本文選題：ZEB2 + 上皮-間充質轉化　； 參考：《天津醫(yī)科大學》2015年博士論文

【摘要】：研究背景和目的肝細胞肝癌(hepatocellular carcinoma,HCC)是一種常見原發(fā)性惡性腫瘤,具有高轉移率和高復發(fā)率。盡管近年來肝癌的治療手段有了明顯進展,但治療效果仍不盡如人意,患者的生存質量、存活時間都沒有顯著改善。不同于傳統(tǒng)的腫瘤血供模式,血管生成擬態(tài)(vasculogenic mimicry,VM)是一種獨特的腫瘤微循環(huán)模式,并且往往關聯(lián)著腫瘤的侵襲、轉移以及不良預后。在VM形成的分子機制中,有較多的觀點支持上皮-間充質轉化(epithelial-mesenchymal transition,EMT)參與其中,但兩者之間的調(diào)控機制仍不明確。本研究旨在通過分析EMT轉錄因子ZEB2在人肝癌中表達和預后價值,初步揭示ZEB2與VM的關系;通過研究細胞因子TGF-β1對體外肝癌細胞的誘導作用,進一步證實EMT參與肝癌VM形成;通過研究ZEB2對細胞功能的影響,探討ZEB2調(diào)控的EMT在肝癌血管生成擬態(tài)形成中的可能作用機制。方法1)通過特殊染色方法—CD31/PAS雙重染色方法標記肝癌組織中VM管道結構。免疫組織化學染色檢測人肝癌組織中ZEB2的表達并分析ZEB2與VM形成、臨床病理參數(shù)的關系以及預后價值。2)選取不能形成管道樣結構的肝癌Hep G2細胞系,用細胞因子TGF-β1誘導培養(yǎng),觀察誘導前后細胞形態(tài)和成管能力的變化。3)Western blot檢測TGF-β1誘導培養(yǎng)后的肝癌細胞EMT轉錄因子Twist1、Snail、Slug和ZEB2的表達情況;檢測EMT相關分子E-cadherin,Vimentin及內(nèi)皮相關分子VE-cadherin的表達情況。4)Western blot檢測四種肝癌細胞系Hep G2,Bel7402,PLC和SMMC7221中ZEB2的表達情況。5)篩選出低表達和高表達ZEB2的肝癌細胞系,分別上調(diào)和下調(diào)ZEB2的表達,并觀察ZEB2高、低表達對細胞成管能力及內(nèi)皮相關分子表達的影響。6)Western blot和免疫熒光染色檢測ZEB2高、低表達對肝癌細胞EMT相關分子E-cadherin、Vimentin表達的影響。7)細胞劃痕和侵襲實驗檢測ZEB2高、低表達對肝癌細胞遷移和侵襲能力的影響。8)明膠酶譜檢測ZEB2高、低表達對肝癌細胞MMP-2和MMP-9活性的影響。結果1)92例人肝癌組織樣本中發(fā)現(xiàn)17例存在VM,其發(fā)生率為18%。免疫組化染色發(fā)現(xiàn)ZEB2細胞漿表達53例(57.6%),ZEB2核表達21例(22.8%)。ZEB2核表達VM形成、臨床分期和肝癌患者的復發(fā)/轉移有關(P0.05)。生存分析提示具有ZEB2細胞核表達或者VM陽性的肝癌患者總生存期均較短。多因素分析結果表明ZEB2是肝癌患者總生存期的獨立預后因素(P0.05)。2)TGF-β1誘導培養(yǎng)肝癌Hep G2細胞,細胞由典型的上皮樣表型轉變?yōu)殚g充質樣表型并獲得了形成管道樣結構的能力,能在體外形成VM樣結構。3)TGF-β1誘導培養(yǎng)后的Hep G2細胞EMT轉錄因子ZEB2的表達顯著增強,而對Twist1、Snail和Slug的影響不明顯;間葉標記分子Vimentin表達增高,而上皮標記分子E-cadherin表達下降;內(nèi)皮相關分子VE-cadherin表達增高。4)Western blot檢測證實在四種肝癌細胞系HepG2,Bel7402,PLC和SMMC7221中,Hep G2細胞低表達ZEB2,Bel7402,PLC和SMMC7221細胞均有較高的ZEB2表達,其中Bel7402細胞表達最高。5)HepG2細胞上調(diào)ZEB2表達后在三維培養(yǎng)下能夠形成管道樣結構并表達內(nèi)皮相關分子VE-cadherin、Flt-1、Flk-1;Bel7402細胞下調(diào)ZEB2表達后形成管道樣結構的能力消失并且VE-cadherin、Flt-1、Flk-1的表達顯著下降。6)上調(diào)ZEB2表達使Hep G2細胞間葉標記分子Vimentin表達增高,而上皮標記分子E-cadherin表達下降;下調(diào)ZEB2表達使Bel7402細胞間葉標記分子Vimentin表達下降,而上皮標記分子E-cadherin表達增高。7)上調(diào)ZEB2表達能夠增強肝癌Hep G2細胞侵襲和遷移能力。下調(diào)ZEB2表達降低肝癌Bel7402細胞的侵襲和遷移能力。8)上調(diào)ZEB2表達能夠增強肝癌Hep G2細胞MMP-2和MMP-9的活性;下調(diào)ZEB2表達降低肝癌Bel7402細胞MMP-2和MMP-9的活性。結論1)通過免疫組化及統(tǒng)計學分析證實ZEB2與VM形成有關;ZEB2還與肝癌復發(fā)、轉移等惡性生物學行為有關,是影響患者生存的獨立預后因素。2)體外實驗通過TGF-β1誘導培養(yǎng)肝癌細胞,證實其能夠通過顯著上調(diào)ZEB2的表達誘導肝癌細胞發(fā)生EMT,并且促進體外形成VM樣結構。3)體外實驗通過構建ZEB2上、下調(diào)細胞模型,證實ZEB2能夠增強肝癌細胞的侵襲、遷移性和三維管狀結構形成能力。4)體外實驗亦證實ZEB2能夠增強肝癌細胞MMP-2和MMP-9的活性,上調(diào)內(nèi)皮相關分子VE-cadherin、Flt-1、Flk-1的表達,從而促進VM形成。
[Abstract]:Background and objective hepatocellular carcinoma (HCC) is a common primary malignant tumor with high metastasis rate and high recurrence rate. Although there has been significant progress in the treatment of liver cancer in recent years, the therapeutic effect is still unsatisfactory. The quality of life and the survival time of the patients have not improved significantly. Vasculogenic mimicry (VM) is a unique model of tumor microcirculation and is often associated with tumor invasion, metastasis and poor prognosis. In the molecular mechanism of VM formation, there are many ideas to support the participation of epithelial-mesenchymal transition (EMT). The regulation mechanism between the two is not clear. This study aims to analyze the relationship between the expression and prognosis of EMT transcription factor ZEB2 in human hepatocellular carcinoma and to reveal the relationship between ZEB2 and VM, and to further confirm that EMT is involved in the formation of VM in liver cancer by studying the cytokine TGF- beta 1, and the effect of ZEB2 on cell function is studied. To explore the possible mechanism of ZEB2 regulated EMT in the formation of angiogenesis in hepatoma. Method 1) the structure of VM pipeline in liver cancer tissue was marked by a special staining method - CD31/PAS double staining method. The expression of ZEB2 in human hepatocellular carcinoma tissue was detected by immunohistochemistry and the formation of ZEB2 and VM was analyzed. The relationship between the clinicopathological parameters and the relationship of the clinicopathological parameters was analyzed. And prognostic value.2) Hep G2 cell line, which can not form pipe like structure, was induced by cytokine TGF- beta 1, and the changes of cell morphology and tubular ability were observed before and after induction. Western blot was used to detect the EMT transcriptional factor Twist1, Snail, Slug and expression of TGF- beta 1 induced by TGF- beta. The expression of E-cadherin, Vimentin and endothelial related molecule VE-cadherin.4) Western blot to detect the expression of the four hepatocellular carcinoma cell lines Hep G2, Bel7402, PLC and SMMC7221) to screen the hepatocellular carcinoma cell lines with low expression and high expression. The effect of Western blot and immunofluorescence staining on.6, ZEB2 high, low expression of EMT related molecules E-cadherin, Vimentin expression,.7) cell scratch and invasion test, the detection of ZEB2 high, low expression on the migration and invasiveness of hepatoma cells,.8) ZEB2 high, low expression of gelatinase detection The effect of MMP-2 and MMP-9 activity in hepatoma cells. Results 1) in 92 cases of human hepatocellular carcinoma, 17 cases of VM were found. The incidence of 18%. immunohistochemical staining showed that ZEB2 cytoplasm was expressed in 53 cases (57.6%), 21 cases of ZEB2 nuclear expression (22.8%).ZEB2 nucleus expressed VM formation, clinical staging was related to the recurrence / metastasis of liver cancer patients (P0.05). Survival analysis hints for survival The total survival time of the patients with ZEB2 nuclear expression or VM positive liver cancer was shorter. The multifactor analysis showed that ZEB2 was an independent prognostic factor (P0.05).2 of the total survival period of the patients with liver cancer. TGF- beta 1 induced the culture of liver cancer Hep G2 cells, and the cells transformed from typical epithelioid phenotype to mesenchymal like phenotype and obtained the ability to form a pipeline like structure. The expression of EMT transcriptional factor ZEB2 in Hep G2 cells was significantly enhanced after TGF- beta 1 was induced in vitro. The effect of TGF- beta 1 on Twist1, Snail and Slug was not obvious. In the four kinds of hepatoma cell lines, HepG2, Bel7402, PLC and SMMC7221, Hep G2 cells have low expression of ZEB2, Bel7402, PLC and SMMC7221 cells with higher ZEB2 expression. The ability of -1, Flk-1, and Bel7402 cells to downregulate the expression of ZEB2 was disappearing and the expression of VE-cadherin, Flt-1, Flk-1 significantly decreased.6) and the expression of ZEB2 expressed in Hep G2 cells increased, but the expression of epithelial marker molecules decreased. The expression of tin decreased and the expression of epithelial marker E-cadherin increased.7). Up regulation of ZEB2 expression enhanced the invasion and migration of Hep G2 cells in liver cancer. Down regulation of ZEB2 expression reduced the invasion and migration of Bel7402 cells of liver cancer. Up regulation of ZEB2 expression could enhance the activity of Hep G2 cells and the activity of hepatocellular carcinoma Hep G2 cells. The activity of 402 cells MMP-2 and MMP-9. Conclusion 1) ZEB2 was related to the formation of VM by immunohistochemical and statistical analysis; ZEB2 was related to the malignant biological behavior of the recurrence and metastasis of liver cancer, which was an independent prognostic factor affecting the survival of the patients,.2). In vitro experiments were conducted to induce the cultured liver cancer cells through TGF- beta 1, which proved to be able to significantly increase ZEB2. The expression induced EMT in hepatoma cells and promoted the formation of VM like structure in vitro.3) in vitro, the cell model was downregulated by constructing ZEB2 in vitro. It was proved that ZEB2 could enhance the invasion, mobility and the ability of three-dimensional tubular structure to form.4). In vitro experiments also confirmed that ZEB2 can enhance the activity of MMP-2 and MMP-9 in liver cancer cells and up regulation of endothelial phase. The expression of molecules VE-cadherin, Flt-1 and Flk-1 promoted VM formation.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7

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