌細(xì)胞的自我更新以及侵襲轉(zhuǎn)移能力,促使肝癌患者預(yù)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(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

【參考文獻(xiàn)】

相關(guān)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

，

本文編號：1897337