【作者】 吕丽虹；

【导师】 陈亚进；

【作者基本信息】 中山大学 ， 外科学， 2012， 博士

【摘要】 【目的】Exosomes为活细胞胞内内体小泡或多泡体（multivesicular bodies，MVBs）通过与细胞膜融合并释放到细胞外环境中的一种直径介于30-100纳米的膜性微囊小体。不同细胞来源的exosomes所携带的蛋白质不同，决定其生物学功能也有差异。近年来，大量的研究证据表明肿瘤细胞来源的exosomes携带大量与免疫相关的蛋白质，并在宿主的抗肿瘤免疫应答和免疫调节方面具有重要作用，提示肿瘤细胞来源的exosomes有望成为一种新型的亚细胞肿瘤疫苗，用于人类多种恶性肿瘤的免疫治疗。原发性肝细胞癌（primary hepatocellular carcinoma，以下简称肝癌）是目前临床上最常见的消化系统恶性肿瘤之一。机体免疫监控功能的失常导致其无法有效地诱导抗肿瘤免疫反应，与肝癌患者术后的高复发率和预后不良等密切相关。在细胞膜上表达或分泌到细胞外的热休克蛋白（heatshock proteins，HSPs）被免疫系统视为重要的“危险信号”之一，一方面能够提高肿瘤细胞的免疫原性，另一方面可通过激活自然杀伤（natural killer，NK）细胞诱导机体产生抗肿瘤免疫反应。热休克蛋白与肿瘤免疫治疗的关系是近年来研究者关注的热点问题之一。本课题旨在研究不同抗肿瘤药物对人肝癌细胞释放exosomes的数量及其表面热休克蛋白的表达量的影响。在此基础上，通过对NK细胞功能的影响进一步研究携带热休克蛋白的exosomes的生物学活性。此项研究结果不仅有望为肝癌的免疫治疗提供合适的肿瘤疫苗来源，也为更深入地开展肝癌化学-免疫联合治疗的临床应用提供理论和实验依据。【方法】1.使用MTT法检测不同抗肿瘤药物对人肝癌细胞株HepG2和PLC/PRF/5生长的抑制作用，筛选出对人肝癌细胞敏感和耐受的抗肿瘤药物。2.人肝癌细胞株HepG2和PLC/PRF/5分别经对人肝癌细胞敏感（依托泊苷和紫杉醇）或耐受（卡铂和盐酸伊立替康）的抗肿瘤药物或热休克处理后，使用ELISA法检测两种细胞培养上清液中HSP60、HSP70及HSP90的含量。3.采用多步超速离心法从人肝癌细胞株HepG2培养上清液中分离exosomes后，使用透射电镜和Western blot法对其进行形态学和分子标记物鉴定。4.人肝癌细胞株HepG2经对人肝癌细胞敏感（依托泊苷和紫杉醇）或耐受（卡铂和盐酸伊立替康）的抗肿瘤药物或热休克处理后，分别应用乙酰胆碱酯酶活性、Western blot法及流式细胞术检测细胞培养上清液中exosomes的数量、exosomes总及其表面HSP60、HSP70及HSP90的表达量。5.人肝癌细胞株HepG2经对热肝癌细胞敏感（紫杉醇）或耐受（卡铂）的抗肿瘤药物处理后，从细胞培养上清液中分离得到的exosomes与NK细胞共培养，分别使用LDH、ELISA法及流式细胞术检测NK细胞的杀伤活性、NK细胞培养上清液中颗粒酶B的含量及NK细胞表面活化性受体CD69、NKG2D及NKp44和NK细胞表面抑制性受体CD94的表达情况。【结果】1.依托泊苷和紫杉醇对人肝癌细胞株HepG2和PLC/PRF/5的生长抑制率高（敏感），卡铂、丝裂霉素及盐酸伊立替康对人肝癌细胞株HepG2和PLC/PRF/5的生长抑制率低（耐受）。2.抗肿瘤药物促进人肝癌细胞株HepG2和PLC/PRF/5分泌HSP60、70及90，并且耐受组细胞培养上清液中HSP60、70及90的含量明显高于敏感组，组间比较显示差别有统计学意义（P＜0.05）。3.通过透射电镜观察其形态结构和Western blot法检测其特征性标志物CD63分子，证实人肝癌细胞株HepG2细胞培养上清液含有exosomes。4.抗肿瘤药物促进人肝癌细胞株HepG2释放exosomes，并且耐受组细胞培养上清液中exosomes的数量明显多于敏感组，组间比较显示差别有统计学意义（P＜0.05）。与此同时，抗肿瘤药物上调人肝癌细胞株HepG2来源的exosomes总和表面HSP60、70及90的表达水平，并且耐受组exosomes总和表面HSP60、70及90的表达水平明显高于敏感组，组间比较显示差别有统计学意义（P＜0.05）。5.抗肿瘤药物处理后的人肝癌细胞株HepG2来源的exosomes可增强NK细胞对人慢性粒细胞白血病急性红白血病变细胞株K562的杀伤活性，促进NK细胞分泌颗粒酶B，上调NK细胞表面活化性受体CD69、NKG2D及NKp44的表达水平并下调NK细胞表面抑制性受体CD94的表达水平。耐受组exosomes对NK细胞免疫功能的促进作用明显强于敏感组，组间比较显示差别有统计学意义（P＜0.05），并且具有明显的剂量依赖性。当exosomes浓度达到20μg/mL时，其促进作用最明显。【结论】通过MTT法从11种常见的抗肿瘤药物中筛选出对人肝癌细胞株HepG2和PLC/PRF/5敏感（依托泊苷和紫杉醇）和耐受（卡铂、丝裂霉素及盐酸伊立替康）的抗肿瘤药物，并且每组各选定两种药物作为应激因素应用于后续的实验。我们目前的研究结果证实，抗肿瘤药物促进人肝癌细胞株HepG2释放exosomes和上调人肝癌细胞株HepG2来源的exosomes表面HSP60、70及90的表达水平，并且耐受组对细胞的促进作用明显强于敏感组。再者，抗肿瘤药物处理后的人肝癌细胞株HepG2来源的exosomes具有更佳的生物学活性，特别是耐受组的exosomes，可显著增强NK细胞的活性。上述实验结果提示携带热休克蛋白的exosomes在肝癌的化学-免疫联合治疗中具有潜在价值。这项研究发现不仅为将来肝癌患者的个性化生物治疗提供安全、高效的肿瘤疫苗来源，也为肝癌的化学-免疫联合治疗的临床应用提供可靠的实验基础与理论依据。更多还原

【Abstract】 【OBJECTIVE】Exosomes are actively released into the extracellular environmentfrom a wide range of living cells via endosomal vesicles/multivesicularbodies (MVBs) pathway by fusion with the plasma membrane, and specialized30-100nanometer (nm)-sized lipid-rich membrane-bound microvesicles. Theexosomal protein composition varies with the cell types from which theyare derived, and accounting for their biological functions. In recentyears, many studies have demonstrated that tumor-derived exosomes (Tex),carrying a variety of immune-related proteins, played an important rolein the host anti-tumor immune response and immune regulation. Tex areexpected to become a novel subcellular tumor vaccine for immunotherapyof a variety of human malignancies. Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Dysfunction of immunesurveillance in tumor-bearing host resulted in ineffective induction ofanti-tumor immune responses, and was closely related to the high rate ofrecurrence and poor prognosis of HCC. Membrane-bound or extracellularlocalized heat shock proteins (HSPs) have been found to provide dangersignals for the host’s cellular immune system. HSPs not only enhance theimmunogenicity of cancer cells, but also induce anti-tumor immunity byactivating natural killer (NK) cells. The relationship between HSPs andtumour immunotherapy has become a research hotspot in recent years.The purpose of the present study was to investigate whether anticancerdrugs may significantly increase exosome secretion by hepatocellularcarcinoma cells, and efficiently up-regulate the surface expression ofHSPs on exosomes. Based on the above results, the biological activitiesof HSPs-bearing exosomes were investigated through influencing NK cellfunctions. These results not only were expected to provide suitablesources of tumor vaccines for HCC immunotherapy, but also providedtheoretical and experimental basis for more in-depth clinicalapplications of HCC chemoimmunotherapy.【METHODS】1. The in vitro growth inhibitory effect of anticancer drugs on the humanhepatocellular carcinoma (HepG2and PLC/PRF/5) cell lines was measuredby the MTT assay.2. Cell culture supernatants were harvested at different time points fromHepG2and PLC/PRF/5cells, exposured to100%test drug concentration(TDC) of paclitaxel, carboplatin, etoposide, irinotecan hydrochloride,heat shock or control, and the concentration of HSP60, HSP70and HSP90in each sample were measured using the ELISA Kit. 3. Conditioned culture medium was collected from HepG2cells, andsubjected to differential centrifugation to obtain the purifiedexosomal pellet. The morphology of exosome was examined bytransmission electron microscopy (TEM), and the exosomal marker CD63was analysed by Western blot.4. We isolated exosomes from cell culture supernatants produced by HepG2cells under both basal and stress-induced conditions at different timepoints. The amount of exosomes was quantified via the determinationof AChE enzymatic activity, HSP60, HSP70and HSP90expression inexosomes were assessed by Western blot, and these molecules expressionon the surface of exosomes were analyzed by flow cytometry.5. NK cells were incubated either with low-dose IL-2alone or with IL-2in combination with exosomal protein for4days, which were isolatedfrom HepG2cells under100%TDC paclitaxel or carboplatin treatment.NK cell-mediated cytotoxic function was analyzed using a standardlactate dehydrogenase (LDH) release assay, granzyme B production wasmeasured by ELISA kit, and the cell surface density of several NK cellreceptors was examined by flow cytometry.【RESULTS】1. The human hepatocellular carcinoma (HepG2and PLC/PRF/5) cell linesshowed remarkably higher sensitivity to paclitaxel and etoposide, andexhibited resistance to irinotecan hydrochloride, carboplatin andmitomycin.2. HSP60, HSP70and HSP90secretion were generally higher afteranticancer drugs treatment, reaching statistical significance (P＜0.05). Moreover, our results demonstrated that HepG2and PLC/PRF/5cells secreted higher levels of HSP60, HSP70and HSP90treated with hepatocellular carcinoma cell-resistant anticancer drugs comparedwith cells exposed to hepatocellular carcinoma cell-sensitiveanticancer drugs.3. A pure exosomal population was present with typical cup-shapedmorphology, surrounded by a two-layer lipid membrane, and varying insize between30nm-100nm, the majority around70nm-90nm. Besidesmorphology and size, we confirmed the presence of exosomes through itsspecific marker by Western blot with anti-CD63antibody.4. There was an increase in exosome release by HepG2cells understress-induced conditions compared with cells under basal condition(P＜0.05). Moreover, hepatocellular carcinoma cell-resistantanticancer drugs could enhance exosome release to a higher degree thanhepatocellular carcinoma cell-sensitive anticancer drugs. HepG2cell-derived exosomes carried more HSP60, HSP70and HSP90undertreatment with anticancer drugs (P＜0.05), and hepatocellularcarcinoma cell-resistant anticancer drugs caused induction ofexosome-carried HSPs release at a level remarkably higher thanhepatocellular carcinoma cell-sensitive anticancer drugs. Elevatedlevels of HSP60, HSP70and HSP90were observed under stress-inducedconditions as compared to basal condition (P＜0.05), andhepatocellular carcinoma cell-resistant anticancer drugs seemed toenhance HSP60, HSP70and HSP90exosome-surface expression to a higherdegree than hepatocellular carcinoma cell-sensitive anticancer drugs.5. Contact of NK cells with the increased amount of HSPs-bearing exosomesaugmented cytolytic activity against K562target cells throughgranzyme B release, down-regulation of activating receptors CD69,NKG2D and NKp44, and up-regulation of inhibitory receptor CD94(P＜0.05). Carboplatin-treated HepG2cells-derived exosomes had astronger effect on NK cell function than paclitaxel-treated HepG2 cells-derived exosomes. Furthermore, the extent of cytotoxic effectof the HSPs-expressing exosomes was correlated with theirconcentrations, reaching a maximum effect at20μg/mL.【CONCLUSION】The in vitro growth inhibitory effect of anticancer drugs on the humanhepatocellular carcinoma (HepG2and PLC/PRF/5) cell lines was measuredby the MTT assay. Hepatocellular carcinoma cell-resistant anticancerdrugs (irinotecan hydrochloride and carboplatin) and hepatocellularcarcinoma cell-sensitive anticancer drugs (paclitaxel and etoposide)were selected for further investigation. In conclusion, our presentresults demonstrated that①HepG2and PLC/PRF/5cells secreted higherlevels of HSP60, HSP70and HSP90treated with hepatocellular carcinomacell-resistant anticancer drugs compared with cells exposed tohepatocellular carcinoma cell-sensitive anticancer drugs;②hepatocellular carcinoma cell-resistant anticancer drugs could enhanceexosome release to a higher degree than hepatocellular carcinomacell-sensitive anticancer drugs, and hepatocellular carcinomacell-resistant anticancer drugs seemed to enhance HSP60, HSP70and HSP90exosome-surface expression to a higher degree than hepatocellularcarcinoma cell-sensitive anticancer drugs;③carboplatin-treated HepG2cells-derived exosomes had a stronger effect on NK cell function thanpaclitaxel-treated HepG2cells-derived exosomes. HSPs-bearing exosomeshave potential values of application in HCC chemoimmunotherapy. Thesefindings not only were expected to provide safe and effective sources oftumor vaccines for HCC immunotherapy, but also provided theoretical andexperimental basis for more in-depth clinical applications of HCCchemoimmunotherapy.更多还原