本文選題：ZEB2 + 上皮-間充質(zhì)轉(zhuǎn)化��； 參考：《天津醫(yī)科大學(xué)》2015年博士論文

【摘要】：研究背景和目的肝細(xì)胞肝癌(hepatocellular carcinoma,HCC)是一種常見原發(fā)性惡性腫瘤,具有高轉(zhuǎn)移率和高復(fù)發(fā)率。盡管近年來肝癌的治療手段有了明顯進(jìn)展,但治療效果仍不盡如人意,患者的生存質(zhì)量、存活時(shí)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(dú)特的腫瘤微循環(huán)模式,并且往往關(guān)聯(lián)著腫瘤的侵襲、轉(zhuǎn)移以及不良預(yù)后。在VM形成的分子機(jī)制中,有較多的觀點(diǎn)支持上皮-間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)參與其中,但兩者之間的調(diào)控機(jī)制仍不明確。本研究旨在通過分析EMT轉(zhuǎn)錄因子ZEB2在人肝癌中表達(dá)和預(yù)后價(jià)值,初步揭示ZEB2與VM的關(guān)系;通過研究細(xì)胞因子TGF-β1對(duì)體外肝癌細(xì)胞的誘導(dǎo)作用,進(jìn)一步證實(shí)EMT參與肝癌VM形成;通過研究ZEB2對(duì)細(xì)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(jī)制。方法1)通過特殊染色方法—CD31/PAS雙重染色方法標(biāo)記肝癌組織中VM管道結(jié)構(gòu)。免疫組織化學(xué)染色檢測(cè)人肝癌組織中ZEB2的表達(dá)并分析ZEB2與VM形成、臨床病理參數(shù)的關(guān)系以及預(yù)后價(jià)值。2)選取不能形成管道樣結(jié)構(gòu)的肝癌Hep G2細(xì)胞系,用細(xì)胞因子TGF-β1誘導(dǎo)培養(yǎng),觀察誘導(dǎo)前后細(xì)胞形態(tài)和成管能力的變化。3)Western blot檢測(cè)TGF-β1誘導(dǎo)培養(yǎng)后的肝癌細(xì)胞EMT轉(zhuǎn)錄因子Twist1、Snail、Slug和ZEB2的表達(dá)情況;檢測(cè)EMT相關(guān)分子E-cadherin,Vimentin及內(nèi)皮相關(guān)分子VE-cadherin的表達(dá)情況。4)Western blot檢測(cè)四種肝癌細(xì)胞系Hep G2,Bel7402,PLC和SMMC7221中ZEB2的表達(dá)情況。5)篩選出低表達(dá)和高表達(dá)ZEB2的肝癌細(xì)胞系,分別上調(diào)和下調(diào)ZEB2的表達(dá),并觀察ZEB2高、低表達(dá)對(duì)細(xì)胞成管能力及內(nèi)皮相關(guān)分子表達(dá)的影響。6)Western blot和免疫熒光染色檢測(cè)ZEB2高、低表達(dá)對(duì)肝癌細(xì)胞EMT相關(guān)分子E-cadherin、Vimentin表達(dá)的影響。7)細(xì)胞劃痕和侵襲實(shí)驗(yàn)檢測(cè)ZEB2高、低表達(dá)對(duì)肝癌細(xì)胞遷移和侵襲能力的影響。8)明膠酶譜檢測(cè)ZEB2高、低表達(dá)對(duì)肝癌細(xì)胞MMP-2和MMP-9活性的影響。結(jié)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(xì)胞漿表達(dá)53例(57.6%),ZEB2核表達(dá)21例(22.8%)。ZEB2核表達(dá)VM形成、臨床分期和肝癌患者的復(fù)發(fā)/轉(zhuǎn)移有關(guān)(P0.05)。生存分析提示具有ZEB2細(xì)胞核表達(dá)或者VM陽性的肝癌患者總生存期均較短。多因素分析結(jié)果表明ZEB2是肝癌患者總生存期的獨(dú)立預(yù)后因素(P0.05)。2)TGF-β1誘導(dǎo)培養(yǎng)肝癌Hep G2細(xì)胞,細(xì)胞由典型的上皮樣表型轉(zhuǎn)變?yōu)殚g充質(zhì)樣表型并獲得了形成管道樣結(jié)構(gòu)的能力,能在體外形成VM樣結(jié)構(gòu)。3)TGF-β1誘導(dǎo)培養(yǎng)后的Hep G2細(xì)胞EMT轉(zhuǎn)錄因子ZEB2的表達(dá)顯著增強(qiáng),而對(duì)Twist1、Snail和Slug的影響不明顯;間葉標(biāo)記分子Vimentin表達(dá)增高,而上皮標(biāo)記分子E-cadherin表達(dá)下降;內(nèi)皮相關(guān)分子VE-cadherin表達(dá)增高。4)Western blot檢測(cè)證實(shí)在四種肝癌細(xì)胞系HepG2,Bel7402,PLC和SMMC7221中,Hep G2細(xì)胞低表達(dá)ZEB2,Bel7402,PLC和SMMC7221細(xì)胞均有較高的ZEB2表達(dá),其中Bel7402細(xì)胞表達(dá)最高。5)HepG2細(xì)胞上調(diào)ZEB2表達(dá)后在三維培養(yǎng)下能夠形成管道樣結(jié)構(gòu)并表達(dá)內(nèi)皮相關(guān)分子VE-cadherin、Flt-1、Flk-1;Bel7402細(xì)胞下調(diào)ZEB2表達(dá)后形成管道樣結(jié)構(gòu)的能力消失并且VE-cadherin、Flt-1、Flk-1的表達(dá)顯著下降。6)上調(diào)ZEB2表達(dá)使Hep G2細(xì)胞間葉標(biāo)記分子Vimentin表達(dá)增高,而上皮標(biāo)記分子E-cadherin表達(dá)下降;下調(diào)ZEB2表達(dá)使Bel7402細(xì)胞間葉標(biāo)記分子Vimentin表達(dá)下降,而上皮標(biāo)記分子E-cadherin表達(dá)增高。7)上調(diào)ZEB2表達(dá)能夠增強(qiáng)肝癌Hep G2細(xì)胞侵襲和遷移能力。下調(diào)ZEB2表達(dá)降低肝癌Bel7402細(xì)胞的侵襲和遷移能力。8)上調(diào)ZEB2表達(dá)能夠增強(qiáng)肝癌Hep G2細(xì)胞MMP-2和MMP-9的活性;下調(diào)ZEB2表達(dá)降低肝癌Bel7402細(xì)胞MMP-2和MMP-9的活性。結(jié)論1)通過免疫組化及統(tǒng)計(jì)學(xué)分析證實(shí)ZEB2與VM形成有關(guān);ZEB2還與肝癌復(fù)發(fā)、轉(zhuǎn)移等惡性生物學(xué)行為有關(guān),是影響患者生存的獨(dú)立預(yù)后因素。2)體外實(shí)驗(yàn)通過TGF-β1誘導(dǎo)培養(yǎng)肝癌細(xì)胞,證實(shí)其能夠通過顯著上調(diào)ZEB2的表達(dá)誘導(dǎo)肝癌細(xì)胞發(fā)生EMT,并且促進(jìn)體外形成VM樣結(jié)構(gòu)。3)體外實(shí)驗(yàn)通過構(gòu)建ZEB2上、下調(diào)細(xì)胞模型,證實(shí)ZEB2能夠增強(qiáng)肝癌細(xì)胞的侵襲、遷移性和三維管狀結(jié)構(gòu)形成能力。4)體外實(shí)驗(yàn)亦證實(shí)ZEB2能夠增強(qiáng)肝癌細(xì)胞MMP-2和MMP-9的活性,上調(diào)內(nèi)皮相關(guān)分子VE-cadherin、Flt-1、Flk-1的表達(dá),從而促進(jìn)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鐘寶元;劉艷秀;;腫瘤中STAT3、P-STAT3的表達(dá)及與EMT關(guān)系的研究進(jìn)展[J];贛南醫(yī)學(xué)院學(xué)報(bào);2013年04期

2 Yin-Sheng Chen;Zhong-Ping Chen;;Vasculogenic mimicry:a novel target for glioma therapy[J];Chinese Journal of Cancer;2014年02期

3 彭璇;;ZEB-1在胃癌組織中的表達(dá)及臨床意義[J];湖北民族學(xué)院學(xué)報(bào)(醫(yī)學(xué)版);2013年03期

4 艾婷;周軍;;乳腺癌血管生成擬態(tài)與超聲分子影像學(xué)相關(guān)性的研究進(jìn)展[J];廣東醫(yī)學(xué);2013年24期

5 流小舟;黎承軍;周光新;趙建寧;;核轉(zhuǎn)錄因子Snail與骨肉瘤關(guān)系的研究進(jìn)展[J];東南國(guó)防醫(yī)藥;2014年01期

6 李f3穎;姜樺;;ZEB家族在上皮性卵巢癌上皮-間質(zhì)轉(zhuǎn)化中的作用[J];國(guó)際婦產(chǎn)科學(xué)雜志;2014年01期

7 吳濤;黃秋林;熊廷剛;高緒照;;MMP-9、Snail蛋白在肝細(xì)胞癌聯(lián)合表達(dá)與術(shù)后復(fù)發(fā)的關(guān)系[J];肝膽胰外科雜志;2014年02期

8 魏慧君;郭麗麗;李林;周清華;吳志浩;;Warburg效應(yīng)及其對(duì)腫瘤轉(zhuǎn)移的影響[J];中國(guó)肺癌雜志;2015年03期

9 陳俊杰;藍(lán)育青;吳共發(fā);林俊汕;區(qū)瑞章;曾宇婷;;血管生成擬態(tài)與翼狀胬肉進(jìn)行期及靜止期的相關(guān)性研究[J];國(guó)際眼科雜志;2015年03期

10 孫鳳丹;崔演;張佰玲;楚佳楠;玄延花;;食管鱗癌組織中Twist1的表達(dá)及其臨床意義[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2013年08期

相關(guān)博士學(xué)位論文 前10條

1 朱玉德;膠質(zhì)瘤干細(xì)胞的生物學(xué)特性及其與膠質(zhì)瘤血管發(fā)生的關(guān)系[D];蘇州大學(xué);2013年

2 陳建敏;CEACAM6介導(dǎo)上皮間質(zhì)轉(zhuǎn)化促進(jìn)胰腺癌侵襲和轉(zhuǎn)移的機(jī)制研究[D];南京醫(yī)科大學(xué);2013年

3 李棟;遺傳變異與食管癌和小細(xì)胞肺癌生存的關(guān)聯(lián)研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

4 丁松明;腫瘤微環(huán)境不同成分在肝癌侵襲轉(zhuǎn)移中的作用研究[D];浙江大學(xué);2013年

5 楊曦;人Fcα/μR配體結(jié)合位點(diǎn)研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

6 孫丹;Slug對(duì)肝癌細(xì)胞干細(xì)胞特性及VM形成影響的研究[D];天津醫(yī)科大學(xué);2013年

7 沈捷;MicroRNA-2抑制肺癌細(xì)胞增殖、侵襲和轉(zhuǎn)移的生物學(xué)作用研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

8 張建超;轉(zhuǎn)錄因子SOX4誘導(dǎo)人乳腺上皮細(xì)胞發(fā)生上皮—間質(zhì)轉(zhuǎn)換并促進(jìn)乳腺癌進(jìn)程研究[D];東北師范大學(xué);2013年

9 尹科;Twist在骨肉瘤轉(zhuǎn)移和上皮間質(zhì)轉(zhuǎn)化中作用及機(jī)制[D];中南大學(xué);2013年

10 周亮;Notch信號(hào)通路參與肝癌細(xì)胞侵襲遷移的機(jī)制研究[D];第四軍醫(yī)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 劉晨輝;塞來昔布對(duì)非小細(xì)胞肺癌A549細(xì)胞增殖、侵襲和黏附的影響及其機(jī)制的研究[D];南京醫(yī)科大學(xué);2013年

2 安昭杰;胃癌ZNF139mRNA表達(dá)及與RUNX3基因甲基化關(guān)系的研究[D];河北醫(yī)科大學(xué);2013年

3 張周;Bmi-1和MMP-2、E-cadherin在膀胱移行細(xì)胞癌中的表達(dá)及相關(guān)性研究[D];河北醫(yī)科大學(xué);2013年

4 曾園園;IL-6/JAK/STAT3與TGF-β/Smad通路介導(dǎo)肺癌細(xì)胞上皮—間充質(zhì)轉(zhuǎn)化過程的交互作用機(jī)制研究[D];蘇州大學(xué);2013年

5 李夢(mèng)婷;鞘氨醇激酶1對(duì)結(jié)腸癌細(xì)胞血管生成擬態(tài)的影響及其機(jī)制[D];廣西醫(yī)科大學(xué);2013年

6 吳雯雯;胃癌肝轉(zhuǎn)移危險(xiǎn)因素的相關(guān)性分析[D];大連醫(yī)科大學(xué);2012年

7 何寧;RNA干擾抑制MACC1對(duì)大腸癌細(xì)胞增殖及其與上皮-間質(zhì)轉(zhuǎn)化關(guān)系的研究[D];福建醫(yī)科大學(xué);2013年

8 李丹華;ZEB1對(duì)上皮間質(zhì)轉(zhuǎn)化及ESRP1表達(dá)的調(diào)控機(jī)制研究[D];天津醫(yī)科大學(xué);2013年

9 Madhav Goit;Twist基因?qū)Y(jié)腸癌細(xì)胞系侵襲轉(zhuǎn)移影響的體外實(shí)驗(yàn)[D];天津醫(yī)科大學(xué);2013年

10 陳思睿;HMGA1在甲狀腺癌SW579細(xì)胞中對(duì)Snail表達(dá)的調(diào)控作用及機(jī)制的初步研究[D];南華大學(xué);2013年

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本文編號(hào)：2009037