【作者】 陈雪红；

【导师】 耿美玉； 王春波；

【作者基本信息】 中国海洋大学 ， 药物化学， 2010， 博士

【摘要】 肝细胞癌(Heptocellular carcinoma,HCC)是发病率最高的原发性肝脏恶性肿瘤。尽管目前对肝癌可以采取手术切除、动脉插管化疗、放疗及局部治疗等方法,但效果欠佳,人们一直在寻找一种有效治疗肝癌的措施。恶性肿瘤的形成与肿瘤细胞增殖、凋亡调控信号失衡有关,诱导肝癌细胞凋亡是治疗肝癌的重要策略。此外,肝癌血供非常丰富,血管生成对肿瘤的生长、侵袭和转移都有决定性的作用,而且在发病早期就有血管侵犯和肝内转移的倾向,这是肝癌预后差的重要原因。诱导肝癌细胞凋亡和抑制肝癌新生血管形成将是治疗肝癌的重要途径。海生素是自海洋贝类毛蚶(Tegillarca granosa Linnaeus)中提取出的抗肿瘤活性物质,在课题组以往的研究中得知海生素对白血病细胞K562、肺癌细胞A549和肾癌细胞OS-RC-2有抑制作用,并能抑制小鼠艾氏腹水瘤。而海生素诱导肝癌细胞的凋亡,以及抑制肝癌新生血管形成则是本次研究的重点。首先,观察海生素对体外肝癌细胞生长的抑制作用,通过对细胞凋亡相关分子、细胞信号转导相关分子的检测探讨其可能的效应机制。以MTT法检测48h不同浓度海生素对人肝癌细胞BEL-7402增殖的OD值,根据OD值计算细胞生长抑制率,实验结果表明海生素能剂量依赖性抑制肝癌细胞BEL-7402的增殖。电镜结果提示海生素能明显诱导BEL-7402细胞凋亡,出现染色质浓缩、碎裂,线粒体空泡化及凋亡小体等凋亡特征性改变。100μg/ml海生素作用48h后,流式细胞仪PI染色凋亡率为29.63±1.09%,能明显诱导细胞凋亡；Hoechst 33258荧光染色法呈现出凋亡核固缩形态,可见典型的凋亡小体。在证明凋亡发生的基础上,进一步进行海生素诱导凋亡的机制研究。RT-PCR和Western Blot检测海生素作用后BEL-7402细胞Fas mRNA和蛋白的表达,随着海生素浓度增加FasmRNA以及蛋白表达量增多。提示海生素诱导BEL-7402凋亡可能和Fas表面受体通路有关。进一步用western blot方法检测其下游信号分子的表达和活化情况,结果表明海生素能引起caspase-8和caspase-3的活化,以及细胞色素C的释放；而用caspase-8抑制剂z-IETD-fmk事先孵育后,caspase-8和caspase-3的活化片段明显减少,表明海生素可能通过Fas通路及线粒体信号通路诱导肝癌细胞BEL-7402的凋亡。其次,由于体外环境的局限性,应用荷瘤裸鼠动物模型进一步研究海生素体内抑瘤效应及其可能的作用机制。用皮下接种BEL-7402细胞的方法建立了裸鼠荷瘤模型,62.5mg/kg、125mg/kg、250mg/kg海生素肠溶胶囊组的抑瘤率分别为50%、58%和60%,相对肿瘤增殖率分别为45%、42%和32%,表明海生素肠溶胶囊对荷人肝癌裸鼠移植瘤BEL-7402的生长有明显的抑制作用。HE结果显示,海生素组的肿瘤组织出现细胞凋亡和坏死现象,电镜结果表明海生素引起移植瘤组织中的肝癌细胞发生凋亡的特征性改变,从而从形态学证实海生素作用后肿瘤细胞发生了凋亡。原位杂交技术检测到移植瘤组织中Fas mRNA的表达在海生素用药后明显增加,免疫组织化学法检测到移植瘤组织中caspase-8、caspase-3蛋白表达增加。进一步证明,海生素通过Fas信号通路诱导BEL-7402细胞凋亡。最后,观察海生素对肝癌新生血管形成的抑制作用。以人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)为研究对象,用MTT实验结果表明海生素抑制HUVEC的增殖活性,并且用Transwell小室法和管腔形成实验观察到海生素能明显抑制HUVEC的迁移和管腔形成,表明海生素能够直接抑制内皮细胞。体内试验选用鸡胚尿囊膜实验,观察到海生素对鸡胚尿囊膜新生血管也有明显的抑制作用。微血管密度(microvessel density,MVD)是反映肿瘤血管形成的比较直观的指标,用CD34分子免疫组织化学法观察到海生素用药组裸鼠移植瘤组织MVD明显减少。与血管形成有关的血管内皮细胞生长因子(VEGF)以及基质金属蛋白酶-2(MMP-2)的免疫组化蛋白表达在海生素用药组都明显降低。提示,海生素不仅能直接抑制血管内皮细胞,还能通过影响VEGF和MMP-2的表达而影响肝癌新生血管的形成。总之,海生素对肝癌的抑制作用是多方面的,不仅通过Fas信号通路诱导肝癌细胞凋亡发挥其抗肿瘤活性,并且可以靶向血管具有抑制肝癌新生血管形成的作用,从而多靶点、多途径抑制肝细胞癌。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. The current treatment options for liver cancer, including liver resection surgery, intra-arterial chemotherapy and radiation therapy, remain disappointing. Scientists are working hard, however, to address this problem.As carcinogenesis is closely related to the control of tumor cell proliferation, and apoptosis, the induction of apoptotic cell death may be the important therapeutic strategy for live cancer treatment. Meanwhile, angiogenesis plays a critical role in tumor growth, invasion and metastasis. Live cancer is rich in blood supply, its high mortality rate is mainly a result of vascular invasion and intra-hepatic metastases occurring during the early stage of cancer formation.Haishengsu, which is extracted from Tegillarca granosa Linnaeus, is an novel antineoplastic agent. Our previous studies showed that Haishengsu can inhibit the growth of human erythroleukemia K562 cell, lung adenocarcinoma epithelial A549 cell line, renal cell carcinoma OS-RC-2 cell, and suppress Ehrlich ascites carcinoma in mice. The aim of the present study is to investigate the apoptotic effect and anti-angiogenesis actions of Haishengsu on hepatocellular carcinoma cells.First, we investigated the inhibitory effect of Haishengsu on the growth of hepatocellular carcinoma cells in vitro and its effect on the apoptosis pathway. MTT metabolism assay showed that Haishengsu treatment for 72h significantly inhibited the human hepatoma BEL-7402 cell proliferation in a dose dependent manner. Electron microscopy analysis was performed to bserve apoptosis of BEL-7402 cells induced by HSS.In these cells, the typical condensed chromatin pulled away from the nuclear envelope and fragmented into several micronuclei.In addition, the nuclear volume decreased markedly and a considerable increase in vacuolisation was observed.FACS analysis showed that treatment with 100μg/ml of Haishengsu for 48h could induce 29.63±1.09% apoptosis. Hoechst 33258 staining also showed that Haishengsu caused condensed nuclei and apoptotic bodies.These data indicated that Haishengsu could induce BEL-7402 cells apoptosis. To reveal its mechanism of apoptosis induction, RT-PCR was performed to test Fas mRNA levels in BEL-7402 cells. Fas mRNA was upregulated by Haishengsu, which indicated that Fas receptor pathway may involve in Haishengsu-induced apoptosis. Western blot assay showed that haishengsu activated caspase-8 and caspase-3.Pretreatment with z-IETD-fmk markedly decreased the activation of caspase-8 and caspase-3.These data suggested that haishengsu may induce BEL-7402 apoptosis via Fas signaling pathway.Next, based on the antiapoptotic effects of Haishengsu, we measured the antitumor activity of haishengsu in null mice inoculated with BEL-7402 cells. Daily feed with 62.5 125 and 250mgkg-1 Haishengsu for 25 days yielded tumor inhibition of about 50%,58% and 60% respectively, relative rate of tumor proliferation was 45%、42% and 32% respectively. This indicated that HSS significantly inhibited tumor growth in vivo.HE staining showed that apoptotic bodies and necrosis appeared in tumor in Haishengsu treated groups.Electron microscopy analysis showed that haishengsu caused the typical apoptotic morphology in tumor. Haishengsu increased Fas mRNA level in tumor, as assessed by in-situ hybridization. Immunohistochemistry assay showed that haishengsu increased caspase-8 and caspase-3 in mice turmor. These results confirmed that haishengsu induced BEL-7402 apoptosis via Fas singnaling pathway.Finally, the antiangiogenic properties of haishengsu were investigated with several in vitro and in vivo models. Possible mechanisms underlying its antiangiogenic activity were also assessed. Haishengsu dose-dependently inhibited proliferation of human umbilical vein endothelial cells (HUVEC).In tube formation and cell migration assays using HUVEC cells, haishengsu significantly inhibited formation of intact tube networks and reduced the number of migratory cells. In the chick chorioallantoic membrane assay, haishengsu reduced new vessel formation compared with the vehicle control. Microvessel density (MVD) in sections of the tumor was reduced after Haishengsu treatment. The levels of VEGF, and MMP-2 were also significantly decreased.These data suggested that Haishengsu could directly inhibit vescular endothelial cells and exert anti-angiogenic effect via downregulation of VEGF and MMP-2.Collectively, Haishengsu could inhibit hepatocellular carcinoma and its effect associated at least in part with Fas singnaling pathway and the antiangiogenesis.更多还原