肝癌SP细胞全抗原负载树突状细胞治疗肝癌的作用和机制研究

Hepatocellular carcinoma (HCC) which was unsensitized to radiotherapy and chemotherapy and easy to recurrence and metastasis companying with poor prognosis has the highest incidence of malignant tumors in the world,especially in China. Based on some reports, inhibition of stem cell-like tumor cells which was the essential reason for the malignant behavior of HCC will be useful to the therapy of HCC and prevent the following metastasis and recurrence.It was found that CD133,the stem cell markers,and AFP,immature hepatocyte marker, were highly expressed in HCC side population (SP) cells with self-renewal and tumorigenicity after the establishment of SP cell sorting methods, and also confirmed that they are a group of "seed" cells with the characteristics of stem cells which play a key role in the development of hepatocellular carcinoma.In addition, the efficient and stable system for the culture and induction of mature dendritic cell has been established.In the current projects, the whole tumor antigens of SP cells will be prepared and loaded with the dendritic cells firstly, and the anti-tumor effects and mechnisam will be observed and pursued in vitro by the methods of LDH and ELISA etc, and evaluated through rat HCC model induced by the diethylnitrosamine (DEN) or nude mice in vivo. Moreover, ELISPOT and MHC-peptide tetramer will be used to determine the important immunological moleculars and dominant antigen epitopes which induce the cytotoxic effect on liver tumor cells. Finally，we will observe theraputic effetcs of DC loaded by sepecific peptide in vitro and in vivo. It will provide the new strategy for clinical immunotherapy of hepatocellular carcinoma in the future.

肝癌是全球及我国发病率最高的恶性肿瘤之一，预后极差，对放、化疗不敏感，易复发转移。肝癌中干细胞样肿瘤细胞的存在是其恶性生物学行为的根本原因，将其杀灭有望从根本上治疗肝癌。我们已建立肝癌边缘群(SP)细胞的分选方法，并发现其高表达干细胞标志物CD133和未成熟肝细胞标志物AFP，同时具有自我更新及成瘤能力，证实是一群具有干细胞特性的"种子"细胞，在肝癌发生发展中起关键作用。此外，我们还建立了稳定的树突状细胞(DC)培养诱导体系。本项目拟在前期研究基础上，制备肝癌SP细胞的全肿瘤抗原，负载DC后以LDH、ELISA等方法，从细胞、动物水平观察对肝癌细胞的杀伤效应；再以ELISPOT和MHC肽四聚体技术分析诱发杀伤效应的重要免疫分子及其优势抗原表位，然后合成鉴定的抗原表位，负载DC后通过体外、体内实验判断特异抗原肽负载DC的治疗作用，最终为肝癌治疗提供新策略。

针对肿瘤干细胞的树突状细胞疫苗能够有效的抑制肿瘤生长，减少肿瘤的转移和复发，本研究主要通过探讨边缘群细胞(SP cells)能否作为一个合适的抗原提供细胞，并利用体内体外实验检测负载了SP细胞表面抗原的树突状细胞针对肝癌的免疫治疗效果。主要研究内容及结果包括：1. 分离并鉴定了人肝癌细胞系MHCC97 和小鼠肝癌细胞系Hepa 1-6 细胞的干细胞特性，结果表明SP细胞高表达肿瘤相关抗原和MHCI类分子，负载抗原前后，树突状细胞分子表型并未发生明显的变化，但是SP细胞抗原负载的树突状细胞能够引起T细胞抗原特异性的反应，例如IFN-γ的产生。2. 通过细胞毒实验检测发现，SP细胞抗原负载的树突状细胞能够刺激T细胞，这种T细胞的反应具有抗原特异性。利用抗原肽段刺激，发现CD133，EpCAM，Survivin及ANAXA3的抗原短肽能够刺激T细胞产生IFN-γ。3.利用体外SP抗原负载树突状细胞诱导的CTL杀伤细胞，对荷瘤裸鼠治疗，发现能够有效的抑制肿瘤的形成。同时为了进一步体内验证，我们通过对负载Hepa1-6肿瘤细胞免疫功能正常的小鼠，进行负载抗原的树突状细胞治疗，发现负载SP抗原的树突状细胞能够有效的抑制肿瘤的生长。4.通过对不同处理组肿瘤内浸润的免疫细胞检测发现，CD8阳性的细胞在SP抗原负载树突状细胞治疗组中阳性程度较高。这表明树突状细胞疫苗能够激活体内的效应细胞发挥功能。综上，我们的结果表明通过利用SP细胞作为抗原来源，负载树突状细胞可能是一种有效的治疗肝癌的手段。