【作者】 李青青；

【导师】 骆海明；

【作者基本信息】 华中科技大学 ， 生物医学光子学， 2021， 硕士

【摘要】 脑胶质瘤是一种常见且致死率较高的恶性肿瘤,目前临床主要通过手术切除结合替莫唑胺化疗。然而,替莫唑胺水溶性差,生物利用率低。因此,开发可以改善替莫唑胺水溶性并能将其靶向运输到肿瘤部位的纳米载体就显得极为重要。与常用的脂质体和聚合纳米颗粒相比,外泌体不仅可作为肿瘤治疗药物的载体,提高疏水药物的溶解性和生物利用率,而且由于它是细胞自身分泌物,生物相容性好且能够主动穿越血脑屏障,有利于脑胶质瘤的靶向治疗。目前,外泌体主要聚焦于从肿瘤细胞中提取,然而肿瘤细胞与肿瘤组织在本质上存在差异。与细胞衍生的外泌体相比,从肿瘤组织中提取的外泌体能够更多地反映出与肿瘤相关的信息。因此,本文基于脑胶质瘤组织中提取的外泌体进行脑胶质瘤成像与治疗的初步探索,其主要研究内容包括以下两个方面:(1)脑胶质瘤组织外泌体的鉴定及其肿瘤靶向能力的评价。通过透射电镜和粒径仪测定显示外泌体的粒径大小在20～300 nm范围;通过Western blot实验证明通过蔗糖密度梯度超高速离心的方法可以成功提取到不同分层的脑胶质瘤组织来源外泌体。共聚焦成像和流式细胞术的结果表明,脑胶质瘤组织来源的外泌体能被GL261肿瘤细胞、U87肿瘤细胞、U251肿瘤细胞和4T1肿瘤细胞等所摄取,表明组织来源外泌体没有肿瘤特异性。DiI标记的脑胶质瘤组织来源外泌体(Exosome-DiI)对GL261、4T1等肿瘤的活体光学成像进一步证明该结论。(2)脑胶质瘤组织来源的外泌体包裹姜黄素(Exosome-CUR)抑制皮下脑胶质瘤生长能力的初步研究。不同浓度的Exosome-CUR对肿瘤细胞具有浓度依赖的杀伤能力。当Exosome-CUR浓度为20μg/mL时,肿瘤细胞的存活率显著降低,这种杀伤能力是通过诱导细胞凋亡的途径进行的。相比较PBS与单独外泌体处理组,尾静脉注射Exosome-CUR(2 mg/Kg)可以对皮下脑胶质瘤的生长进行显著的抑制。综上所述,本文从胶质瘤组织中成功提取了外泌体,并初步实现了对脑胶质瘤的活体荧光成像和治疗应用,为后续相关研究奠定基础。更多还原

【Abstract】 Glioma is a common malignant tumor with a high fatality rate.At present,surgical resection combined with temozolomide chemotherapy is mainly used in clinical practice.However,temozolomide has poor water solubility and low bioavailability.Therefore,it is extremely important to develop a nanocarrier system that can improve the water solubility of temozolomide and target it to the tumor site.Compared with the common liposomes and polymeric nanoparticles,exosomes can not only be used as a carrier of tumor treatment drugs to improve the solubility and bioavailability of hydrophobic drugs but also because they are cell secretions and are biocompatible and able to actively cross the blood-brain barrier,which is conducive to the targeted therapy of glioma.At present,the source of exosomes is focused on extracting from tumor cells,but tumor cells and tumor tissues are essentially different.Compared with cell-derived exosomes,exosomes extracted from tumor tissues can reflect more tumor-related information.Therefore,this article is based on the exosomes extracted from the brain glioma tissue for the preliminary exploration of brain glioma imaging and treatment.The main research includes the following two aspects:(1)The identification of glioma tissue exosomes and the evaluation of tumor targeting ability.Transmission electron microscopy and particle size analyzer showed that the particle size of exosomes was in the range of 20～300 nm;Western blot experiments proved that the sucrose density gradient ultra-high-speed centrifugation method can successfully extract different layers of glioma tissue Source exosomes.The results of confocal imaging and flow cytometry showed that exosomes derived from glioma tissue can be taken up by GL261 tumor cells,U87 tumor cells,U251 tumor cells and 4T1 tumor cells,indicating that tissue-derived exosomes have no tumor Specificity.The in vivo optical imaging of DiI-labeled glioma tissue-derived exosomes(Exosome-DiI)on GL261,4T1 and other tumors further proves this conclusion.(2)Preliminary study on the ability of curcumin encapsulated in exosomes derived from glioma tissue to inhibit the growth of subcutaneous glioma.Different concentrations of Exosome-CUR have a concentration-dependent killing ability on tumor cells.When the concentration of Exosome-CUR is 20 μg/mL,the survival rate of tumor cells is significantly reduced.This killing ability is carried out by inducing cell apoptosis.Compared with PBS and exosomes alone treatment group,tail vein injection of Exosome-CUR(2 mg/Kg)can significantly inhibit the growth of subcutaneous glioma.In summary,this article successfully extracted exosomes from glioma tissue,and initially realized the in vivo fluorescence imaging and therapeutic application of glioma,laying the foundation for subsequent related research.更多还原