【摘要】：研究目的肝癌是病死率最高的惡性腫瘤之一,也是威脅人類(lèi)健康的重大疾病,其發(fā)病率一直很高。在我國(guó),肝癌的死亡率位列第二、發(fā)病率位列第四。對(duì)于肝癌的診斷,主要是對(duì)腫瘤標(biāo)志物的檢測(cè)和影像學(xué)檢查。對(duì)于肝癌的治療的方法有很多,例如手術(shù)切除等。但是,盡管經(jīng)過(guò)了有效的治療,肝癌仍然有復(fù)發(fā)和轉(zhuǎn)移的可能。因此,需要進(jìn)一步闡明肝癌發(fā)生的分子機(jī)制,發(fā)現(xiàn)肝癌發(fā)生時(shí)有差異的分子和異常的信號(hào)通路,尋找有效治療肝癌的新方法,為臨床治療提供新的方向。近年來(lái)的研究中有學(xué)者發(fā)現(xiàn),在肝癌的發(fā)生發(fā)展過(guò)程中有異常活化的信號(hào)通路。值得我們重視的是,在肝癌發(fā)生發(fā)展過(guò)程中有Wnt經(jīng)典信號(hào)通路的活化現(xiàn)象。Wnt信號(hào)通路是一條高度保守的信號(hào)通路,其在生物體的胚胎發(fā)育,組織修復(fù)與再生,調(diào)控生物體內(nèi)與細(xì)胞生長(zhǎng)、凋亡、自我更新和生存基因的表達(dá)發(fā)揮著重要的作用。根據(jù)所激活信號(hào)通路的不同,Wnt信號(hào)通路若依賴(lài)于β-catenin分子發(fā)揮作用則為經(jīng)典的信號(hào)通路;若不依賴(lài),則為非經(jīng)典通路。關(guān)于Wnt與肝臟的研究主要集中于Wnt信號(hào)通路在肝纖維化過(guò)程中發(fā)揮的作用。研究表明,當(dāng)經(jīng)典的Wnt信號(hào)通路發(fā)生活化時(shí)可激活肝星狀細(xì)胞而導(dǎo)致肝纖維化。但是,對(duì)于Wnt信號(hào)通路與肝癌的研究比較少,且目前對(duì)于Wnt家族分子的研究主要集中于Wnt3a與Wnt5a。在前期工作中我們發(fā)現(xiàn),與正常肝組織相比,病變肝臟中Wnt2b分子呈現(xiàn)高表達(dá)趨勢(shì),并且與疾病嚴(yán)重程度有一定相關(guān)性。在TLR4促進(jìn)HCC發(fā)生、TLR2抑制HCC發(fā)生的前提下,我們發(fā)現(xiàn)TLR4激動(dòng)劑刺激人肝癌細(xì)胞系(HepG2,H7702)和鼠肝癌細(xì)胞系(Hepa1-6)Wnt2b表達(dá)上調(diào),TLR2激動(dòng)劑刺激則表達(dá)下調(diào)。這些結(jié)果提示我們,在肝癌的發(fā)生發(fā)展過(guò)程中,Wnt2b可能起到關(guān)鍵的作用。根據(jù)上述的實(shí)驗(yàn)現(xiàn)象,本研究在體外實(shí)驗(yàn)中利用分子生物學(xué)手段,探討了 Wnt2b分子對(duì)肝癌細(xì)胞的腫瘤生物學(xué)特征的影響,并探討相關(guān)機(jī)制;并通過(guò)體內(nèi)實(shí)驗(yàn)證明,Wnt2b促進(jìn)小鼠腫瘤的生長(zhǎng),且抑制腫瘤組織中NK細(xì)胞的數(shù)量和比例。另外,本研究初步探討了抑制Wnt2b的表達(dá)輔助治療肝癌的應(yīng)用,為肝癌的治療提供新的靶點(diǎn)和實(shí)驗(yàn)依據(jù)。研究方法1.以幾種常見(jiàn)的人和鼠肝癌細(xì)胞系為模型,采用MTT法、細(xì)胞平板克隆實(shí)驗(yàn)檢測(cè)TLR2激動(dòng)劑Pam3CSK4和TLR4激動(dòng)劑LPS對(duì)肝癌細(xì)胞增殖能力的影響。2.采用免疫熒光、免疫組化及western blot方法,檢測(cè)TLR4對(duì)肝癌細(xì)胞系和肝癌組織中β-catenin的影響。3.以不同的TLR缺失小鼠為模型,采用半定量RT-PCR法檢測(cè)Wnts mRNA的基礎(chǔ)表達(dá)水平。采用半定量RT-PCR法,檢測(cè)TLR2和TLR4對(duì)人肝癌細(xì)胞HepG2所有Wnts mRNA水平的影響。4.采用半定量RT-PCR法和western blot,檢測(cè)TLR2和TLR4對(duì)人肝癌細(xì)胞HepG2、H7402和鼠肝癌細(xì)胞系Hepa1-6中Wnt2b mRNA的影響。5.采用MTT法檢測(cè)Wnt2b對(duì)肝癌細(xì)胞增殖的影響。采用細(xì)胞集落形成方法和Real-time PCR探究Wnt2b對(duì)肝癌細(xì)胞系干性的影響。采用PI單染、Annexin V/PI雙染法分別檢測(cè)Wnt2b分子對(duì)肝癌細(xì)胞周期和凋亡的影響。6.采用western blot、定量PCR、MTT法檢測(cè)Wnt2b對(duì)肝癌細(xì)胞的作用是否通過(guò)活化Wnt信號(hào)通路。7.動(dòng)物實(shí)驗(yàn)采用皮下腫瘤組織荷瘤,細(xì)胞內(nèi)轉(zhuǎn)染質(zhì)粒并瘤內(nèi)注射的方法觀察Wnt2b對(duì)腫瘤的影響,并用流式檢測(cè)腫瘤組織和肝臟中淋巴細(xì)胞亞群的變化。研究結(jié)果1.TLR2抑制肝癌細(xì)胞系的增殖,TLR4則促進(jìn)肝癌細(xì)胞系的增殖。TLR2抑制肝癌細(xì)胞的干性,TLR4則促進(jìn)肝癌細(xì)胞的干性。2.TLR4促進(jìn)肝癌細(xì)胞系中β-catenin的表達(dá)及入核。與TLR4缺失小鼠的肝癌組織相比,WT小鼠肝癌組織中β-catenin表達(dá)上調(diào),且入核β-catenin增多。3.不同TLR缺失小鼠的肝組織中,Wnts的表達(dá)不同。TLR2激動(dòng)劑刺激人肝癌細(xì)胞系HepG2,Wnt2分子,Wnt2b分子表達(dá)下調(diào)。在TLR4激動(dòng)劑LPS刺激下,Wnt5a分子表達(dá)下調(diào),Wnt2b分子表達(dá)上調(diào)。4.TLR2抑制肝癌細(xì)胞系中Wnt2b的表達(dá),TLR4促進(jìn)肝癌細(xì)胞系中Wnt2b的表達(dá)。5.Wnt2b促進(jìn)肝癌細(xì)胞系的增殖。Wnt2b促進(jìn)肝癌細(xì)胞的干性。Wnt2b抑制肝癌細(xì)胞系的凋亡,促進(jìn)抗凋亡基因Bcl-2和Mcl-1的表達(dá),但不影響肝癌細(xì)胞系的周期。6.Wnt2b對(duì)肝癌的促進(jìn)作用是通過(guò)活化Wnt/β-catenin信號(hào)通路實(shí)現(xiàn)的;沉默β-catenin后,Wnt2b則不能發(fā)揮促進(jìn)肝癌的作用。7.Wnt2b促進(jìn)腫瘤的生長(zhǎng)并通過(guò)抑制腫瘤組織中NK細(xì)胞的數(shù)量。結(jié)論及意義1本文首次發(fā)現(xiàn)TLR2分子和TLR4分子在肝癌過(guò)程中對(duì)Wnt2b表達(dá)的影響,發(fā)現(xiàn)TLR4分子促進(jìn)β-catenin的活化,為T(mén)LR信號(hào)通路與Wnt信號(hào)通路建立了聯(lián)系。2首次發(fā)現(xiàn)Wnt2b對(duì)肝細(xì)胞癌細(xì)胞的促進(jìn)作用,并闡明了其內(nèi)在機(jī)制,發(fā)現(xiàn)通過(guò)活化Wnt信號(hào)通路,介導(dǎo)了肝癌細(xì)胞的增殖。在體內(nèi),Wnt2b促進(jìn)腫瘤的生長(zhǎng)通過(guò)抑制NK細(xì)胞,為今后腫瘤的治療提供新的方向。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

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