ZEB2在肝癌血管生成拟态形成中的作用研究

发布时间：2018-06-12 08:27

本文选题：ZEB2 + 上皮-间充质转化　；参考：《天津医科大学》2015年博士论文

【摘要】：研究背景和目的肝细胞肝癌(hepatocellular carcinoma,HCC)是一种常见原发性恶性肿瘤,具有高转移率和高复发率。尽管近年来肝癌的治疗手段有了明显进展,但治疗效果仍不尽如人意,患者的生存质量、存活时间都没有显著改善。不同于传统的肿瘤血供模式,血管生成拟态(vasculogenic mimicry,VM)是一种独特的肿瘤微循环模式,并且往往关联着肿瘤的侵袭、转移以及不良预后。在VM形成的分子机制中,有较多的观点支持上皮-间充质转化(epithelial-mesenchymal transition,EMT)参与其中,但两者之间的调控机制仍不明确。本研究旨在通过分析EMT转录因子ZEB2在人肝癌中表达和预后价值,初步揭示ZEB2与VM的关系;通过研究细胞因子TGF-β1对体外肝癌细胞的诱导作用,进一步证实EMT参与肝癌VM形成;通过研究ZEB2对细胞功能的影响,探讨ZEB2调控的EMT在肝癌血管生成拟态形成中的可能作用机制。方法1)通过特殊染色方法—CD31/PAS双重染色方法标记肝癌组织中VM管道结构。免疫组织化学染色检测人肝癌组织中ZEB2的表达并分析ZEB2与VM形成、临床病理参数的关系以及预后价值。2)选取不能形成管道样结构的肝癌Hep G2细胞系,用细胞因子TGF-β1诱导培养,观察诱导前后细胞形态和成管能力的变化。3)Western blot检测TGF-β1诱导培养后的肝癌细胞EMT转录因子Twist1、Snail、Slug和ZEB2的表达情况;检测EMT相关分子E-cadherin,Vimentin及内皮相关分子VE-cadherin的表达情况。4)Western blot检测四种肝癌细胞系Hep G2,Bel7402,PLC和SMMC7221中ZEB2的表达情况。5)筛选出低表达和高表达ZEB2的肝癌细胞系,分别上调和下调ZEB2的表达,并观察ZEB2高、低表达对细胞成管能力及内皮相关分子表达的影响。6)Western blot和免疫荧光染色检测ZEB2高、低表达对肝癌细胞EMT相关分子E-cadherin、Vimentin表达的影响。7)细胞划痕和侵袭实验检测ZEB2高、低表达对肝癌细胞迁移和侵袭能力的影响。8)明胶酶谱检测ZEB2高、低表达对肝癌细胞MMP-2和MMP-9活性的影响。结果1)92例人肝癌组织样本中发现17例存在VM,其发生率为18%。免疫组化染色发现ZEB2细胞浆表达53例(57.6%),ZEB2核表达21例(22.8%)。ZEB2核表达VM形成、临床分期和肝癌患者的复发/转移有关(P0.05)。生存分析提示具有ZEB2细胞核表达或者VM阳性的肝癌患者总生存期均较短。多因素分析结果表明ZEB2是肝癌患者总生存期的独立预后因素(P0.05)。2)TGF-β1诱导培养肝癌Hep G2细胞,细胞由典型的上皮样表型转变为间充质样表型并获得了形成管道样结构的能力,能在体外形成VM样结构。3)TGF-β1诱导培养后的Hep G2细胞EMT转录因子ZEB2的表达显著增强,而对Twist1、Snail和Slug的影响不明显;间叶标记分子Vimentin表达增高,而上皮标记分子E-cadherin表达下降;内皮相关分子VE-cadherin表达增高。4)Western blot检测证实在四种肝癌细胞系HepG2,Bel7402,PLC和SMMC7221中,Hep G2细胞低表达ZEB2,Bel7402,PLC和SMMC7221细胞均有较高的ZEB2表达,其中Bel7402细胞表达最高。5)HepG2细胞上调ZEB2表达后在三维培养下能够形成管道样结构并表达内皮相关分子VE-cadherin、Flt-1、Flk-1;Bel7402细胞下调ZEB2表达后形成管道样结构的能力消失并且VE-cadherin、Flt-1、Flk-1的表达显著下降。6)上调ZEB2表达使Hep G2细胞间叶标记分子Vimentin表达增高,而上皮标记分子E-cadherin表达下降;下调ZEB2表达使Bel7402细胞间叶标记分子Vimentin表达下降,而上皮标记分子E-cadherin表达增高。7)上调ZEB2表达能够增强肝癌Hep G2细胞侵袭和迁移能力。下调ZEB2表达降低肝癌Bel7402细胞的侵袭和迁移能力。8)上调ZEB2表达能够增强肝癌Hep G2细胞MMP-2和MMP-9的活性;下调ZEB2表达降低肝癌Bel7402细胞MMP-2和MMP-9的活性。结论1)通过免疫组化及统计学分析证实ZEB2与VM形成有关;ZEB2还与肝癌复发、转移等恶性生物学行为有关,是影响患者生存的独立预后因素。2)体外实验通过TGF-β1诱导培养肝癌细胞,证实其能够通过显著上调ZEB2的表达诱导肝癌细胞发生EMT,并且促进体外形成VM样结构。3)体外实验通过构建ZEB2上、下调细胞模型,证实ZEB2能够增强肝癌细胞的侵袭、迁移性和三维管状结构形成能力。4)体外实验亦证实ZEB2能够增强肝癌细胞MMP-2和MMP-9的活性,上调内皮相关分子VE-cadherin、Flt-1、Flk-1的表达,从而促进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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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