肝细胞核因子-1β对肝癌细胞干性特征的调控及机制研究

发布时间：2018-05-16 15:02

本文选题：肝细胞核因子-1β + 肝癌细胞　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究目的及背景:世界卫生组织(WHO)将原发性肝癌分为肝细胞癌(hepatocullular carcinoma,HCC)、肝内胆管细胞癌(intrahepatic cholangiocarcinoma,ICC)和兼具前两者病理学特征的混合型肝细胞-胆管细胞癌(combined hepatocellular-cholangiocarinoma,CHC)。由于肿瘤异质性,不同病理类型的肝癌之间的预后存在很大的差异,ICC的预后要明显比HCC差。许多研究表明肿瘤异质性与肿瘤干细胞(cancer stem cells,CSCs)相关。CSCs是存在于肿瘤组织中的一类细胞,它们具有干细胞的特征,即具有无限的自我增殖和分化潜能,从而维持肿瘤细胞活力。而肝癌中CSCs可能是导致肝癌复发转移的原因之一。有研究表明,部分肝细胞癌同时也表达干细胞表型,如CK7和CK19等,并且这部分肝细胞癌的预后较CK7、CK19阴性的肝细胞癌的预后更差,治疗后的复发率更高。肝细胞核因子-1β(HNF-1β)是肝细胞核因子家族的成员,其参与调节脂质,碳水化合物和蛋白质的代谢,并且在调节肝脏发育和肝细胞分化中起着重要的作用。而原发性肝癌的发生发展又和肿瘤干细胞密切相关。我们前期研究发现,HNF-1β表达水平与原发性肝癌的不同病理类型相关,ICC肿瘤组织中的HNF-1β表达高于HCC。因此,我们推测,HNF-1β高表达可能促使HCC干性能力增强,从而提高HCC的恶性程度,影响HCC预后。本课题回顾性地分析肝细胞癌患者肿瘤中HNF-1β的表达和预后的关系,并通过建立HNF-1β过表达的肝癌细胞系来观察肝癌细胞干性的变化,最后探讨HNF-1β对于肝癌细胞干性特征的影响机制。研究方法:(1)收集临床资料:回顾性地收集90例肝细胞癌患者的蜡块标本和临床资料,该90例患者均行手术治疗,术后随访以上患者的复发及生存情况。利用免疫组织化学方法检测以上患者肿瘤组织中的HNF-1β表达水平,由两位病理医师分别独立进行主观评价HNF-1β表达水平高低,将90例患者分为HNF-1β高表达组和HNF-1β低表达组,用统计学方法分析患者癌组织中HNF-1β表达水平与患者预后的相关性。(2)在人类肝癌细胞系中稳定转染HNF-1β,使其过表达HNF-1β,利用Western-Blotting、RT-PCR和细胞免疫荧光方法检测肝癌细胞和过表达HNF-1β的肝癌细胞中HNF-1β和干系标志物(CK7、CK19、SOX9和CD133)的表达水平差异。利用细胞克隆形成实验观察肝癌细胞和过表达HNF-1β的肝癌细胞在HNF-1β表达水平差异的情况下细胞的克隆形成能力。(3)利用transwell侵袭实验观察HNF-1β对肝癌细胞侵袭能力的影响。利用Western-Blotting、RT-PCR和细胞免疫荧光方法检测HNF-1β对肝癌细胞上皮间质转化(EMT)的影响。(4)进一验证HNF-1β对体内肿瘤形成能力的影响,将肝癌细胞和过表达HNF-1β的肝癌细胞分别种植于裸鼠皮下,观察不同HNF-1β表达水平的肝癌细胞的成瘤能力,利用免疫组织化学方法观察肿瘤组织中干性标志物(CK7、CK19、SOX9和CD133)的表达水平差异。(5)进一步研究HNF-1β增强肝癌细胞干性特征的作用机制。利用Western-Blotting、RT-PCR方法检测HNF-1β与Notch信号通路相关基因的表达。研究结果:(1)HNF-1β表达水平较高的HCC患者的无病生存期较HNF-1β低表达组更短,HNF-1β高表达提示更差的预后。(2)HNF-1β过表达的肝癌细胞的干性标志物(CK7、CK19、SOX9和CD133)的表达水平较对照组明显增高。这表示HNF-1β阳性细胞可能保留一些干/祖细胞样特征,肝癌的恶性程度和HNF-1β之间的相关性可能是由于肝脏祖细胞标志物的上调和肿瘤细胞的干性增强。(3)Transwell侵袭实验发现HNF-1β过表达的肝癌细胞侵袭转移能力较对照组明显升高。(4)HNF-1β可以促进肝癌细胞的裸鼠皮下成瘤能力,并且免疫组织化学染色发现HNF-1β过表达形成的肿瘤组织中,干性标志物(CK7,CK19,SOX9和CD133)表达较对照组高。(5)HNF-1β过表达可以增强肝癌细胞中Notch信号通路相关分子(Notch1和Hes1)的表达水平。这说明HNF-1β可以通过Notch通路增强肝癌细胞的干性。结论:我们的研究结果表明,HNF-1β可以通过激活Notch信号通路从而调控肝癌细胞的干性特征,并且HNF-1β还可以促使肝癌细胞上皮间质转化,进而增强肝癌细胞的自我更新以及侵袭转移能力,促使肝癌患者预后更差。
[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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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