【作者】 周瑜；

【导师】 丁晓毅；

【作者基本信息】 上海交通大学 ， 影像医学与核医学， 2019， 博士

【摘要】 7研究背景:胰腺癌是最难治疗的肿瘤之一,其5年生存率低于6%。化疗耐药性的增加和以基质、炎症因子丰富为特点的肿瘤微环境是减弱胰腺癌化疗效果的主要原因。纳米靶向药物制剂在增加药物渗透、提高有效药物浓度并克服耐药中有一定优势。基于“骨髓-胰腺癌轴”学说,我们提出BM-MSCs来源外泌体作为载体能够高效靶向胰腺癌,并有效穿透基质的科学假设。为克服胰腺癌化疗耐药,吉西他滨单磷酸酯和紫杉醇被包载至外泌体中以构建外泌体共递药系统。以期此生物型靶向纳米药物能够实现解除基质对药物渗透限制、抗基质并克服化疗耐药的三重功能。研究目的:探讨不同分离方法是否影响外泌体载药;BM-MSCs来源外泌体是否能高效靶向胰腺癌,并有效穿透细胞外基质;外泌体共递药系统能否有效抑制胰腺癌,并降解基质;外泌体共递药系统能否克服胰腺癌化疗耐药性。研究方法:（1）不同方法分离外泌体,采用不同电穿孔条件载药,通过载药量和细胞药效评估分离方法是否对外泌体载药有影响。（2）建立胰腺癌裸鼠原位模型,考察BM-MSCs来源的外泌体是否有胰腺癌靶向性,并通过免疫荧光切片和肿瘤球模型评价其穿透能力。（3）构建外泌体共递药系统,通过细胞抑制及分析胞内产物含量,考察其是否能克服胰腺癌化疗耐药。（4）通过动物活体药效及治疗后肿瘤组织病理评价,考察外泌体共递药系统能否降解基质及其方式,并再次于活体水平验证能否克服耐药。研究结果:（1）密度梯度离心分离外泌体,粒径较小且纯度高,结合Opti Prep^TM缓冲液电穿孔载药效率最高,能有效抑制细胞增殖。（2）BM-MSCs来源的外泌体能够高效靶向胰腺癌区域,并能穿透胰腺癌基质,到达肿瘤深部。（3）外泌体共递药系统能够提高胞内GEMP含量,克服耐药。（4）外泌体共递药系统能有效抑制肿瘤生长,并以“由内往外”的方式降解基质。研究结论:密度梯度离心分离外泌体有利于其电穿孔载药;BM-MSCs来源外泌体共递药系统能够高效靶向胰腺癌并有效穿透,降解基质并克服耐药。更多还原

【Abstract】 BACKGROUND:Pancreatic ductal adenocarcinoma(PDAC)is one of the most intractable malignancy,with an only 6% 5-year survival rate.The dismal therapeutic effect is attributed to the chemotherapy resistance and unique pathophysiology with abundant inflammatory cytokines and abnormal hyperplasia of extracellular matrix.Nanotechonology based tagert drug delivery has advantegous on facilitating penetrating and accumulationg of drugs and overcoming chemoresistance.Based on the theory of “bone marrow mesenchymal stem cells and PDAC axis”,we employed exosomes(Exo)derived from BM-MSCs as PDACtargeting vehicles to surpass the restrictions of pathological ECM and increase the accumulation of therapeutics in tumor site.To overcome chemoresistance of PDAC,paclitaxel(PTX)and gemcitabine monophosphate(GEMP)– an intermediate product of GEM metabolism – were loaded in/on the exosomes to construct a co-delivery system.We hope this bio-nanomedicine can perform triple functions on removing the limitation of matrix,anti-matrix and overcoming chemoresistance.OBJECTIVE:To investigate whether different isolation methods of exosomes affect drug loading;to investigate whether exosomes derived from BM-MSCs could tagert and penetrate into PDAC effectively;to evaluate whether exosomal co-delivery system could inhibite tumor,degrade extracellular matrix and overcome chemoresistance.METHODS:(1)To investigate whether different isolation methods of exosomes affect drug loading by analyzing the loading dose and cell proliferation inhibition after employing diverse isolation methods and electroporation.(2)To investigate tagerting and penetrating capabilities of exosomes in PDAC orthotopic models by immunofluorescence staining and tumor spheroids.(3)Constructing exosomal co-delivery system and evaluating inhibitory effect on Mia Paca-2 cells and overcoming chemoresistance or not.(4)To investigate whether exosomal co-delivery system could inhibit tumor,degrade matrix and overcome chemoresistance.RESULTS:(1)We harvested exosomes with high purity and little diameter which exhibited preferable efficiency of drug loading and inhibition of cell proliferation.(2)Exosomes derived from BM-MSCs could tagert and penetrate in PDAC situ effectively.(3)Exosomal co-delivery system could overcome chemoresistance by enhancing accumulation of GEMP.(4)Exosomal co-delivery system could inhibit tumor and degrade matrix effectively by inside-out way.CONCLUSIONS:Density gradiant method benefited drug loading of exosomes employing electroporation;exosomal co-delivery system exhibited triple functions on tagerting and penetrating in PDAC effectively,degrading matrix and overcoming chemoresistance.更多还原