【作者】 龙淼云；

【导师】 郑启昌；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 背景众所周知,恶性肿瘤最重要的生物特性是侵袭和转移,这也是肿瘤病人的主要临床致死原因。据不完全统计,约70%的肿瘤病人在临床确诊时已经存在肿瘤转移。尤其是死亡率占前几位的肿瘤,如肺癌,胃癌,肝癌等,临床病人确诊时,往往已经处于中晚期,通常肿瘤一旦发生转移,常规临床治疗效果不佳,死亡率极高。早在1971年,Folkman首先发现了肿瘤血管形成因子(TAF) ,并认为抑制肿瘤血管生成(angiogenesis)能有效的抑制肿瘤的生长、转移和复发。目前这一观点已经得到广泛的证实。近年的研究表明,实体肿瘤生长至1mm3以上时即需要新生血管形成;否则肿瘤停止生长,静止于休眠状态(dormantstate),瘤体维持在2～3mm3以内,细胞数在107以内;并且肿瘤维持在原位(insitu)达数月或数年,不会发生转移。而一旦肿瘤细胞获得血管生成表型(angiogenicphenotype),便可诱导血管生成,肿瘤建立起新生血管网,从而促进肿瘤快速生长,其转移潜能也迅即出现。因此,抗血管生成,切断肿瘤恶性生长的供养途径,并阻断肿瘤侵袭转移的血行通道,致使肿瘤静止于休眠状态,可有效遏制肿瘤的恶性行为,是治疗肿瘤的有效策略。目前,血管生成抑制剂的研究主要有如下4种策略:(1)阻断内皮细胞降解周围基质的能力;(2)直接抑制内皮细胞的功能;(3)阻断血管生成因子的合成和释放,拮抗其作用;(4)阻断内皮细胞表面整合素的作用。arresten是Colorado等发现的一种强效的血管生成抑制因子,它属于Ⅳ型胶原α1链的羧基末端NC1结构域多肽片段,其分子量为26kd。研究发现,arresten抑制血管生成的作用显著强于内皮素,而且其蛋白结构稳定,因此有望成为肿瘤血管生成抑制剂。本课题组在先前的研究中,对原核表达的arresten抑制血管生成作用进行了一些探讨。原核表达虽产量高,但产物的重复性差、活性低,而中国仓鼠卵巢细胞(CHO细胞)是目前常用的真核表达体系,它可较长时间地表达所携带的基因而不衰减,并能稳定地大量表达,其表达产生的蛋白质具有糖基化、磷酸化等修饰,具有良好的生物活性,因此被广泛用于蛋白表达和疫苗生产。本研究通过CHO细胞株真核表达并纯化arresten蛋白,探讨其抑制血管生成的分子生物学机制,并观察arresten基因转染对结肠癌肝转移的抑制作用。目的建立arresten的真核表达体系,获得纯化的arresten蛋白;观察arresten对人脐静脉内皮细胞增殖、迁移及血管腔形成的影响,了解其对内皮细胞与肿瘤细胞黏附的影响;并探讨其相关的分子生物学机制;观察arresten基因转染对裸鼠实验性结肠癌肝转移的抑制效果,探讨arresten在肿瘤转移治疗中的应用前景。方法用脂质体Lipofectamine2000通过基因转染将arresten基因导入CHO细胞,建立arresten蛋白的真核表达体系,经过纯化获得arresten蛋白;观察arresten对huvec细胞增殖、迁移、细胞黏附和管腔形成的影响;应用RT-PCR、Western-blot等实验方法,检测VEGF和VCAM-1的表达;通过基因转染将arresten基因导入结肠癌LOVO细胞株,建立裸鼠实验性结肠癌肝转移的模型,观察arresten基因转染对肿瘤转移的抑制作用,FⅧRag多克隆抗体染色的免疫组化方法检测肿瘤组织的微血管密度(MVD),进一步证实arresten抑制肿瘤转移的作用途径。结果RT-PCR及Western-blot证实arresten成功导入CHO细胞并在mRNA及蛋白水平高表达,表明成功建立arresten的真核表达体系,经过纯化得到arresten蛋白。体外实验表明,arresten能抑制内皮细胞的增殖和迁移,进而抑制血管腔形成,而其相关作用机制可能与arresten降低huvec细胞的VEGF表达有关。arresten还能通过抑制内皮细胞VCAM-1的表达进而抑制内皮细胞与肿瘤细胞的黏附。体内实验表明,arresten基因转染能抑制实验性裸鼠结肠癌肝转移,抑制肿瘤血管生成是其作用途径。结论arresten能抑制血管生成,并能抑制细胞黏附,此两种方式可能是其抑制肿瘤转移的作用途径;arresten作用机制与其降低内皮细胞VEGF和VCAM-1表达有关。Arresten能抑制肿瘤转移,有望成为有效抑制肿瘤转移的生物制剂。更多还原

【Abstract】 Background The process of cancer metastasis consists of a series of sequential steps, all of which must be successfully completed to give rise to new metastatic foci.Folkman noted in the 1970s that blood supply was necessary for tumor growth、metastasis and . Tumors will stop growing or die when it exceeds 2 to 3 mm in diameter if new blood vessels are not formed. Numerous studies have also shown that inhibition of tumor growth could be achieved by inhibiting angiogenesis. Moreover, genes for endothelial cells, targeted by antiangiogenic factors, have stable genetic properties and a low mutation rate. As a result, acquired drug resistance to antiangiogenic factors rarely developed during antiangiogenic therapy. These results point to anti-angiogenesis as being one of the most effective and promising ways to cure and control the development of tumors in the future.The development of new blood vessels from preexisting ones is generally referred to as angiogenesis. In the adult, new blood vessels arise via angiogenesis,a process critical foe normal physiological events such as wound repair, the menstrual cycle, and endometrium remodeling. In the last decades, considerable research has been conducted documenting that tumor growth and metastasis repair angiogenesis. This process is pivotal to the survival and subsequent growth of solid tumors beyond a few cubic millimeters in size. Vascular basement membrane constitutes an insoluble structural wall of newly formed capillaries and undergoes several changes during tumor-induced angiogenesis. Initially, the membrane is degraded and disassembled but is finally reorganized to a native state around a newly formed capillary. Such vascular matrix changes during angiogenesis are associated with the expression of matrix proteins that can interact with vascular endothelium and provide endogenous angiogenic and anti- angiogenic signals. Basement membranes are composed of macromolecules such as typeⅣcollagen, laminin, HSPG, fibronectin, and entactin. typeⅣcollagen is composed of six genetically distinct gene products, namely,α1-α6.theα1 andα2 isoforms are ubiquitously present in human basement membranes. The other four isoforms exhibit restricted distributions. TypeⅣcollagen promotes cell adhesion, migration, differentiation, and growth. It is thought to play a crucial role in endothelial cell proliferation and behavior during the angiogenic process. Several studies have shown the anti- angiogenic properties with inhibitoes of collagen metabolism, supporting the notion that basement membrane collagen synthesis and deposition are crucial for blood vessel formation and survival. Additionally, the COOH-terminal globular NC1 domain of typeⅣcollagen is speculated to play an important role in the assembly of typeⅣcollagen suprastructure, basement membrane organization, and modulation of cell behavior. Recently, the NC1 domain ofα2 chain of typeⅣcollagen (canstatin) was identified as an angiogenesis inhibitor. In the present study, we demonstrate the pivotal role of arresten, the NC1 domain ofα1 chain of typeⅣcollagen, in modulating the function of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angiogenesis and tumor growth..In this report, we identify an important anti-angiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of theα1 chain of typeⅣcollagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and CHO cells. We demonstrate that arresten functions as an anti- angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neo-vascularization. Arresten inhibits the development of tumor metastases in nude mice. Additionally, we show that the anti-angiogenic activity of arresten is protentially mediated via mechanisms involve cell surface proteoglycans and theα1β1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.Object To evaluate the specifically inhibition tumor metastases of arresten anti- angiogenic activity in huvec and nude mice and its mechanisms.Methods By gene transfected way, pSecTag2-arresten was transfected into CHO cell line ,the expression of arresten was measure by PCR、Western-blot, now arresten protein was determinated .Then the result of arresten inhibited of huvec proliferation, migration, tube formation, and Matrigel neo-vascularization was searched by MTT、cell migration and anchor to three-dimensional vascular structures. The correlation mechanism of arresten molecule biology was discussed by PCR、Western-blot. Moreover the inhibition of arresten of LOVO cell adhesive huvec was probed. Then in the nude mice pSecTag2-arresten was transfected into human colonic cancer cell line LOVO using Lipofectamine 2000.RT-PCR and Western-blot method was used to examine the expression of arresten mRNA and arresten protein, the biological behaviors of genetically modified LOVO cell clone was further investigated with MTT. At last, the LOVO cell expressing arresten were implanted into nude mice to investigate the prevention of hepatic metastasis of colon cancer, and the weights of tumor were recorded and analyzed,in the cancer ,MVD was detected by immunohistochemistry.Result Arresten gene was expressed successfully in transfected CHO cell, and the positive CHO cell clones expressing human arresten gene stable obtained, it can offer arresten protein. Arresten can inhibit huvec proliferation, migration, tube formation, and Matrigel neo-vascularization by inhibiting the expression of VEGF. Moreover it can inhibit the adhesive of LOVO cell to huvec by inhibiting expression of VCAM-1. The LOVO cell line with the character of expression of arresten was obtained, the biological behaviors of it was not modified. Both the MVD and the growth of colonic cancer with hepatic metastasis in nude mice of LOVO cell transfected arresten gene were suppressed.Conclusion Arresten can inhibit angiogenesis and adhesive of tumor cell to huvec, by these ways it can inhibit tumor metastasis. It is hoped to be a new therapeutic agent for treating the metastasis of tumor.更多还原