【作者】 李稳；

【导师】 张春清；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 [目的]肝细胞肝癌(Hepatocellular carcinoma, HCC)是最常见的恶性肿瘤之一,严重影响着人类的健康,在我国每年肝癌的发病率约为20/10万,其死亡率高居所有癌症死亡率第二位。目前,最有效的治疗手段为手术切除和肝移植,但疗效欠佳,术后肿瘤的复发和肝内及远处转移发生率较高。除此之外,化学药物治疗是肝脏恶性肿瘤一种重要的治疗方法,一般来讲可作为不能手术切除的肿瘤及肝癌术后的辅助治疗、术后复发治疗的重要的补充治疗手段。化疗药物杀伤肿瘤细胞主要是通过促进肿瘤细胞凋亡,从而起到治疗肿瘤的目的。在化疗过程中肿瘤细胞对化疗药物诱导的肿瘤细胞凋亡具有抵抗力,某些肿瘤甚至可在化疗治疗后出现耐药,严重影响化学药物治疗效果。一方面化疗药物诱导肿瘤细胞凋亡,另一方面化疗药物激活肿瘤细胞内的一系列生物信号,使得肿瘤细胞对化疗药物的抵抗能力增加。抑制和促进肿瘤细胞凋亡的各种因素之间的相互作用决定了细胞的凋亡。肝细胞肝癌的肿瘤新生血管生成被认为与HCC的侵袭、转移等生物学行为密切相关。血管内皮细胞生长因子(vasculaur endothelial growth factor, VEGF)是肿瘤新生血管生成中最主要的刺激因子,与HCC的侵袭、转移和肿瘤新生血管生成密切相关,VEGF被认为是HCC血管生成中最主要的刺激因子。Vasohibin-1是一个可以被VEGF诱导的血管生成负反馈抑制因子,因此研究vasohibin-1参与肿瘤新生血管生成异常疾病的发生、发展备受关注。核因子-Kb (nuclear factor-Kb, NF-κb)是一类广泛存在的转录因子,与凋亡、免疫反应有着密切的联系。近来研究发现能被多种化疗药物所激活,尤其是干扰核酸生成的抗肿瘤药物,并且通过多种机制参与了肿瘤细胞耐药的形成,与肿瘤的化疗密切相关[5]。除此之外,还研究发现NF-κb与胚胎发育中的细胞凋亡也有相关性。人们逐渐认识到肿瘤的发生,以及肿瘤细胞先天形成、后天获得的对化疗药物的耐药性与NF-κb所调节的细胞凋亡密切相关,即肿瘤细胞中NF-κb可能与相关抑制因子表达缺失、结构性激活等因素导致NF-κb过度激活。NF-κb的作用及其在实践中的应用成为当前研究的热点内容之一。激活后的NF-κb在肿瘤细胞中主要发挥抑制肿瘤细胞凋亡的作用,并且抗调亡作用具有多样性,在肝癌、淋巴瘤、胃癌等多种肿瘤中均已经被证实[6]。淫羊藿苷(Icariin ICA)作为淫羊藿主要的提取物,是一种天然化合物,有抗肿瘤等多种药理学功效[7]。ICA作为一种常见中药,近年来研究发现,ICA具有活血化瘀、增强免疫、扶正培本且无明显不良反应,而且对心血管、脑血管系统、骨代谢、免疫系统及抗肿瘤治疗等方面具有广泛的药理作用,它能抑制肿瘤细胞生长、诱导肿瘤细胞凋亡[8]。本课题旨在探讨NF-Kb和Vasohibin-1在肝细胞肝癌中的表达情况及其在肿瘤发生、发展过程中的作用,他们之间是否存在相关性。通过实验研究淫羊藿苷能否加强三氧化二砷在对肝细胞肝癌的治疗中的抗肿瘤活性,为阐明肝癌的发病机理和提高肝癌的化疗效果进行有益的探索。[方法]1. Vasohibin-1蛋白、NF-kb蛋白在肝细胞肝癌组织中的表达：选取山东省省立医院及济宁市第一人民医院病理科106例肝癌组织及相应癌旁组织,应用免疫组织化学方法检测vasohibin-1、NF-κb的表达,分析vasohibin-1、NF-κb在HCC组织中的表达有无相关性。2.通过MTT试验及流式细胞仪分别检测细胞增殖和细胞凋亡。通过荧光信号来分析ROS水平和线粒体膜电位的变化。蛋白免疫印迹检测蛋白质的表达,酶联免疫吸附试验检测NF-Kb活性。[结果]1. Vasohibin-1蛋白在肝癌组织中的表达：免疫组化检测结果显示vasohibin-1蛋白在106例肝癌组织中的高表达阳率为68.90%(73/106),癌旁组织中高表达阳性率16.04%(17/106),经统计学分析,vasohibin-1在HCC中的高表达阳性率高于非肿瘤癌旁组织,二者间有显著性差异(x2=60.55,P<0.001)。NF-κb蛋白在肝癌组织中的表达：免疫组化检测结果显示NF-κb蛋白在106例肝癌组织中的高表达阳性率为71.70%(76/106),癌旁组织中高表达阳性率23.58(25/106)。经统计学分析,NF-κb在HCC中的高表达阳性率高于癌旁组织,二者间有显著性差异(x2=49.19,P<0.001)。Vasohibin-1和NF-κB表达水平之间的相互关系：76例NF-κb高阳性表达的HCC组织中Vasohibin-1低阳性表达19例,Vasohibin-1高阳性表达57例,30例NF-κb低表达阳性的HCC组织中Vasohibin-1高阳性表达16例, Vasohibin-1低阳性表达14例,经统计学分析,NF-κB及Vasohibin-1在HCC中的表达无显著性相关(x2=4.70,P>0.05)。2.淫羊藿苷在所有的肝细胞肝癌细胞组中对肝癌细胞的抑制增殖、诱导凋亡作用存在剂量依赖性。无论体内实验还是体外实验,淫羊藿苷都能加强三氧化二砷的抗肿瘤活性。淫羊藿苷抗肿瘤活性和它对三氧化二砷的加强作用与细胞内ROS的产生及NF-κb活性抑制相关。[结论]1. Vasohibin-1蛋白在肝癌组织中高表达,在癌旁组织中弱表达,vasohibin-1可作为临床检测的有重要参考价值的标志物,对判断肝癌发生、发展及判断预后有重要意义；NF-κb在肝癌组织中高表达,可能与其抑制肿瘤细胞凋亡,促进肿瘤细胞增殖有关,但是NF-κb与Vasohibin-1蛋白在肝细胞肝癌组织中的表达之间不存在相关性。2.我们的研究结果表明淫羊藿苷加强三氧化二砷对肝细胞肝癌的抗肿瘤活性。因此我们认为淫羊藿苷和三氧化二砷联合治疗能促进三氧化二砷对肝细胞肝癌患者的优化治疗。更多还原

【Abstract】 Background and aimHepatocellular carcinoma (HCC) is one of the most common solid tumors throughout the world. Surgical ablation and liver transplantation are the most effective measures for treating HCC, but it is not satisfactory. Metastasis and recurrence of HCC are the maximal barrier influencing therapeutic effect. Chemical therapy is the most important rescue treatment for metastasis and recurrence of HCC. Chemotherapy drugs kill cancer cells mainly through promoting tumor cell apoptosis, which is the purpose of the treatment of HCC. On the one hand, chemotherapy drugs induced tumor cells apoptosis, on the other hand chemotherapy drugs activate a series of biological signals within the tumor cells, which enhance the resistance of tumors to the chemotherapy drugs. All the factors, which restrain or promote tumor cell apoptosis, determine the apoptosis of the tumor cells.As we have know, vascular endothelial growth factor (VEGF) plays a very important role in mediating tumor angiogenesis, associated with tumor progression, invasion, metastasis, and poorer survival of patients with HCC. VEGF is considered to be the main stimulating factor angiogenesis in HCC. Recently, vasohibin-1was identified, and it is a different type of endogenous inhibitor of angiogenesis. Therefore, some researchers pay attention to the vasohibin-1, which participates in the occurrence and development of tumor angiogenesis new disorders.Nuclear factor-KB (nuclear factor-κb, NF-Kb) is a kind of widespread presence of transcription factors, which is closely related to apoptosis, immune response. Recent study found that the NF-κb can be activated by a variety of chemotherapy drugs, especially interference the nucleic acid generated antitumor drugs, and through a variety of mechanisms involved in the formation of drug-resistant tumor cells and is closely related to tumor chemotherapy. Besides, the study found that the NF-κb also has correlation with the apoptosis in embryonic. people gradually realized that the chemotherapy drug resistance of the tumor is closely related to the apoptosis, which regulated by the NF-Kb, NF-Kb in tumor cells may be associated with loss of inhibiting factor expression, structural activation factors leading to excessive activation of NF-Kb. The role of NF-Kb and its application in practice is becoming the focus at present. Activated NF-Kb plays the role of inhibiting tumor cell apoptosis, and the resistance to apoptosis is very various, which has been confirmed in liver cancer, lymphoma, gastric cancer and other tumors.Icariin, as a major extract of epimedium, is a kind of natural compounds, it has anti-tumor and other pharmacologic effect. The ICA, as a kind of common traditional Chinese medicine, has the function of promoting blood circulation and enhancing the immunity and no obvious adverse reaction, and has a wide range of pharmacological effects on the cardiovascular system, cardiovascular system and bone metabolism and the immune system and anti-tumor treatment. It can inhibit the growth of tumor cells and induce the apoptosis of tumor cells. We investigate the expression of NF-Kb and Vasohibin-1in HCC and their roles in the tumorigenesis and development process, at the same time, we investigate the correlation between of them. We study whether ICA can strengthen the antitum or activity of arsenic trioxide in the treatment of HCC, and carry on the beneficial exploration to elucidate the pathogenesis of liver cancer and improve the effect of liver cancer chemotherapy through the experiment.Methods1.The expression of vasohibin-1and NF-Kb in HCC:Formalin-fixed and paraffin-embedded106HCC tissues and corresponding adjacent non-tumorous liver tissue (ANLT) were retrieved from the Department of Pathology, Shandong Provincial Hospital and Jining NO1people’s hospital. The expression of NF-Kb and vasohibin-1in HCC were detected by immunochemistry. We investigate the correlation between the vasohibin-1and NF-kb expressed in the HCC.2. Based on determined by MTT test and flow cytometry instrument to detect cell proliferation and apoptosis, respectively. The changes of the level of ROS and mitochondrial membrane potential were analyzed by the fluorescent signals.The expression of protein immunoblot assay protein, enzyme-linked immunosorbent assay to detect the activity of NF-Kb.Results1.The expression of vasohibin-1in HCC:Cytoplasm high expression of vasohibin-1was detected in68.9%(73/106) of the HCC tissues, which was significantly higher than that in16.04%(17/106) of adjacent non-tumorous liver tissue (ANLT)(P<0.001). According to the results of chi-square analysis, the high expression of Vasohibin-1in HCC is higher than the adjacent to carcinoma non-tumorous liver tissues(χ2=60.55, P<0.001).The expression of NF-κb in HCC:Cytoplasm high expression of NF-Kb was detected in71.70%(76/106) of the HCC tissues, which was significantly higher than that in23.58%(25/106) of adjacent non-tumorous liver tissue (ANLT)(P<0.001). According to the results of chi-square analysis, the high expression of NF-Kb in HCC is higher than the adjacent to carcinoma non-tumorous liver tissues(χ2=49.19, P<0.001).The relationship between the expression level Of Vasohibin-1and NF-Kb:There were19cases which the expression of Vasohibin-1were low positive in76cases of NF-κb high positive expression in HCC tissues, There were16cases which the expression of Vasohibin-1were high positive in30cases of NF-Kb low positive expression in HCC tissues. According to the results of chi-square analysis, the expression of NF-kb has not correlated with the Vasohibin-1in the HCC (χ2=4.70, P>0.05).2. Icariin could inhibit the proliferation of HCC and induce apoptosis in all of the HCC, which depended on the dose of the treatment. Both in vivo and in vitro experiments, Icariin could strengthen the antitumor activity of arsenic trioxide. The antitumor activity and the effect on the strengthening of arsenic trioxide have correlated with the generation of ROS and the activity of NF-Kb in HCC.Conclusion 1. The expression of vasohibin-1protein was high in HCC, and it was low in adjacent non-tumorous liver tissue. Vasohibin-1protein may be used as an important reference value for clinical markers in HCC, which to judge the occurrence, development and prognosis of HCC. The expression of NF-xb protein was high in HCC, and NF-Kb inhibited tumor cell apoptosis, promoted tumor cell proliferation perhaps, but there is no correlation between the expression of NF-Kb protein and vasohibin-1protein in HCC.2. Icariin could strengthen the anti-tumor activity of arsenic trioxide in HCC in our research work. So we think that the combination therapy of Icariin and arsenic trioxide could optimize the treatment of arsenic trioxide in HCC.更多还原