【作者】 李伟栩；

【导师】 杨迪生；

【作者基本信息】 浙江大学 ， 骨科学， 2010， 博士

【摘要】 骨肉瘤是儿童及青少年最常见的骨的原发性恶性肿瘤,其发生率为每百万人口中4至5例,各种族间无差异。目前临床治疗骨肉瘤的化疗药物仍以阿霉素(ADM)、顺铂(DDP)、和大剂量的甲氨蝶岭(MTX)为主,其他还有近年来应用的异环磷酰胺(IFO)等。虽然化疗明显改善骨肉瘤患者的预后,并且近年来骨肉瘤的治疗取得了一定进展。但近二十多年来骨肉瘤患者的5年生存率仍徘徊在60%上下。在过去的20年中,新的化疗药层出不穷,如肺癌在新药物的使用下疗效明显改善。但是少有发现抗骨肉瘤的新药。仅Navid F发现联合应用吉西他滨和紫杉萜治疗骨与软组织肉瘤转移患者,有良好的抗肿瘤作用及耐受性。因此,开发新的高效低毒的抗骨肉瘤药物或新的化疗增效药,是目前骨肉瘤治疗中的重要课题。小檗胺(Berbamine)是从小檗属植物的根茎中提取的一种药物。近年来,研究发现小檗胺对肝癌细胞(SMMC7721).宫颈癌细胞(Hela).白血病细胞(K562和HL-60)等都有抑制增殖作用,但是小檗胺对骨肉瘤作用的研究还未见报道。为近一步提高抗肿瘤作用,通过结构修饰化学合成了新型小檗胺衍生物BBD24。因此,本论文以小檗胺衍生物BBD24为切入点,旨在研究小檗胺衍生物BBD24体内外抗骨肉瘤的作用及其机制。MTT实验结果表明,BBD24能显著抑制骨肉瘤细胞系MNNG/HOS和MG63的生长,并且呈剂量依赖性和时间依赖性。进一步研究了BBD24对骨肉瘤细胞增殖抑制的分子机制发现,可以诱导骨肉瘤细胞发生凋亡、坏死和自噬。为了明确小檗胺类化合物在细胞内的分布,我们应用生物素标记的小檗胺和免疫荧光技术对小檗胺在人MNNG/HOS骨肉瘤细胞内的分布进行动态示踪和观察后发现,随着作用时间的延长,小檗胺的分布逐渐向细胞核周边的胞浆集中,并且48小时在细胞核内也可见红色荧光,提示小檗胺有可能进入细胞核内,发挥抗肿瘤活性。进一步对增殖相关的多个信号通路的研究发现,BBD24下调人骨肉瘤MNNG/HOS细胞Wnt/p-catenin信号通路和NF-κB信号通路。对BBD24和临床常用的骨肉瘤化疗药物顺铂联合作用骨肉瘤细胞系MNNG/HOS发现,二者具有协同作用。本实验收集了8例骨肉瘤患者原代标本,通过MTT测定后发现,小檗胺衍生物BBD24可以有效杀伤原代骨肉瘤细胞,且呈现明显的剂量依赖性,IC50基本在1.0-2.0μg／ml。我们同时收集了4例正常人外周血单个核细胞标本,BBD24对正常细胞的毒性实验提示IC50基本在8.0-10.0μg／ml,明显低于骨肉瘤患者原代细胞。小檗胺衍生物BBD24对裸鼠骨肉瘤移植模型的作用实验,以3×106／0.2ml细胞浓度接种于背部肩胛附近皮下,造模成功率为100%,接种24h后开始给药,给药时间及剂量为小檗胺衍生物BBD24 10mg／kg,灌胃,一天两次,连续给药20天。结果显示,BBD24可以显著抑制MNNG/HOS移植瘤,且对BALB/c-nu/nu裸鼠没有任何可见的毒副反应。本研究表明小檗胺衍生物BBD24具有显著地体内外抗骨肉瘤作用,提示其具有一定的临床应用价值和开发前景。更多还原

【Abstract】 Osteosarcoma is one of the most common types of primary bone tumors for children and adolescents with the incidence of 4-5*10-6 and no difference among various ethnic groups. Current common chemotherapeutic agents for osteosarcoma are adoxorubicin (ADM), cisplatin (DDP), methotrexate (MTX) and the recently-introduced ifosfamide (IFO). Chemotherapy improves outcome of patients with osteosarcoma and to some extent regimens of osteosarcoma have been progressing recently. However, the 5-year survival rate is about 60% during last 2 decades. An endless stream of new drugs emerged in the past 20 years. Patients with lung cancer, for example,have been greatly relieved by new agents. However, few novel effective candidates for osteosarcoma can used for osteosarcoma treatment, except that the combination of Gemcitabine and Yew terpene displays anti-tumor activity for patients with metastasis of bone and soft tissue sarcomas with good tolerance. So the major concern in osteosarcoma treatment is to develop novel chemotherapy agents with more potent anti-tumor activity and low toxicity.Recent studies showed that berbamine, a natural product extracted from Berber is plants, inhibited the cell growth of a variety of human tumors such as SMMC7721 (hepatoma), HeLa (cervical cancer) and K562 (chronic myeloid leukemia, CML) and HL-60(acute myeloid leukemia, AML), but little is known about the effects of berbamine and derivatives on osteosarcoma. In this study, we investigated the effect and mechanism of BBD24, a novel berbamine derivative, on osteosarcoma in vivo and in vitro. MTT assay showed that BBD24 remarkably inhibited the growth of cell lines MNNG/HOS and MG63 with time-and dose-dependent manners. Cell cycle analysis showed that treatment of tumor cells with BBD24 resulted in cell arrest at G2/M phase. Interestingly, BBD24 could trigger a variety of cell death pathways including apoptosis, necrosis and autophagy. Subcellular distribution investigation using immunofluorescence assay (IFA) with biotinylated berbamine revealed that berbamine was mainly distributed in cytoplasm at 24 hours, but berbamine was predominantly localizated in nucleus at 48 hours. Studies of tumor-related signaling pathways indicated that berbamine derivative BBD24 activated caspase-dependent pathway, and down-regulated NF-kB, AKT, and ERK pathways. Combined treatments of BBD24 with cisplatin (DDP), a commomly-used chemotherapeutic agent for osteosarcoma, on cell line MNNG/HOS confirmed their synergistic effect.Primary osteosarcoma cells were collected from eight untreated patients and MTT assay showed BBD24 effectively killed these primary cells in a obvious dose-dependent manner with IC50 1.0-2.0μg/ml, meanwhile, PBMCs were also collected from 4 health adults, and MTT assay showed that the IC50 was up to 8.0-10.0μg/ml in general, suggesting that BBD24 selectively inhibit the growth of osteosarcoma cells.In this study, ostersarcoma xenograft model of nude mice (BALB/c-nu/nu) was used to evaluate the anti-tumor activity of BBD24 in vivo.3×106/0.2ml MNNG/HOS cells were injected subcutaneously near the scapular of mice, and osteosarcoma appeared in all experimental animals at 5 days after injection. BBD24 was orally administered 24 hours after being implanted with MNNG/HOS at 10mg/kg bid for 20 days. Experimental results showed that xenografts of MNNG/HOS were significantly inhibited and no obviously toxic or side effects were observed during experiments.Taken together, our study results demonstrate berbamine derivative BBD24 posses a potent antitumor activity against osteosarcoma in vivo and in vitro, suggesting that it might be a potential agent for osteosarcoma, and deserving further investigation.更多还原