【作者】 郑瑾；

【导师】 刘文超；

【作者基本信息】 第四军医大学 ， 肿瘤学， 2010， 博士

【摘要】 研究背景上皮细胞与内皮细胞、成纤维细胞、神经内分泌细胞、炎性细胞、细胞外基质等构成组织微环境,细胞间的相互作用可调控细胞的增值、分化、凋亡、分泌活化的可溶性因子及细胞外基质组份,这些组份的协调使细胞和组织达到动态平衡。但是在肿瘤微环境中[1],这些细胞可以被肿瘤细胞诱导,在其周围产生大量的生长因子、细胞趋化因子、基质降解酶等,从而有利于肿瘤细胞的增殖和侵袭。肝星型细胞(Hepatic stellate cell HSC)是重要的肝基质细胞,肝癌细胞可诱导HSC活化,而活化的HSC又可促进肝细胞癌(Hepatic carcinoma HCC)的浸润和转移。本研究拟了解肝癌细胞与HSC的相互作用对HCC转移的影响。NDRG2属于NDRG家族,被定义为抑癌候选基因的它可在许多正常组织中表达。最近的许多研究表明NDRG2除了能抑制肿瘤增值,增加细胞凋亡外,还能抑制肿瘤转移。有研究表明NDRG2表达缺失可引起HCC具有高转移潜力和不良预后,针对100例肝癌患者的免疫组化提示NDRG2降低与淋巴结转移、肿瘤分化程度、门静脉栓塞、肿瘤浸润生长方式及肿瘤复发相关,将肝癌细胞系转染NDRG2后可明显抑制其浸润及转移能力。NDRG2过表达可抑制由TGF-β1引起的肝癌细胞MMP-2及PAI-1表达的增加,从而抑制肿瘤转移。乳腺癌细胞系中过表达NDRG2可诱导BMP4抑制MMP9表达,从而使乳腺癌细胞侵袭及转移能力下降。另外在侵袭性脑膜瘤、黑色素瘤和大肠癌鼠模型中亦发现NDRG2能够抑制肿瘤转移。HCC引起高复发、高转移的特性一直是肿瘤治疗的难点,NDRG2在其转移中的机制研究十分重要。已有文献提示NDRG2与MMPs间具有一定相关性,这引起我们对NDRG2是否参与了分泌MMPs的重要间质细胞HSC活化的兴趣。CD44是细胞膜上广泛表达的糖蛋白,在细胞与细胞、细胞与基质间作用、淋巴细胞归巢以及肿瘤转移中起重要作用。CD44与透明质酸(hyaluronic acid HA)结合可调节肿瘤细胞的运动信号。通过有效手段抑制CD44可降低肿瘤细胞的侵袭及转移能力。CD24是与P选择素结合的粘附分子与肿瘤生长和转移相关,CD24通过活化alpha3beta1和alpha4beta1整合素促进肿瘤细胞增值及增强对纤连蛋白、I和IV型胶原、层粘连蛋白的粘附,CD24表达会促进细胞快速播散、运动及侵袭。同时HSC活化也会引起粘附分子的表达变化,所以我们也想进一步明确NDRG2是否参与对肝癌细胞及HSC的粘附分子表达调控。目的和内容:研究NDRG2与HCC转移相关粘附分子间的作用机制,研究NDRG2是否参与HSC的相关活化基因调节和对粘附分子的表达调控,进一步了解NDRG2是否也参与了HSC的活化与肝癌细胞间的相互作用及对肝癌转移的影响。方法:1)运用RT-PCR及Western blot方法检测不同转移能力肝癌细胞系的NDRG2表达;通过划痕、侵袭、粘附实验观察不同转移能力细胞系的迁移、侵袭、粘附能力;运用NDRG2基因的重组腺病毒及siRNA感染和转染肝癌细胞系使NDRG2过表达或下调后,观察细胞迁移、侵袭、粘附能力的改变。2)运用NDRG2基因的重组腺病毒及siRNA感染和转染肝癌细胞后,通过Real Time PCR观察转移相关粘附分子的改变,进一步通过Weatern blot检测蛋白水平的改变。3)继续运用NDRG2基因的重组腺病毒及siRNA感染和转染肝星形细胞系LX2后,通过Real Time PCR观察HSC活化相关分子的改变,进一步通过Weatern blot检测蛋白水平的改变。4)建立体外肝癌细胞与HSC的胶内共培养模型和裸鼠荷瘤三维共培养模型,给予NDRG2干扰后,通过HE染色及免疫组化等实验技术观察两细胞间的粘附、趋化性及相应基因的改变。结果:1)NDRG2随肝癌细胞系转移能力的增强而减弱,细胞迁移、侵袭、粘附能力随NDRG2表达增高而减弱,进一步过表达NDRG2可观察到细胞迁移、侵袭、粘附能力的减弱,下调NDRG2可观察到细胞迁移、侵袭、粘附能力的增强。2)过表达NDRG2可观察到粘附分子CD44、CD24在mRNA及蛋白水平的表达减弱;下调NDRG2可观察到粘附分子CD44、CD24在mRNA及蛋白水平的增强;在肝癌患者癌组织病理切片免疫组化也提示NDRG2减低及CD44和CD24表达增高。3)HSC中NDRG2过表达后,HSC活化相关基因α-SMA、CD44在mRNA及蛋白水平表达减弱,同时细胞的侵袭能力减弱;下调HSC上的NDRG2表达可使α-SMA、CD44表达增加。4)通过肝癌细胞和HSC的共培养模型,发现肝癌细胞过表达NDRG2后减弱了它们对HSC的趋化、粘附;下调肝癌细胞上的NDRG2增加了它们对HSC的趋化、粘附。过表达HSC上的NDRG2使得α-SMA、CD44表达减弱;同时发现过表达NDRG2可使TGF-β1作用HSC后增加的CD44的配体透明质酸含量降低。结论:在HCC微环境中NDRG2具有重要的调节作用,NDRG2的过表达和下调会引起肝癌细胞上CD44、CD24表达的相应减弱及增强,会引起HSC活化基因α-SMA、CD44等的相应减弱及增强。NDRG2过表达可减弱TGF-β1诱导的细胞透明质酸的增加。进一步影响两种细胞间的粘附、趋化作用而调控转移。更多还原

【Abstract】 Background The interaction of epithelial and endothelium, mesenchymal basal cells, fibroblasts, neuroendocrine cells, smooth muscle cells, inflammatory cells and extracellular matrix (ECM) which composes microenvironment regulates cells activities as proliferation, differentiation, and apoptosis as well as secretion and activation of soluble factors and/or deposition of extracellular matrix (ECM) components. The balance of these components results the homeostasis of cells and tissues. But, in tumor microenvironment, the increase of matrix metalloproteinase, growth factors, chemotactic factors induced by many cells promotes cancer progression, metastasis. Our study focused on the interaction of hepatoma carcinoma and stellate cells in tumor microenvironment. Hepatoma carcinoma cells can induce the activation of hepatoma stellate cells which can promote the invasion and metastasis of HCC.NDRG2 belongs to the NDRG (N-myc downstream-regulated genes) family where it has been reported to function as a tumor suppressor gene. NDRG2, a candidate of tumor suppressor gene (TSG), is expressed in many normal tissues. The aggressive cancers have Low and undetectable levels of NDRG2, along with the poor prognosis of cancer patients. NDRG2 is a gene inhibiting proliferation of tumors can enhance apoptosis of some tumor cell lines. Immunohistochemistry showed that NDRG2 expression was reduced at a late stag. This provoked the attention of the connection of metastasis and NDRG2. In 100 HCC patient tissues, Immunohistochemical analysis of NDRG2 protein indicated that loss of NDRG2 expression is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis stage, differentiation grade, portal vein thrombi, infiltrative growth pattern, nodal/distant metastasis, and recurrent tumor, as well as shorter patient survival rates. NDRG2 could antagonize transforming growth factorβ1–mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and PAI-1, with concomitant suppression of Rho GTPase activity. It has been demonstrated that loss of NDRG2 expression in hepatocellular carcinomas is significantly correlated with aggressiveness and NDRG2 expression contributes to the suppression of metastatic potential in Hepatic Carcinoma cells. In our study, we want to confirm that low expression levels of NDRG2 increased invasion, metastasis, adhesion ability of hepatic cacinoma cells.NDRG2 significantly suppresses tumor invasion by inhibiting MMPs activities. NDRG2 playing an major role in suppressing HCC metastasis can inhibit extracellular matrix– based, Rho-driven tumor cell invasion and migration. BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity. Our study also suggested loss of NDRG2 expression in HCC is correlated with the increase of metastatic potential. NDRG2 expression will be a useful and functionally relevant biomarker to predict aggressive behavior in patients with meningioma、colorectal carcinogenesis and melanoma. It is urgent to study NDRG2 in metastasis of HCC.CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. CD44, a hyaluronic acid receptor, can promote or inhibit motogenic signaling in tumor cells. It may be possible to suppress CD44 by effective means, and thus potentially cause a reduction in invasive capability and metastasis.CD24, an adhesion molecule for P-selectin, was related with tumor growth and metastasis. CD24 expression increased tumor cell proliferation, adhesion to fibronectin, collagens I and IV, and laminin through the activation of alpha3beta1 and alpha4beta1 integrin activity. Moreover, CD24 expression supported rapid cell spreading and strongly induced cell motility and invasion. We want to know if NDRG2 can control tne expression of CD44 and CD24.Objecyives Studies indicated that NDRG2 have relationship with MMPs, PAI-1 and integrins. There isn’t a study revealed the relationship between NDRG2 and adhesion molecules influencing metastasis of HCC. NDRG2 influences not only tumorigenesis, progression, metastasis of cancer cells but also differentiation, stress of cells. So, we want to know if NDRG2 effects the activation of HSC. If NDRG2 participates gene regulation of HSC activation.Methods 1) hepatic cacinama cells have defferent metastasis ability. The NDRG2 expression of hepatic cacinama cells were detected by RT-PCR and Western blot. We also detected invasion, metastasis, adhesion ability of defferent hepatic cacinoma cells. Subsequently, we investigated the change of invasion, metastasis, adhesion ability of defferent hepatic cacinama cells using Ad-NDRG2, siRNA-NDRG2 to overexpress, knockdown NDRG2 expression.2) We investigated the adhesion molecules influencing metastasis of hepatic cacinoma cells infected by Ad-NDRG2, transfected by NDRG2 siRNA by Real Time PCR and Western blot.3) We investigated molecules having relationship with HSC activation of HSC infected by Ad-NDRG2, transfected by NDRG2 siRNA by Real Time PCR and Western blot.4) This study investigated whether NDRG2 contributes to the tumor microenviroment. A model of inoculation of tumor cells and activated HSC of mixing glum (including Fibxinogen, Thrombin and Aprotinin) and (nu/nu) mice were created. Changing NDRG2 by Ad-NDRG2 and siRNA-NDRG2, we investigated the ashesion, chemotaxis and gene regulation of two kinds of cells.Results 1) hepatic cacinoma cells have defferent metastasis ability. Decrease of NDRG2 expression was compared with increase of invasion, metastasis and adhesion ability by RT-PCR and Western blot. Invasion, metastasis and adhesion ability decreased when the defferent hepatic cacinoma cells were infected by Ad-NDRG2. Invasion, metastasis and adhesion ability increased when the different hepatic cacinoma cells were tansfected by siRNA-NDRG2. Overexpression NDRG2 suppressed adhesion, invasion and migration of different invasive cell line, whereas small interfering RNA-mediated knockdown resulted in increased invasion and migration of different cell lines.2)CD44 and CD24 mRNA and protein decreased when the hepatic cacinoma cells were infected by Ad-NDRG2. CD44 and CD24 mRNA and protein increased when the hepatic cacinoma cells were tansfected by siRNA-NDRG2 by Real Time PCR and Western blot. The down-regulation of NDRG2 and increase of CD44, CD24 expression in the HCC cases were detected by immunohistochemisty.3)α-SMA、CD44 are activation marker of HSC.α-SMA、CD44 mRNA and protein decreased when the HSC were infected by Ad-NDRG2. At the same time, invasion ability was reduced.α-SMA、CD44 mRNA and protein increased when the hepatic stellate cells were infected by Ad-NDRG2. At the same time, the invasion ability was raised by siRNA-NDRG24)We investigated that overexpression or knockdown NDRG2 of hepatic cacinoma cells decreased or increased their chemotaxis and adhesion to HSC comparing with reduction or augmentation ofα-SMA and CD44. At the same time, the result indicated that overexpression of NDRG2 could reduce hyaluronic acid(HA) induced by TGF-β1.Conclusion As NDRG2 is reported to be a candidate tumor-suppressor gene in a wide variety of cancers, NDRG2 has effect on tumor microenviroment. Our study investigated that the decrease of NDRG2 raised the expression of CD44 and CD24 on hepatic cacinoma cells. NDRG2 Overexpression could decrease CD44 and CD24 on hepatic cacinoma cells. On HSC, reduction or augmentation ofα-SMA and CD44 were detected by NDRG2 Overexpression or knockdown. NDRG2 overexpression could reduce HA induced by TGF-β1 too. These results suggest that NDRG2 can inhibit hepatic cacinoma cells, activated HSC adhesion, invasion, migration and thereby play important role in suppressing HCC metastasis.更多还原