本文選題：腎細(xì)胞癌 + 石蒜堿��； 參考：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：近年來,關(guān)于腎癌的處理經(jīng)歷很大變化,如對于局部腎癌,腎部分切除術(shù)即可達(dá)到滿意的臨床結(jié)果,而對于轉(zhuǎn)移性腎癌,則以血管內(nèi)皮生長因子或雷帕霉素信號通路的靶向治療占主導(dǎo)。另外,以免疫檢測點為靶點的腫瘤免疫治療,正受到高度關(guān)注,細(xì)胞毒性T細(xì)胞相關(guān)抗原4(CTLA-4)阻斷劑作為此類療法的代表,其臨床研究結(jié)果顯示出較強(qiáng)的抗腫瘤能力。石蒜堿(Lycorine)屬異喹啉類生物堿,提取自石蒜科植物,現(xiàn)有的研究證實其具有多種生物藥理活性,因近年來許多研究表明其具有抗腫瘤作用,關(guān)于石蒜堿的抗腫瘤能力日益受到重視,其抗腫瘤機(jī)制包括:誘導(dǎo)細(xì)胞凋亡,細(xì)胞周期阻滯,誘導(dǎo)程序性壞死及抑制血管生成擬態(tài)等。然而,尚無關(guān)于免疫檢查點阻斷劑聯(lián)合石蒜堿對腎臟惡性腫瘤作用效應(yīng)的研究。因此,在本研究中我們采用熒光素酶報告基因技術(shù)標(biāo)記及構(gòu)建小鼠腎癌細(xì)胞:Renca-Luc,并以此細(xì)胞株建立免疫功能正常的小鼠原位腎細(xì)胞癌及肺轉(zhuǎn)移模型,通過IVIS 2000動物活體熒光成像系統(tǒng)實現(xiàn)無創(chuàng)、動態(tài)監(jiān)測活體水平下腫瘤的生長情況,從而了解免疫檢查點阻斷劑及石蒜堿對腎臟惡性腫瘤的作用效應(yīng),并探討石蒜堿是否可提高CTLA-4阻斷劑的治療效應(yīng),降低其不良事件的發(fā)生率。通過體外實驗,我們明確鹽酸石蒜堿可有效抑制多種人源腎癌細(xì)胞株及鼠源腎癌細(xì)胞Renca的增殖能力,而且Renca最為敏感;同時可抑制Renca的遷移及侵襲能力。為進(jìn)一步探討其作用機(jī)制,我們檢測經(jīng)鹽酸石蒜堿作用后Renca各細(xì)胞周期的分布情況,結(jié)果顯示sub-G1期細(xì)胞含量明顯增高,伴隨G2/M期細(xì)胞含量降低,且呈濃度相關(guān)。免疫印跡技術(shù)檢測細(xì)胞周期相關(guān)蛋白的表達(dá)情況后,得出鹽酸石蒜堿可抑制Renca的細(xì)胞周期相關(guān)蛋白cyclinD1,cyclinD3及CDK2等表達(dá)。隨后通過穩(wěn)定表達(dá)熒光素酶的小鼠腎癌細(xì)胞(Renca-luc)構(gòu)建小鼠原位腎及肺轉(zhuǎn)移模型確認(rèn),聯(lián)合應(yīng)用鹽酸石蒜堿及免疫檢測點阻斷劑可產(chǎn)生增強(qiáng)的抗腫瘤效應(yīng)。除此之外,在本研究中,我們首次報道聯(lián)合應(yīng)用鹽酸石蒜堿及免疫檢測點阻斷劑可以通過下調(diào)T調(diào)節(jié)細(xì)胞伴隨效應(yīng)T細(xì)胞及記憶T細(xì)胞的上調(diào),從而增強(qiáng)免疫系統(tǒng)對惡性腫瘤細(xì)胞的免疫應(yīng)答來實現(xiàn)其增強(qiáng)的抗腫瘤作用。綜上所述,聯(lián)合應(yīng)用鹽酸石蒜堿及免疫檢測點阻斷劑在腎細(xì)胞癌及肺轉(zhuǎn)移動物模型中具有增強(qiáng)的抗腫瘤效應(yīng),且無明顯的毒副作用。這一發(fā)現(xiàn)為腎惡性腫瘤,特別是轉(zhuǎn)移性腎細(xì)胞癌的處理提供新的可能的策略,同時亦可為此類腫瘤免疫治療的相關(guān)基礎(chǔ)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.11

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