免疫检测点阻断剂联合石蒜碱治疗肾癌的作用及机制研究

发布时间：2018-06-24 11:54

本文选题：肾细胞癌 + 石蒜碱　；参考：《南方医科大学》2017年博士论文

【摘要】：近年来,关于肾癌的处理经历很大变化,如对于局部肾癌,肾部分切除术即可达到满意的临床结果,而对于转移性肾癌,则以血管内皮生长因子或雷帕霉素信号通路的靶向治疗占主导。另外,以免疫检测点为靶点的肿瘤免疫治疗,正受到高度关注,细胞毒性T细胞相关抗原4(CTLA-4)阻断剂作为此类疗法的代表,其临床研究结果显示出较强的抗肿瘤能力。石蒜碱(Lycorine)属异喹啉类生物碱,提取自石蒜科植物,现有的研究证实其具有多种生物药理活性,因近年来许多研究表明其具有抗肿瘤作用,关于石蒜碱的抗肿瘤能力日益受到重视,其抗肿瘤机制包括:诱导细胞凋亡,细胞周期阻滞,诱导程序性坏死及抑制血管生成拟态等。然而,尚无关于免疫检查点阻断剂联合石蒜碱对肾脏恶性肿瘤作用效应的研究。因此,在本研究中我们采用荧光素酶报告基因技术标记及构建小鼠肾癌细胞:Renca-Luc,并以此细胞株建立免疫功能正常的小鼠原位肾细胞癌及肺转移模型,通过IVIS 2000动物活体荧光成像系统实现无创、动态监测活体水平下肿瘤的生长情况,从而了解免疫检查点阻断剂及石蒜碱对肾脏恶性肿瘤的作用效应,并探讨石蒜碱是否可提高CTLA-4阻断剂的治疗效应,降低其不良事件的发生率。通过体外实验,我们明确盐酸石蒜碱可有效抑制多种人源肾癌细胞株及鼠源肾癌细胞Renca的增殖能力,而且Renca最为敏感;同时可抑制Renca的迁移及侵袭能力。为进一步探讨其作用机制,我们检测经盐酸石蒜碱作用后Renca各细胞周期的分布情况,结果显示sub-G1期细胞含量明显增高,伴随G2/M期细胞含量降低,且呈浓度相关。免疫印迹技术检测细胞周期相关蛋白的表达情况后,得出盐酸石蒜碱可抑制Renca的细胞周期相关蛋白cyclinD1,cyclinD3及CDK2等表达。随后通过稳定表达荧光素酶的小鼠肾癌细胞(Renca-luc)构建小鼠原位肾及肺转移模型确认,联合应用盐酸石蒜碱及免疫检测点阻断剂可产生增强的抗肿瘤效应。除此之外,在本研究中,我们首次报道联合应用盐酸石蒜碱及免疫检测点阻断剂可以通过下调T调节细胞伴随效应T细胞及记忆T细胞的上调,从而增强免疫系统对恶性肿瘤细胞的免疫应答来实现其增强的抗肿瘤作用。综上所述,联合应用盐酸石蒜碱及免疫检测点阻断剂在肾细胞癌及肺转移动物模型中具有增强的抗肿瘤效应,且无明显的毒副作用。这一发现为肾恶性肿瘤,特别是转移性肾细胞癌的处理提供新的可能的策略,同时亦可为此类肿瘤免疫治疗的相关基础研究提供了新思路。
[Abstract]:In recent years, there have been great changes in the management of renal cell carcinoma. For local renal cell carcinoma, partial nephrectomy can achieve satisfactory clinical results, while for metastatic renal cell carcinoma, The target therapy of vascular endothelial growth factor or rapamycin signaling pathway is dominant. In addition, tumor immunotherapy targeting immunological detection sites is receiving high attention. Cytotoxic T cell associated antigen-4 (CTLA-4) blocker as a representative of this kind of therapy, its clinical research results show strong anti-tumor ability. Lycorine, an isoquinoline alkaloid, was extracted from Lycorine plants. Existing studies have proved that Lycorine has a variety of biological pharmacological activities, because in recent years, many studies have shown that Lycorine has anti-tumor effect. More and more attention has been paid to the antitumor ability of Lycorine. Its mechanisms include inducing cell apoptosis, cell cycle arrest, inducing programmed necrosis and inhibiting angiogenesis mimicry. However, there is no study on the effect of immune checkpoint blocker combined with Lycorine on renal malignancy. In this study, we used luciferase reporter gene technique to label and construct mouse renal cancer cell line: Renca-Luc. and establish mouse model of normal immune function in situ renal cell carcinoma and lung metastasis. In order to understand the effect of immunological checkpoint blockers and Lycorine on renal malignancy, IVIS 2000 animal in vivo fluorescence imaging system was used to dynamically monitor tumor growth at living level. To explore whether Lycorine can improve the therapeutic effect of CTLA-4 blocker and reduce the incidence of adverse events. Through in vitro experiments, we confirmed that Lycorine hydrochloride could effectively inhibit the proliferation of Renca in various human renal cell lines and murine renal cancer cells, and Renca was the most sensitive, and could also inhibit the migration and invasion of Renca. In order to further study its mechanism, we detected the distribution of Renca cell cycle after treated with Lycorine hydrochloride. The results showed that the cell content in sub-G1 phase was significantly increased, and the cell content in G 2 / M phase was decreased in a concentration-dependent manner. After detecting the expression of cell cycle associated protein by Western blotting, it was concluded that Lycorine hydrochloride could inhibit the expression of cyclinD1, cyclin D3 and CDK2 of Renca. Then mouse renal cell carcinoma cells (Renca-luc) which expressed luciferase stably were used to establish in situ renal and lung metastasis model of mice. It was confirmed that the combination of Lycorin hydrochloride and immunoassay point blocker could produce enhanced antitumor effect. In addition, in this study, we reported for the first time that the combination of Lycorine hydrochloride and immunoassay antagonists could down-regulate the up-regulation of T cells associated with T cells and memory T cells. In order to enhance the immune response of the immune system to malignant tumor cells to achieve its enhanced anti-tumor effect. To sum up, combined use of Lycorine hydrochloride and immunoassay point blockers in renal cell carcinoma and lung metastasis animal models have enhanced anti-tumor effect, and no obvious side effects. This finding provides a new strategy for the treatment of renal malignant tumors, especially metastatic renal cell carcinoma, and also provides a new idea for the basic research of immunotherapy of these tumors.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.11

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