Wnt2b对肝细胞癌生物学功能的影响及相关机制探究

发布时间：2018-07-26 06:30

【摘要】：研究目的肝癌是病死率最高的恶性肿瘤之一,也是威胁人类健康的重大疾病,其发病率一直很高。在我国,肝癌的死亡率位列第二、发病率位列第四。对于肝癌的诊断,主要是对肿瘤标志物的检测和影像学检查。对于肝癌的治疗的方法有很多,例如手术切除等。但是,尽管经过了有效的治疗,肝癌仍然有复发和转移的可能。因此,需要进一步阐明肝癌发生的分子机制,发现肝癌发生时有差异的分子和异常的信号通路,寻找有效治疗肝癌的新方法,为临床治疗提供新的方向。近年来的研究中有学者发现,在肝癌的发生发展过程中有异常活化的信号通路。值得我们重视的是,在肝癌发生发展过程中有Wnt经典信号通路的活化现象。Wnt信号通路是一条高度保守的信号通路,其在生物体的胚胎发育,组织修复与再生,调控生物体内与细胞生长、凋亡、自我更新和生存基因的表达发挥着重要的作用。根据所激活信号通路的不同,Wnt信号通路若依赖于β-catenin分子发挥作用则为经典的信号通路;若不依赖,则为非经典通路。关于Wnt与肝脏的研究主要集中于Wnt信号通路在肝纤维化过程中发挥的作用。研究表明,当经典的Wnt信号通路发生活化时可激活肝星状细胞而导致肝纤维化。但是,对于Wnt信号通路与肝癌的研究比较少,且目前对于Wnt家族分子的研究主要集中于Wnt3a与Wnt5a。在前期工作中我们发现,与正常肝组织相比,病变肝脏中Wnt2b分子呈现高表达趋势,并且与疾病严重程度有一定相关性。在TLR4促进HCC发生、TLR2抑制HCC发生的前提下,我们发现TLR4激动剂刺激人肝癌细胞系(HepG2,H7702)和鼠肝癌细胞系(Hepa1-6)Wnt2b表达上调,TLR2激动剂刺激则表达下调。这些结果提示我们,在肝癌的发生发展过程中,Wnt2b可能起到关键的作用。根据上述的实验现象,本研究在体外实验中利用分子生物学手段,探讨了 Wnt2b分子对肝癌细胞的肿瘤生物学特征的影响,并探讨相关机制;并通过体内实验证明,Wnt2b促进小鼠肿瘤的生长,且抑制肿瘤组织中NK细胞的数量和比例。另外,本研究初步探讨了抑制Wnt2b的表达辅助治疗肝癌的应用,为肝癌的治疗提供新的靶点和实验依据。研究方法1.以几种常见的人和鼠肝癌细胞系为模型,采用MTT法、细胞平板克隆实验检测TLR2激动剂Pam3CSK4和TLR4激动剂LPS对肝癌细胞增殖能力的影响。2.采用免疫荧光、免疫组化及western blot方法,检测TLR4对肝癌细胞系和肝癌组织中β-catenin的影响。3.以不同的TLR缺失小鼠为模型,采用半定量RT-PCR法检测Wnts mRNA的基础表达水平。采用半定量RT-PCR法,检测TLR2和TLR4对人肝癌细胞HepG2所有Wnts mRNA水平的影响。4.采用半定量RT-PCR法和western blot,检测TLR2和TLR4对人肝癌细胞HepG2、H7402和鼠肝癌细胞系Hepa1-6中Wnt2b mRNA的影响。5.采用MTT法检测Wnt2b对肝癌细胞增殖的影响。采用细胞集落形成方法和Real-time PCR探究Wnt2b对肝癌细胞系干性的影响。采用PI单染、Annexin V/PI双染法分别检测Wnt2b分子对肝癌细胞周期和凋亡的影响。6.采用western blot、定量PCR、MTT法检测Wnt2b对肝癌细胞的作用是否通过活化Wnt信号通路。7.动物实验采用皮下肿瘤组织荷瘤,细胞内转染质粒并瘤内注射的方法观察Wnt2b对肿瘤的影响,并用流式检测肿瘤组织和肝脏中淋巴细胞亚群的变化。研究结果1.TLR2抑制肝癌细胞系的增殖,TLR4则促进肝癌细胞系的增殖。TLR2抑制肝癌细胞的干性,TLR4则促进肝癌细胞的干性。2.TLR4促进肝癌细胞系中β-catenin的表达及入核。与TLR4缺失小鼠的肝癌组织相比,WT小鼠肝癌组织中β-catenin表达上调,且入核β-catenin增多。3.不同TLR缺失小鼠的肝组织中,Wnts的表达不同。TLR2激动剂刺激人肝癌细胞系HepG2,Wnt2分子,Wnt2b分子表达下调。在TLR4激动剂LPS刺激下,Wnt5a分子表达下调,Wnt2b分子表达上调。4.TLR2抑制肝癌细胞系中Wnt2b的表达,TLR4促进肝癌细胞系中Wnt2b的表达。5.Wnt2b促进肝癌细胞系的增殖。Wnt2b促进肝癌细胞的干性。Wnt2b抑制肝癌细胞系的凋亡,促进抗凋亡基因Bcl-2和Mcl-1的表达,但不影响肝癌细胞系的周期。6.Wnt2b对肝癌的促进作用是通过活化Wnt/β-catenin信号通路实现的;沉默β-catenin后,Wnt2b则不能发挥促进肝癌的作用。7.Wnt2b促进肿瘤的生长并通过抑制肿瘤组织中NK细胞的数量。结论及意义1本文首次发现TLR2分子和TLR4分子在肝癌过程中对Wnt2b表达的影响,发现TLR4分子促进β-catenin的活化,为TLR信号通路与Wnt信号通路建立了联系。2首次发现Wnt2b对肝细胞癌细胞的促进作用,并阐明了其内在机制,发现通过活化Wnt信号通路,介导了肝癌细胞的增殖。在体内,Wnt2b促进肿瘤的生长通过抑制NK细胞,为今后肿瘤的治疗提供新的方向。
[Abstract]:Objective liver cancer is one of the most fatal malignant tumors and is also a major disease which threatens human health. The incidence of liver cancer is always high. In China, the mortality rate of liver cancer ranks second and the incidence is fourth. The diagnosis of liver cancer is mainly the detection of tumor markers and the imaging examination. The methods for the treatment of liver cancer are very important. Many, such as surgical excision, etc., however, although effective treatment, liver cancer still has the possibility of recurrence and metastasis. Therefore, it is necessary to further clarify the molecular mechanism of the occurrence of liver cancer, find different molecular and abnormal signal pathways in the occurrence of liver cancer, find new ways to effectively treat liver cancer, and provide new directions for clinical treatment. In recent years, some scholars have found that there are abnormal signaling pathways in the development of liver cancer. It is worthy of our attention that the activation of the classical Wnt signaling pathway.Wnt signaling pathway is a highly conserved signaling pathway during the development and development of liver cancer, which is in the development of the embryo, tissue repair and regeneration in the organism. Regulation of cell growth, apoptosis, self renewal and expression of survival genes plays an important role. According to the different signaling pathways that are activated, the Wnt signaling pathway is a classic signaling pathway if it depends on the role of beta -catenin molecules; if not dependent, it is a nonclassical pathway. The research on Wnt and the liver is mainly focused on Wnt signaling pathway plays a role in the process of liver fibrosis. Studies have shown that when the classic Wnt signaling pathway is activated, hepatic stellate cells can be activated to lead to liver fibrosis. However, there are few studies on the Wnt signaling pathway and the liver cancer, and the research on the Wnt family is mainly focused on the early work of Wnt3a and Wnt5a.. We found that, compared with normal liver tissue, the Wnt2b molecule in the liver is highly expressed and has a certain correlation with the severity of the disease. On the premise of TLR4 promoting HCC and TLR2 inhibition of HCC, we found that TLR4 activator stimulates human hepatoma cell line (HepG2, H7702) and rat hepatoma cell line (Hepa1-6) Wnt2b expression up-regulated, TL These results suggest that Wnt2b may play a key role in the development of liver cancer. According to the experimental phenomena mentioned above, the effects of Wnt2b on the tumor biological characteristics of hepatoma cells are discussed in this study in vitro, and the mechanism of Wnt2b is discussed. In vivo experiments have proved that Wnt2b promotes the growth of tumor in mice and inhibits the number and proportion of NK cells in tumor tissues. In addition, this study has preliminarily explored the application of inhibiting the expression of Wnt2b to assist in the treatment of liver cancer, and provides new targets and experimental basis for the treatment of liver cancer. Method 1. study several common human and rat hepatoma cells The effects of TLR2 agonist Pam3CSK4 and TLR4 agonist LPS on the proliferation of hepatoma cells were measured by MTT method, and.2. using immunofluorescence, immunohistochemistry and Western blot method to detect the effect of TLR4 on the beta -catenin in liver cancer cell lines and liver cancer tissues,.3. with different TLR deletion mice was used as a model. Semi quantitative RT-PCR method was used to detect the basic expression level of Wnts mRNA. Semi quantitative RT-PCR method was used to detect the effect of TLR2 and TLR4 on the mRNA level of all Wnts in human hepatoma cell HepG2.4. using semi quantitative RT-PCR method and Western limitation. The effect of Wnt2b on the proliferation of hepatoma cells was detected by TT. Cell colony formation and Real-time PCR were used to investigate the effect of Wnt2b on the stem of liver cancer cell lines. PI single staining and Annexin V/PI double staining were used to detect the effect of Wnt2b molecules on the cell cycle and apoptosis of hepatoma cells respectively..6. used Western blot. Whether the function of the cell was activated by the activation of Wnt signaling pathway in.7. animal experiment, the tumor was charged by subcutaneous tumor tissue, the transfection of plasmids and intratumoral injection were used to observe the effect of Wnt2b on the tumor, and the changes of lymphocyte subsets in the tumor tissues and liver were detected by flow cytometry. The results of the study were that 1.TLR2 inhibited the proliferation of the liver cancer cell lines, and TLR4 promoted the proliferation of the liver cancer cell lines. The proliferation of hepatoma cell line.TLR2 inhibits the dry nature of hepatoma cells, and TLR4 promotes the expression and nucleation of beta -catenin in liver cancer cell lines by promoting the dry.2.TLR4 of hepatoma cells. The expression of beta -catenin in the liver tissues of WT mice is up to up, and the nucleated beta -catenin increases in the liver tissues of the mice lacking TLR in.3. and TLR. Wnts expression of different.TLR2 agonists stimulated the human hepatoma cell line HepG2, Wnt2 molecules, and Wnt2b molecules down regulated. The expression of Wnt5a molecules was down regulated by TLR4 agonist LPS, and Wnt2b molecule expression up.4.TLR2 inhibited the Wnt2b expression in hepatoma cell lines. TLR4 promoted the expression of the hepatocellular carcinoma cell line to promote the liver cancer cell line The proliferation of.Wnt2b stimulates the dry.Wnt2b of hepatoma cells to inhibit the apoptosis of hepatoma cell lines and promote the expression of anti apoptotic gene Bcl-2 and Mcl-1, but does not affect the promoting effect of.6.Wnt2b on liver cancer by activating Wnt/ beta -catenin signaling pathway. After silent beta -catenin, Wnt2b can not play a role in promoting liver cancer. .7.Wnt2b promotes the growth of tumor and inhibits the number of NK cells in tumor tissue. Conclusion and significance 1, this paper first discovered the effect of TLR2 and TLR4 molecules on the expression of Wnt2b in the process of liver cancer, and found that TLR4 molecules promote the activation of beta -catenin, and the first discovery of Wnt2b to the liver for TLR signaling pathway and Wnt signaling pathway is the first discovery of Wnt2b on the liver. The promoting effect of cell cancer cells, and elucidates its intrinsic mechanism, is found to mediate the proliferation of hepatoma cells by activating the Wnt signaling pathway. In vivo, Wnt2b promotes tumor growth by inhibiting NK cells and provides a new direction for the treatment of cancer in the future.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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