【作者】 袁日明；

【导师】 陈填烽；

【作者基本信息】 暨南大学 ， 无机化学， 2020， 硕士

【摘要】 癌症作为非传染性慢性疾病,是威胁人类生命健康的三大杀手之一,已成为全人类亟待解决的公共卫生问题。其中,肺癌发病率和死亡率在近50年不断地迅速上升,已经被卫生部列为癌症防治的重点。靶向治疗和免疫治疗被认为是肺癌有效的治疗手段。其中,吉非替尼(Gefitinib,GFT)是有效靶向非小细胞肺癌(NSCLC)表皮生长因子(EGFR)的药物。然而,GFT产生的耐药性导致其癌症治疗效果减弱,同时引起机体毒性,最终影响GFT的临床使用。自然杀伤细胞(Natural Killer Cell,NK)免疫疗法对治疗NSCLC具有较高的潜力。但由于肿瘤微环境的影响,NK细胞对肿瘤的识别与杀伤能力下降,导致治疗效果不佳。因此,开发GFT和NK细胞免疫治疗的增敏剂是提高肺癌治疗效果的重要策略。近年来,人们发现天然冰片(Natural Borneol,NB)能增强抗肿瘤药物对癌症的治疗效果。然而,由于NB易升华、水溶性极差等缺点,NB的进一步应用受到了限制。目前,纳米技术在生物医药领域的进展飞快,为提高和拓展药物的药用价值提供了新策略。其中,水包油(Oil in water,O/W)纳米乳能有效提高脂溶性药物的溶解性,增强药物的细胞吸收和生物利用率。在此,我们采用简便的高压均质方法合成O/W冰片纳米粒子(NBNPs),增强NB的稳定性和水溶性,并联合GFT以及NK细胞应用于肺癌的治疗。具体研究内容如下:1.合成水包油NBNPs纳米体系并探究其对抗NSCLC药物GFT的增敏作用。实验结果显示,NBNPs在A549非小细胞肺癌和WI38正常胚肺成纤维细胞之间具有良好的选择性杀伤作用。相比于NB,NBNPs更能增强GFT的细胞吸收和细胞毒性。蛋白组学结果显示,NBNPs能特异性识别A549肺癌细胞中的8种靶标蛋白,从而增强NBNPs在A549细胞和WI38细胞之间的选择性杀伤作用。而NBNPs对GFT的增敏作用主要通过特异性结合并抑制EHD1表达,诱导产生FADD凋亡信号,引起了癌细胞显著凋亡。安全性评价结果显示,NBNPs联合GFT治疗对机体并未出现明显的毒副作用。此外,NBNPs能降低GFT对肝脏的毒性。2.我们拓展了NBNPs在增敏NK细胞治疗NSCLC方面的研究。发现NBNPs能够增敏不同病人NK细胞的抗肿瘤活性,提高NK细胞在A549肿瘤球中的渗透能力。机制研究表明NBNPs与NK细胞联合使用会增强A549细胞中ULBP1和ULBP3的m RNA表达量,有利于提高免疫反应,促进NKG2D配体结合NK细胞的NKG2D受体,从而增强抗肿瘤效果。更多还原

【Abstract】 As a non-communicable chronic disease,cancer has been one of the three killers threatening human life and health,which has become an urgent public health problem.And lung cancer has been listed as the focus of cancer prevention and treatment by the Ministry of Health,the morbidity and mortality of which have been increasing rapidly in the past 50 years.Targeted therapy and immunotherapy are considered to be effective strategies for lung cancer therapy.Gefitinib(GFT)is an effective drug targeting epidermal growth factor(EGFR)in non-small cell lung cancer(NSCLC).However,GFT will produce drug resistance,which weakens the therapeutic effect of GFT and causes toxicity to the body,thus affecting the clinical use of GFT.Natural killer(NK)cells for cancer immunotherapy exerts high potential for the treatment of NSCLC.Nevertheless,NK cells therapy is affected by tumor microenvironment,which influences the interaction between NK cells and tumor cells,leading to the reduction of tumor treatment effect.Therefore,developing sensitizers for GFT and NK cells immunotherapy are considered to be important strategies for enhancing therapeutic effect of lung cancer.In recent years,natural borneol(NB)has been studied to enhance the efficacy of antitumor drugs against cancer.However,the further application of NB has been limited due to its disadvantages,such as easy sublimation and poor water solubility.Nanotechnology has been making rapid progress in the field of biomedicine,which provides a new strategy to improve and expande the medicinal value of drugs.And oil-in-water(O/W)nanoemulsion can effectively improve solubility,uptake efficiency and bioavailability of hydrophobic drugs.Here,we synthesized O/W nanoparticles(NBNPs)to enhance the stability and water solubility of NB by using high-pressure homogenization.And then we applied it as a sensitizer to combine with GFT and NK cells in the treatment of NSCLC.The research contents were as follows:1.Oil-in-water NBNPs nanosystem was synthesized and used as a sensitizer to enhance the treatment effect of GFT against NSCLC.NBNPs displayed excellent cancer-targeted ability between A549 NSCLC cells and WI38 normal lung fibroblast cells,and outperformed NB by drastically enhancing the uptake efficiency and cytotoxicity of GFT against NSCLC growth in both cell and tumor xenograft model.Proteomic results showed that NBNPs could specifically identify einght target proteins in A549 lung cancer cells,thus enhancing the specific selectivity of NBNPs between A549 cells and WI38 cells.In addition,the sensitizing effect of NBNPs on GFT was attributed to specific binding and inhibition of EHD1 expression,which activated FADD apoptosis signal,thus causing significant apoptosis of A549 cells.The results of biosafety evaluation indicated that NBNPs in combination with GFT showed no obvious side effects on the body.More importantly,NBNPs could reduce the toxic effect of GFT on liver.2.Simultaneously,the sensitization of NBNPs to NK cells against NSCLC was studied here.It was found that NBNPs could sensitize the antitumor activity of NK cells from different patients and enhance the penetration ability of NK cells in A549 tumor spheroids.Mechanism studies showed that the combination of NBNPs and NK cells could upregulate the m RNA expression of ULBP1 and ULBP3 in A549 cells,which was beneficial to promote immunoreaction and improve the binding of NKG2 D ligands and NKG2 D,thus enhancing the NK cells antitumor effect.更多还原