【作者】 金涛；

【导师】 彭昊；

【作者基本信息】 武汉大学 ， 骨外科学， 2016， 博士

【摘要】 背景：骨肉瘤是最常见的原发性恶性骨肿瘤,青少年多发,青少年的发病率可达8-11/100万。上个世纪80年代之前,骨肉瘤患者5年生存率大约为20%,应用化疗可以大幅度降低患者肺部转移率,患者5年生存率可提高到60%～80%,近20年以来,虽然全球范围内众多研究机构进行了大量的研究,但是骨肉瘤患者的生存率仍然无明显提高,且目前用于骨肉瘤的化疗药物毒副作用很大,因此,开发新的抗骨肉瘤的化疗药物具有非常重要的意义。免疫原性细胞死亡(ICD)指理化因素在诱导肿瘤细胞凋亡的同时,肿瘤细胞由不具备免疫原性转变为具有免疫原性,并由此激活特异性T淋巴细胞,诱发机体产生特异性细胞免疫应答,达到清除肿瘤的目的。免疫原性细胞死亡伴有一系列特征性的分子事件发生,包括钙网蛋白(CRT)从细胞质转位到细胞膜表面,细胞外ATP的释放及高迁移率族蛋白1(HMGB1)的分泌。到目前为止,只有少数化疗药物可以诱导肿瘤细胞发生免疫原性细胞死亡,而且国内外还没有关于诱导骨肉瘤细胞免疫原性死亡的报道。目的：研究辣椒素与顺铂对骨肉瘤细胞的生长抑制及凋亡作用,在药物诱导细胞凋亡的基础上,进一步探讨辣椒素与顺铂能否诱导人骨肉瘤细胞免疫原性死亡,为开发新的抗骨肉瘤化疗药物奠定基础。方法：第一部分：分别用辣椒素和顺铂作用于人骨肉瘤细胞MG-6324小时后用MTT检测辣椒素和顺铂对MG-63细胞的生长抑制作用,确定半数致死量,接下来分别用DNA片段化分析、线粒体膜电位及western blot等方法分析辣椒素和顺铂能否诱导MG-63细胞凋亡。第二部分：用辣椒素和顺铂作用于MG-63细胞12及24小时后,流式细胞术检测细胞膜上CRT表达量的变化,荧光素酶法检测细胞外ATP的释放,ELISA检测细胞上清中HMGB1的分泌。进一步将辣椒素或顺铂作用后的MG-63细胞分别与人外周血单核细胞诱导的树突状细胞(DC)一起孵育,然后检测DC细胞对辣椒素或顺铂作用后的MG-63细胞的吞噬效率,最后检测其诱导T淋巴细胞分泌细胞因子IL-4及IFN-γ的能力,由此判断辣椒素能否诱导骨肉瘤细胞发生免疫原性细胞死亡。结果：第一部分：MTT结果显示,辣椒素及顺铂作用MG-63细胞24小时后,能抑制MG-63细胞增殖,且呈剂量依赖性。DNA片段化分析、线粒体膜电位的检测及Western blot等分析结果显示,辣椒素和顺铂均可诱导MG-63细胞凋亡。第二部分：MG-63细胞被辣椒素处理后,在凋亡早期,CRT由内质网转移到细胞膜表面；在凋亡晚期,细胞外ATP及HMGB1的释放增加。而顺铂处理MG-63细胞后,不能诱导CRT的外翻、细胞外ATP分泌及HMGB1的释放。且辣椒素处理的MG-63细胞更易于被DC细胞吞噬,将T淋巴细胞与经辣椒素处理肿瘤细胞所致敏的DC细胞共同孵育后,T淋巴细胞分泌IFN-γ的能力明显增强。结论：辣椒素可作为抗肿瘤制剂诱导人骨肉瘤细胞凋亡,且促进骨肉瘤细胞的免疫原性转化,使辣椒素作用的骨肉瘤细胞更易于被DC细胞所摄取,由此介导骨肉瘤细胞的免疫原性死亡。更多还原

【Abstract】 Background:Osteosarcoma, which is the most common primary malignant bone tumor, occurs most frequently in adolescent. The incidence of adolescent is 8-11 cases/million population/year. Prior to the 1980s, the relative 5-year survival rate of patients with osteosarcoma was approximately 20%. With the introduction of chemotherapy into the multi-modal treatment for osteosarcoma, the survival rate is greatly improved to approximately 60%～80%. However, improvements in osteosarcoma survival during the last decade have been limited and the side effect of osteosarcoma chemotherapy drug is very serious. Clearly, the development of new osteosarcoma chemotherapy drugs has very important significance.Immunogenic cell death (ICD) of tumor cells means a cell death modality, which is not only able to cause cell apoptosis but also stimulate response against homologous tumor cells. ICD is characterized by a series of alterations that usually do not occur in the context of apoptosis: (i) the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, (ii) release of ATP during the blebbing phase of apoptosis, and (iii) post-apoptotic exodus of the chromatinbinding protein high mobility group B1 (HMGB1). Up to now only a few kinds of anti-cancer chemicals were found to induce ICD, so it has important clinical significance to explore the new chemicals that can induce ICD of osteosarcoma.Objective:The purpose of this research is to study the growth inhibition and apoptosis effect of capsaicin and cisplatin on osteosarcoma cells, and then study the mechanism of actions, further investigate whether capsaicin and cisplatin can induce osteosarcoma cells immunogenic cell death or not. This lay a good foundation for the development of new osteosarcoma chemotherapy drugs.Methods:Part Ⅰ:In this study, MTT assay was used to examine the growth inhibiting effects of MG-63 cells when they were treated by capsaicin or cisplatin. DNA ladder, Mitochondrial membrane potential (MMP) and western blot analysis were used to investigate the capsaicin- and cisplatin- induced apoptotosis.Part Ⅱ:Capsaicin and cisplatin acted on the MG-63 cells 12 or 24 hours later, flow cytometry was used to detect the expression of CRT on the cell membrane, fluorescein enzymatic method was used to detect the release of ATP, and ELISA was used to detect the secretion of HMGB1. After co-incubation of capsaicin or cisplatin treated MG-63 cells with DC cells, the phagocytic efficiency of the phagocytes on MG-63 cells was detected and induced T cell’s ability in secreting IL-4 and IFN-y was analyzed. These data can used to identify whether capsaicin induce ICD in human osteosarcoma cells or not.Results:Part Ⅰ:MTT results showed that 200μM capsaicin and 32μg/ml cisplatin can prevent 50% of MG-63 cells proliferation. DNA ladder, Mitochondrial membrane potential (MMP) and western blot analysis were showed that capsaicin and cisplatin can induce MG-63 cells apoptotosis and by Bcl-2 pathway.Part Ⅱ:In early apoptosis, capsaicin induced translocation of CRT from endoplasmic reticulum to the cell surface. In late apoptosis, capsaicin induced secretion of extracellular ATP and HMGB1. Capsaicin-treatment increased phagocytosis of MG-63 cells by dendritic cells (DCs) and these MG-63-loaded DCs could efficiently upregulate the secretion of IFN-y in the lymphocytes.Conclusion: These data demonstrate that capsaicin can induce apoptosis and immunogenic cell death in osteosarcoma cells.更多还原