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

【摘要】：肝細胞癌是我國最常見的惡性腫瘤之一,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,它高表達于多種惡性腫瘤,參與調(diào)節(jié)腫瘤的生物學行為,影響腫瘤患者的預后,有望成為肝細胞癌的治療靶點。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)以及腫瘤干細胞(cancer stem cells,CSCs)特性三方面的關(guān)系,將有助于闡明肝細胞癌的轉(zhuǎn)移復發(fā)機制,為患者的早期診斷、靶向治療以及預后改善研究提供理論基礎(chǔ)。1、FZD2在肝細胞癌組織中的表達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100例肝細胞癌患者癌組織和癌旁組織FZD2的表達水平,發(fā)現(xiàn)其在癌組織的表達量顯著高于癌旁組織(P0.05);統(tǒng)計學分析表明,FZD2表達量與腫瘤大小、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高表達的患者術(shù)后無瘤生存時間和總生存時間明顯縮短(P0.05),提示FZD2可能是肝細胞癌潛在的預后指標。2、FZD2調(diào)控肝癌細胞的增殖、遷移和侵襲能力為了探究FZD2在肝細胞癌中的促癌作用,我們分別構(gòu)建siRNA瞬轉(zhuǎn)FZD2低表達肝癌細胞模型和慢病毒穩(wěn)轉(zhuǎn)FZD2過表達細胞模型,采用CCK-8法、平板克隆形成法、流式細胞儀、Transwell法以及Westernblot檢測發(fā)現(xiàn),敲低FZD2表達抑制肝癌細胞增殖、遷移侵襲能力以及EMT進程,誘導肝癌細胞凋亡,而過表達FZD2則促進細胞增殖、遷移侵襲以及EMT特性;采用FZD2低表達穩(wěn)轉(zhuǎn)細胞株構(gòu)建裸鼠皮下移植瘤模型證實,FZD2也可以調(diào)控肝癌細胞體內(nèi)增殖能力和EMT進程。3、FZD2調(diào)控肝細胞癌的VM形成能力VM是肝細胞癌轉(zhuǎn)移復發(fā)的危險因素,其與FZD2的關(guān)系尚未明確。我們采用CD34/PAS雙染法觀察肝細胞癌和裸鼠移植瘤組織VM的表達情況,發(fā)現(xiàn)FZD2與VM表達呈正相關(guān)(P0.05);細胞三維培養(yǎng)條件下,敲低FZD2抑制肝癌細胞的成管能力,而過表達FZD2則增強這種能力,提示FZD2介導肝細胞癌VM形成。4、FZD2調(diào)控肝癌細胞的CSCs特性除VM外,CSCs特性也與肝細胞癌轉(zhuǎn)移復發(fā)密切相關(guān)。流式細胞儀檢測到,敲低FZD2表達后肝癌細胞干細胞亞群CD44(+)細胞減少(P0.05),Westernblot和免疫組化染色進一步證明,肝癌細胞和移植瘤組織中CSCs轉(zhuǎn)錄因子Nanog和SOX2表達下調(diào),而過表達FZD2上調(diào)肝癌細胞上述干細胞表型。因此,FZD2參與調(diào)節(jié)肝癌細胞CSCs特性。結(jié)論:FZD2可能通過調(diào)控EMT、VM以及CSCs特性促進肝細胞癌的轉(zhuǎn)移復發(fā),有望成為肝細胞癌新的預測指標和治療靶點。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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本文編號：2070343