【作者】 冉娜；

【导师】 杨泽然；

【作者基本信息】 浙江大学 ， 病理学和病理生理学， 2005， 硕士

【摘要】 肿瘤的血管新生(angiogenesiS)在肿瘤的生长、侵袭和转移中起着重要的作用。血管新生是一个复杂的多步骤的过程,包括血管内皮细胞的增殖、血管内皮细胞的趋化、血管内皮细胞在细胞外基质中管样结构的生成。研究证明阻断血管新生过程的一个或多个步骤,就有可能达到抑制肿瘤血管生成的目的。通过抑制肿瘤内的血管新生,就可以抑制肿瘤的生长侵袭和转移。血管内皮细胞的基因较肿瘤细胞要稳定,不易产生耐药性。所以通过抗血管新生来治疗肿瘤已经成为肿瘤治疗的新的研究热点。热疗作为肿瘤的重要治疗手段,正引起人们越来越多的关注。实验研究和临床观察证明热疗联合化疗、放疗和免疫治疗等可以产生明显的协同作用,其在体内抗肿瘤血管新生已有文献报道。一氧化氮(Nitric Oxide,NO)是一种具有多种生理功能的信息分子,参与肿瘤的发生、发展和转移过程。NO在肿瘤生物学方面可能有双重作用。一方面,NO既是巨噬细胞、内皮细胞等杀伤肿瘤细胞的效应分子,可以诱导肿瘤细胞凋亡;另一方面,又可通过促进血管生成,增加血流量等方式促进肿瘤的生长。目前认为NO对肿瘤作用的两面性主要由NO的浓度决定。通过给予外源性的NO供体,则可以干预体内NO水平。NO供体硝酸异山梨酯(Isosorbide dinitrate,ISDN)为血管舒张剂,在临床上广泛用于心血管疾病的治疗,其在体内抗血管新生从而抑制肿瘤生长已有文献报道。然而,热疗联合一氧化氮供体对肿瘤血管形成的抑制是否起到协同作用,机理如何,尚不清楚。本研究通过观察热疗联合一氧化氮供体硝酸异山梨酯,体外条件下对血管内皮细胞增殖、迁移、小管形成和释放一氧化氮量的影响以及诱导其凋亡的效果,以此探索热疗联合硝酸异山梨酯抑制体外血管形成的的疗效及可能机理,为临床寻找有效的以血管为靶点治疗肿瘤的方法提供实验依据。更多还原

【Abstract】 Tumor angiogenesis plays a significant role in the growth, invasion and metastasis of Tumor. Angiogenesis is a complicated and multiple-step process, including proliferation, migration and tubular morphogenesis of vascular endothelial cells. Researchers proved that inhibiting one or more steps in the process of angiogenesis could inhibit angiogenesis. By inhibiting angiogenesis in tumor, it is possible to inhibit the growth, invasion and metastasis of tumor. The genes of endothelial cells are more stable than that of tumor cells, so the endothelial cells are not inclined to developing drug resistance. By inhibiting angiogenesis to cure tumor have been the new object. Hyperthermia is under investigation as a important treatment for tumor. In addition, chemotherapy, irnmunotherapy and radiotherapy combined with hyperthermia may produce synergistic anticancer effects. Recently researchers found that hyperthermia inhibits angiogenesis of tumor in vivo. Nitric oxide(NO) is a multi-faced molecule with double regulatory roles in many areas of biology, moreover, NO is closely related with carcinogenesis, progression and metastasis of tumor. On the one hand, NO can induce apoptosis in tumor cells as effector molecule of macrophage, on the other hand, NO accelerates tumor growth by angiogenesis and increasing blood flow. The complexity of its biological effects can be does-dependent and cell-type specific. Understanding the roles of NO in carcinogenesis will provide important clues for the treatment of tumor by NO-modulating drugs. Nitric Oxide donor (Isosorbide dinitrate, ISDN) is a kind of drugs to relax blood vessel. It is used to cure cardiovascular disease. To date, however, little is known about that the therapeutic effectic effects of hyperthermia combined with ISDN for antiangiogenesis of tumor and its mechanism. Theaim of this experiment is to elucidate the synergistic effects of hyperthermia and ISDN on the bioactivity of vascular endothelial cells(ECV-304) and induction apoptosis in endothelial cells.Methods1. The effct of hyperthermia and ISDN on the angiogenesis in vitroECV-304 cells were routinely cultured in RPMI-1640 culture medium containing 10% FCS, 100U/ml penicillin and 100U/ml streptomycin at 37℃ in a humidified atmosphere of 5% CO2. The ECV-304 cells in this study were incubated with 0.2 mg/ml ISDN(the final concentration), Heating was carried out in a water bath at 43 ℃, which is used clinically on the human body, for 25 min and the control group was given only culture medium. The growth, cytotoxicity migration and formation of tubular morphogenesis of endothelial cells were assayed by microculture tetrzoalium assay(MTT), phase contrast microscope, scratch wound closure assay and tubular morphogenesis test.2. The effect of hyperthermia and ISDN on the NO levels of culture supernatant of endothelialcellsThe ECV-304 cells were treated as above mentioned. NO levels of culture supernatant of endothelial cells were assyed by Griss method.3. The effect of hyperthermia and ISDN on apoptosis in endothelial cellsThe ECV-304 cells were treated as above mentioned. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe ultrastructural changes of cells, and flow cytometry was performed to detect the apoptotic ratio.Results1. The effect of hyperthermia and ISDN on the angiogenesis in vitroStronger inhibitory effects were observed on abilities of growth and migration in three experimental groups than that of control, and hyperthermia combined with ISDN group had better inhibitory effect than other groups (p < 0.01) . The cytotoxicity on ECV-304 cells of hyperthermia and ISDN was more obvious than that three groups. We could see the cytotoxicity clearly: the number of cells was decreased, the detachment of cell happened, and the obvious cells falling off. Hyperthermia and ISDN obviously inhibited the formationof tubular morphogenesis of ECV-304 cells. In positive control, the tubules were integrate and the walls of the tubules were comprised by many layers of ECV-304 cells更多还原