發(fā)布時(shí)間：2018-06-26 12:11

  本文選題：肝細(xì)胞癌 + FZD2　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：肝細(xì)胞癌是我國(guó)最常見(jiàn)的惡性腫瘤之一,新型治療方法逐步增多,但根治性手術(shù)和肝移植仍是主要治療手段,其高復(fù)發(fā)率和高死亡率仍然為得到明顯改善。卷曲蛋白2(frizzled2,FZD2)是一種新近發(fā)現(xiàn)的腫瘤相關(guān)分子,它高表達(dá)于多種惡性腫瘤,參與調(diào)節(jié)腫瘤的生物學(xué)行為,影響腫瘤患者的預(yù)后,有望成為肝細(xì)胞癌的治療靶點(diǎn)。本文旨在探究FZD2與上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)、擬態(tài)血管形成(vasculogenic mimicry,VM)以及腫瘤干細(xì)胞(cancer stem cells,CSCs)特性三方面的關(guān)系,將有助于闡明肝細(xì)胞癌的轉(zhuǎn)移復(fù)發(fā)機(jī)制,為患者的早期診斷、靶向治療以及預(yù)后改善研究提供理論基礎(chǔ)。1、FZD2在肝細(xì)胞癌組織中的表達(dá)及其臨床意義我們采用熒光定量PCR和免疫組化染色分別從基因轉(zhuǎn)錄水平和蛋白水平檢測(cè)100例肝細(xì)胞癌患者癌組織和癌旁組織FZD2的表達(dá)水平,發(fā)現(xiàn)其在癌組織的表達(dá)量顯著高于癌旁組織(P0.05);統(tǒng)計(jì)學(xué)分析表明,FZD2表達(dá)量與腫瘤大小、病理分化程度、微癌栓、TNM分期、BCLC分期、轉(zhuǎn)移以及早期復(fù)發(fā)等多種病理參數(shù)有關(guān)(P0.05);生存分析顯示,FZD2高表達(dá)的患者術(shù)后無(wú)瘤生存時(shí)間和總生存時(shí)間明顯縮短(P0.05),提示FZD2可能是肝細(xì)胞癌潛在的預(yù)后指標(biāo)。2、FZD2調(diào)控肝癌細(xì)胞的增殖、遷移和侵襲能力為了探究FZD2在肝細(xì)胞癌中的促癌作用,我們分別構(gòu)建siRNA瞬轉(zhuǎn)FZD2低表達(dá)肝癌細(xì)胞模型和慢病毒穩(wěn)轉(zhuǎn)FZD2過(guò)表達(dá)細(xì)胞模型,采用CCK-8法、平板克隆形成法、流式細(xì)胞儀、Transwell法以及Westernblot檢測(cè)發(fā)現(xiàn),敲低FZD2表達(dá)抑制肝癌細(xì)胞增殖、遷移侵襲能力以及EMT進(jìn)程,誘導(dǎo)肝癌細(xì)胞凋亡,而過(guò)表達(dá)FZD2則促進(jìn)細(xì)胞增殖、遷移侵襲以及EMT特性;采用FZD2低表達(dá)穩(wěn)轉(zhuǎn)細(xì)胞株構(gòu)建裸鼠皮下移植瘤模型證實(shí),FZD2也可以調(diào)控肝癌細(xì)胞體內(nèi)增殖能力和EMT進(jìn)程。3、FZD2調(diào)控肝細(xì)胞癌的VM形成能力VM是肝細(xì)胞癌轉(zhuǎn)移復(fù)發(fā)的危險(xiǎn)因素,其與FZD2的關(guān)系尚未明確。我們采用CD34/PAS雙染法觀察肝細(xì)胞癌和裸鼠移植瘤組織VM的表達(dá)情況,發(fā)現(xiàn)FZD2與VM表達(dá)呈正相關(guān)(P0.05);細(xì)胞三維培養(yǎng)條件下,敲低FZD2抑制肝癌細(xì)胞的成管能力,而過(guò)表達(dá)FZD2則增強(qiáng)這種能力,提示FZD2介導(dǎo)肝細(xì)胞癌VM形成。4、FZD2調(diào)控肝癌細(xì)胞的CSCs特性除VM外,CSCs特性也與肝細(xì)胞癌轉(zhuǎn)移復(fù)發(fā)密切相關(guān)。流式細(xì)胞儀檢測(cè)到,敲低FZD2表達(dá)后肝癌細(xì)胞干細(xì)胞亞群CD44(+)細(xì)胞減少(P0.05),Westernblot和免疫組化染色進(jìn)一步證明,肝癌細(xì)胞和移植瘤組織中CSCs轉(zhuǎn)錄因子Nanog和SOX2表達(dá)下調(diào),而過(guò)表達(dá)FZD2上調(diào)肝癌細(xì)胞上述干細(xì)胞表型。因此,FZD2參與調(diào)節(jié)肝癌細(xì)胞CSCs特性。結(jié)論:FZD2可能通過(guò)調(diào)控EMT、VM以及CSCs特性促進(jìn)肝細(xì)胞癌的轉(zhuǎn)移復(fù)發(fā),有望成為肝細(xì)胞癌新的預(yù)測(cè)指標(biāo)和治療靶點(diǎn)。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China, but radical surgery and liver transplantation are still the main treatment methods. The high recurrence rate and high mortality rate are still significantly improved. Frizzled2FZD2 (frizzled2FZD2) is a newly discovered tumor-related molecule, which is highly expressed in many kinds of malignant tumors. It is involved in regulating the biological behavior of tumors and affecting the prognosis of tumor patients. It is expected to be a therapeutic target for hepatocellular carcinoma. The purpose of this study was to explore the relationship between FZD2 and the characteristics of epithelial-mesenchymal transition, vasculogenic mimicrysma VM and (cancer stem cells of tumor stem cells, which would be helpful to elucidate the mechanism of metastasis and recurrence of hepatocellular carcinoma. The expression and Clinical significance of FZD2 in Hepatocellular carcinoma We detected 100 cases of Hepatocellular carcinoma by fluorescence quantitative PCR and Immunohistochemical staining from Gene transcription level and protein level Expression of FZD2 in hepatocellular carcinoma tissues and adjacent tissues, It was found that the expression of FZD2 in cancer tissues was significantly higher than that in adjacent tissues (P0.05). Statistical analysis showed that FZD2 expression was correlated with tumor size, pathological differentiation, TNM stage and BCLC stage. Survival analysis showed that tumor free survival time and total survival time of patients with high expression of FZD2 were significantly shortened (P0.05), suggesting that FZD2 might be a potential prognostic index of hepatocellular carcinoma. Proliferation of hepatoma cells, In order to investigate the role of FZD2 in the promotion of hepatocellular carcinoma, we constructed a cell model of low expression of FZD2 by siRNA and a model of overexpression of lentivirus into FZD2 by using CCK-8 method and plate clone formation method. Flow cytometry and Western blot analysis showed that low expression of FZD2 inhibited proliferation, migration, invasion and EMT process of HCC cells, and induced apoptosis of HCC cells, while overexpression of FZD2 promoted cell proliferation, migration and invasion, and EMT characteristics. It was proved that FZD2 could also regulate the proliferation of hepatoma cells in vivo and the VM formation ability of hepatocellular carcinoma regulated by EMT process by using FZD2 low expression stably transformed cell line in nude mice, which was a risk factor for metastasis and recurrence of hepatocellular carcinoma (HCC). Its relationship with FZD2 is not clear. We used CD34 / pas double staining method to observe the expression of VM in hepatocellular carcinoma and xenografts in nude mice, and found that FZD2 was positively correlated with VM expression (P0.05). Overexpression of FZD2 enhances this ability, suggesting that FZD2 mediates the formation of VM in hepatocellular carcinoma. 4FZD2 regulates the characteristics of CSCs in hepatocellular carcinoma cells. Besides VM, the characteristics of CSCs are also closely related to the metastasis and recurrence of HCC. Flow cytometry showed that the expression of CSCs transcription factors Nanog and SOX2 was down-regulated in hepatoma cells and transplanted tumor tissues, and the CD44 (P05) cell subsets were decreased by Western blot and immunohistochemical staining after knockout FZD2 expression was lowered, and the expression of CSCs transcription factors Nanog and SOX2 were down-regulated in HCC cells and transplanted tumor tissues. Overexpression of FZD2 upregulated the above stem cell phenotype. Therefore, FZD2 is involved in regulating the characteristics of CSCs in hepatoma cells. Conclusion: FZD2 may promote the metastasis and recurrence of hepatocellular carcinoma by regulating the characteristics of EMTV and CSCs, and may be a new predictor and therapeutic target of HCC.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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本文編號(hào)：2070343