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以疟原虫为载体表达 GPC3 蛋白的肝癌疫苗及其免疫机理的研究
Alternative TitlePlasmodium parasite as an effective hepatocellular carcinoma antigen GPC3 protein delivery vector and its immunological mechanism
刘权
Subtype博士
Thesis Advisor陈小平
2017-05-01
Degree Grantor中国科学院大学
Place of Conferral广州生物院
Degree Name理学博士
Degree Discipline生物化学与分子生物学
KeywordGPC3 载体 疟原虫 肝细胞肝癌 靶向 免疫治疗
Abstract肝细胞肝癌(hepatocellular carcinoma,HCC)是一种全球高发病率和高死亡率的恶性肿瘤,占原发性肝癌的80-90%。传统的治疗方式(包括手术,放疗和化疗)对肝细胞肝癌的治疗效果十分有限。我们之前的研究表明疟原虫免疫Lewis肺癌小鼠后,小鼠肿瘤受到明显抑制,荷瘤小鼠的生存时间得到显著提高,这与疟原虫免疫激活小鼠的天然和获得性免疫相关。近期,利用微生物和单细胞原生动物作为肿瘤抗原表达载体的研究取得很大进步。本实验证实了利用疟原虫作为肝脏肿瘤抗原表达载体的可能,为肝脏肿瘤患者的免疫治疗提供一种新的方法。利用基因重组的方法将鼠磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)表达基因gpc3稳定插入野生型约氏疟原虫基因组中 (wildtypeofPlasmodium yoelii 17XNL,P.y-WT),构建非分泌型表达GPC3蛋白的重组疟原虫单克隆虫株(P.y-GPC3)及分泌型表达GPC3蛋白的疟原虫单克隆虫株(P.y-eGPC3)。使用自身高表达GPC3蛋白的Hepa1-6细胞建立小鼠皮下肝脏肿瘤模型,我们检测了表达GPC3蛋白的疟原虫对肝脏肿瘤的治疗效果。结果表明,经P.y-GPC3免疫后,小鼠肿瘤受到抑瘤的效果明显,荷瘤小鼠生存时间也显著延长,与未免疫的对照组及野生型(P.y-WT)免疫的对照组比较,差异显著。免疫组化检测结果表明,P.y-GPC3免疫小鼠的肿瘤组织中Ki-67表达水平显著低于其他对照组。然而,我们检测到P.y-eGPC3的抑瘤效果仅与P.y-WT的抑瘤效果相当。进一步研究表明疟原虫感染早期能够显著提高小鼠血清中Th1相关细胞因子的浓度,包括TNF-α、 IFN-γ 和 IL-2。同时,疟原虫感染能够提高小鼠脾脏内CD8α+ DC在脾CD11c+ DC中的比例,CD8α+DC较D8α-DC显著高表达CD80及CD86分子。与未免疫对照组和野生型免疫对照比较,P.y-GPC3免疫组荷瘤小鼠的CD8α+DC也显著高表达CD80及D86分子。通过IFN-γ-ELISAPOT方法检测到P.y-GPC3感染能够显著激活针对GPC3蛋白的特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)反应,与各对照组比较,差异具有显著性。我们检测到P.y-eGPC3免疫组小鼠体内产生针对GPC3蛋白的特异性CTL反应仅与野生型疟原虫免疫组小鼠的CTL反应相当,这可能与它们抑制肝脏肿瘤的效果也相当有直接关系。疟原虫子孢子能够天然靶向和感染肝细胞,我们研究了其靶向和感染肝脏肿瘤细胞的能力。体外实验表明,鼠伯氏疟原虫能够感染Hepa1-6与HC-04细胞,也能够感染人宫颈癌Hela细胞。小鼠体内实验表明,鼠伯氏疟原虫并没有靶向和感染由Hepa1-6细胞建立的皮下肝脏肿瘤组织。疟原虫作为肝脏肿瘤抗原的有效表达载体可能为疟原虫免疫疗法治疗肝脏肿瘤病人提供新的方法。考虑到利用疟原虫治疗肿瘤患者可能带来的伦理问题,减毒疟原虫或减毒的疟原虫子孢子值得进一步研究,这样以肝内期和红内期疟原虫为载体的肝脏肿瘤疫苗的前景值得期待。
Other AbstractHepatocellular carcinoma (HCC), which accounts for 80-90% of primary liver cancers, is one of the most common malignancies with the high incidence and mortality in the world. The traditional treatments, including surgery, radiotherapy and chemotherapy, are often ineffective in controlling HCC. Our previous research demonstrated that malaria parasite infection has an anti-tumor effect through induction of innate and adaptive immunity in a mouse Lewis lung cancer model. A great achievement has been made recently in many antigen-based tumor vaccine vectors, including microorganisms and intracellular protozoans. This study aimed to investigate the possibility of using Plasmodium parasite as a novel vaccine vector for HCC immunotherapy, providing a noval tumor therapy for patients with HCC. We constructed two strains of Plasmodium yoelii 17XNLexpressing murine glypican-3 (GPC3) protein (P.y-GPC3) within the parasite or exported GPC3 (P.y-eGPC3) from the parasite by the gene recombination technology, and examined their therapeutic potency in a murine Hepa1-6-induced HCC model that highly expressed GPC3 proteinwhile compared with their wild-type parasite (P.y-WT). Results showed that P.y-GPC3-based vaccination dramatically inhibited hepatoma growth in the implanted HCC and prolonged the survival of HCC-bearing mice. However, P.y-eGPC3-based vaccination only had a limited effect on the tumor growthas that of the P.y-WT vaccination. The prerequisites for invoking a CD8+ T cell response were assessed after P.y-based immunization, which included obviously increased concentrations of T helper cell type 1 (Th1)-associated cytokines, such as TNF-α,IFN-γ and IL-2, in serum and preferential expansion of the CD8α+ dendritic cell (DC) subset with higher expression of CD80 and CD86 molecules in total splenic CD11c+ DCs. Furthermore, we found that P.y-GPC3-vaccinated mice had a higher expression of CD80 and CD86 molecules in CD8α+ DCs than control mice and even P.y-WT-vaccinated mice. Compared with uninfected and wild-type P.y-infected mice, a significant GPC3-specific cytotoxic T lymphocyte (CTL) response was detected in P.y-GPC3 vaccinated mice. At the same time, we did not check a significant GPC3-specific CTL in P.y-eGPC3 vaccinated mice.Plasmodium sporozoites have a natural ability to target into hepatocytes and finish a liver infection, and we further checked this ability to target into liver cancer cells. Sporozoites of Plasmodium berghei (P.b) could infect Hepa1-6 and HC-04 cellsand evenHela cells in vitro. However, we did not detect the sporozoite infection in Hepa1-6-induced tumor tissue in mice. The possibility of using Plasmodium parasite as an effective HCC antigen glypican-3 delivery may be applicable to tumor immunotherapy for patients with HCC. Lastly, the ethical consideration of using Plasmodium parasites to treat patients with HCC has been envisaged. The parasites at least need to be attenuated or attenuated Plasmodium sporozoites could be tried as live vectors. Combined the blood stage and liver stage of Plasmodium parasite, Plasmodium-basedimmunotherapy is worth to pursue.
Subject Area生物化学与分子生物学
Pages106
Language中文
Document Type学位论文
Identifierhttp://ir.foo.ac.cn/handle/2SETSVCV/1146
Collection中国科学院广州生物医药与健康研究院
Affiliation中国科学院广州生物医药与健康研究院
First Author AffilicationGUANGZHOU INSTITUTES OF BIOMEDICINE AND HEALTH,CHINESE ACADEMY OF SCIENCES
Recommended Citation
GB/T 7714
刘权. 以疟原虫为载体表达 GPC3 蛋白的肝癌疫苗及其免疫机理的研究[D]. 广州生物院. 中国科学院大学,2017.
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