【作者】 李明；

【导师】 李刚；

【作者基本信息】 山东大学 ， 外科学（神经外科）（专业学位）， 2024， 博士

【摘要】 研究背景胶质母细胞瘤(Glioblastoma,GBM)系中枢神经系统内最为常见且极具侵袭性的恶性原发性肿瘤,占成人脑肿瘤的60%以上。它具有高度异质性、高耐药性、高复发率和高死亡率等特点。尽管手术、放疗、化疗和电场治疗等手段被广泛采用,但GBM的治疗效果并不理想。患者的生存率没有得到显著改善,诊断后的平均生存期约为15个月,无进展生存期为7.4个月,5年的相对生存率仅为6%左右。GBM的治疗面临多重困难:肿瘤的异质性促进了耐药亚群的形成;血脑屏障的存在阻碍了药物的有效递送;GBM诱导免疫抑制性微环境的形成,促进了免疫逃逸。影响胶质瘤治疗效果的主要障碍之一是免疫抑制性肿瘤微环境(Tumor microenvironment,TME),TME在神经胶质瘤的发生、发展和治疗中扮演着重要的角色。GBM的TME由多种细胞类型组成,包括肿瘤细胞、免疫细胞、神经元和基质细胞,其特征是T细胞浸润减少,耗竭和肿瘤浸润淋巴细胞上抑制受体的高水平表达,这表明抗肿瘤免疫活性受损。因此,需要进一步研究肿瘤微环境网络中细胞间的功能关系,以获得新的治疗方法,实现理想的治疗效果。随着分子生物学技术的突飞猛进,如空间转录组学及单细胞测序技术等,胶质瘤细胞中的基因表达程序受到肿瘤微环境的相互影响的机制正在逐渐被揭示。胶质瘤相关巨噬细胞(Glioma-associated macrophages,GAMs)是肿瘤微环境中分布最广泛的免疫细胞,亦为主要发挥免疫功能的细胞群之一。单核巨噬细胞的募集与激活,多依赖于特定的细胞因子,其来源主要分为两种细胞类型:脑组织常驻的巨噬细胞与骨髓衍生的活化巨噬细胞。GAMs主要包含功能及形态各异的两种活化类型,即经典活化的M1型巨噬细胞与替代性活化的M2型巨噬细胞。两者功能迥异,M1型巨噬细胞主要发挥抑癌及免疫激活作用,而M2型巨噬细胞则主要发挥促癌及免疫抑制作用。研究表明,GAMs已被证实对神经胶质瘤的多种生物学功能具有显著影响,包括但不限于促进肿瘤的生长与侵袭、血管生成、能量代谢及抗药性等。M1和M2巨噬细胞均具有较高的可塑性,能在多种恶性环境下发生极化或相互转化。经过深入研究与技术创新,先进的诊断技术和新发现的生物标志物将为胶质瘤的检测与治疗提供更精确的路径。目前,随着基于细胞囊泡的液体活检技术的快速进步,研究者正积极探索胶质瘤细胞与巨噬细胞间互动的机制,旨在明确肿瘤负担和治疗效果。值得一提的是,细胞外囊泡(Extracellular vesicles,EVs)作为癌症生物标记物的潜在来源,由于具有低侵入性的特点,正成为一个新兴的研究领域。EVs是一类存在于多种体液中的异质性含膜小囊泡群体。它们由各种细胞类型释放,包括癌细胞,携带的内容包括核酸、蛋白质和代谢物,这些成分会根据分泌细胞的病理生理学状态而变化。因此,EVs被视为癌症生物标志物的可靠来源之一。由于可以实现微创采样,EVs正逐渐成为液体活检中一种具有广泛应用价值的方法,不仅能够弥补现有癌症诊断技术的不足,还可以实现疾病的持续监测与评估。非编码 RNA(ncRNAs),包括 microRNAs(miRNAs)、lncRNAs 以及 circRNAs,在细胞外囊泡中担任至关重要的角色,对胶质瘤的进展起着关键性的调控作用。这些分子可调节参与肿瘤细胞增殖、侵袭迁移的蛋白质表达水平,进而促进血管生成和细胞分化。同时,部分非编码RNA分子还具有肿瘤抑制因子的功能。因此,准确识别这些分子在肿瘤组织、血液或脑脊液中的表达水平变化,将为我们提供一种精准而有力的手段,以实现对胶质瘤的精确诊断与个性化治疗。本研究旨在深入探究胶质瘤恶性进展与肿瘤微环境之间的关联。通过先进的测序分析技术,我们发现miR-3591-3p在胶质瘤组织中的表达相较于正常脑组织呈现出明显的下降趋势,然而在脑脊液外泌体中的表达水平却相对较高,这一发现暗示了 miR-3591-3p可能在胶质瘤进展中扮演重要角色。我们的研究从两个方面对这一现象进行了深入探讨。首先,外泌体中的miR-3591-3p能够被巨噬细胞所吞噬,通过激活JAK2/PI3K/AKT/mTOR和STAT3信号通路诱导巨噬细胞向免疫抑制表型转变,进而促进了胶质瘤的侵袭和迁移能力。其次,我们发现miR-3591-3p对胶质瘤细胞周期具有调控作用,能够诱导细胞周期阻滞并促进细胞凋亡,这一作用是通过抑制MAPK通路来实现的,从而有助于抑制胶质瘤的恶性进展。本研究揭示了一种胶质瘤细胞的新型免疫逃避机制。肿瘤细胞通过选择性地将特定的肿瘤抑制分子分泌至外泌体中,进而传递给巨噬细胞,诱导其转变为免疫抑制表型。这一过程有助于创建一个有利于胶质瘤恶性进展的抑制性免疫微环境,为我们深入理解和治疗胶质瘤提供了新的视角和思路。目的通过对胶质瘤组织、脑脊液及正常脑组织中的基因表达水平进行深度测序分析,探究胶质瘤外泌体miRNAs在肿瘤恶性进展及其免疫微环境形成过程中的潜在机制。方法1.对胶质瘤患者肿瘤组织、术前术后脑脊液外泌体,以及开颅手术的脑外伤或脑出血患者正常脑组织进行miRNA测序分析,筛选出具有差异表达的miRNA分子。2.利用qRT-PCR反应、流式细胞术、酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)等技术检测miR-3591-3p对巨噬细胞Ml与M2型标志物表达的影响。3.通过测序分析及数据库预测,确定miR-3591-3p在巨噬细胞的靶基因及下游信号通路。在巨噬细胞中进行敲减和过表达实验,并通过Western Blot、qRT-PCR、流式细胞技术、ELISA等手段对其进行验证。4.通过流式细胞术检测细胞周期、细胞凋亡等探究miR-3591-3p对胶质瘤细胞恶性生物学行为的影响,并利用ELISA实验检测细胞因子水平、Transwell实验检测胶质瘤细胞穿透力来验证巨噬细胞对胶质瘤细胞恶性进展的作用。5.通过测序及数据库预测,确定miR-3591-3p在胶质瘤细胞中的靶基因及下游信号通路。在胶质瘤细胞中进行敲减和过表达实验,通过流式细胞术、Western Blot等实验对其功能进行验证。6.利用裸鼠原位成瘤模型在体内验证miR-3591-3p对巨噬细胞及胶质瘤细胞的影响。通过动物发光成像系统及HE染色技术检测肿瘤生长的大小。利用免疫组化方法检测胶质瘤细胞增殖及M2型巨噬细胞标志物表达水平。结果1.miR-3591-3p在术后脑脊液外泌体中表达量显著下降,且其在肿瘤组织中表达水平低于正常脑组织。2.胶质瘤细胞通过排出含有miR-3591-3p的外泌体促进巨噬细胞向M2型转化。3.miR-3591-3p可以在体外及体内诱导巨噬细胞M2极化并促进胶质瘤侵袭迁移。4.miR-3591-3p 通过靶向 CBLB 调控 JAK2/PI3K/AKT/mTOR 和 JAK2/STAT3 信号通路诱导巨噬细胞M2转化。5.miR-3591-3p可诱导胶质瘤细胞周期阻滞,促进细胞凋亡,抑制肿瘤生长。6.miR-3591-3p通过靶向MAPK1进而调节MAPK信号通路来抑制胶质瘤的生长。结论1.胶质瘤细胞中miR-3591-3p可以被包裹进外泌体中并被巨噬细胞所吞噬,诱导巨噬细胞向M2型转化,并促进胶质瘤的恶性进展。2.外泌体中miR-3591-3p通过抑制巨噬细胞中CBLB的表达,并激活JAK2/PI3K/AKT/mTOR和JAK2/STAT3信号通路促进巨噬细胞M2型转化。3.miR-3591-3p具有抑制胶质瘤细胞周期阻滞、促进细胞凋亡的作用,其机制是通过靶向MAPK1,进而抑制MAPK分子通路实现的。更多还原

【Abstract】 BackgroundGlioblastoma(GBM)is the most aggressive and common malignant primary tumor in the central nervous system,making up over 60%of adult brain tumors.It is characterized by high heterogeneity,drug resistance,recurrence,and mortality rates.Despite the widespread use of surgical resection,radiotherapy,chemotherapy,and electric field therapy,the treatment outcomes of GBM remain unsatisfactory.Patient survival has not significantly improved,with a median survival time of approximately 15 months,a progression-free survival of 7.4 months,and a relative 5-year survival rate of only around 6%.The treatment of GBM faces multiple challenges:tumor heterogeneity promotes the formation of drug-resistant subpopulations;the presence of the blood-brain barrier hinders effective drug delivery;and GBM induces the formation of an immunosuppressive microenvironment,facilitating immune evasion.One of the main obstacles affecting the treatment outcomes of glioma is the immunosuppressive tumor microenvironment(TME),which plays a crucial role in the genesis,progression,and treatment of glioma.The TME of GBM is composed of various cell types,including tumor cells,immune cells,neurons,and stromal cells.It is characterized by reduced T-cell infiltration,exhaustion,and high levels of inhibitory receptors on tumor-infiltrating lymphocytes,indicating impaired antitumor immune activity.Therefore,further research is needed to investigate the functional relationships among cells within the tumor microenvironment network to develop new therapeutic strategies and achieve ideal treatment outcomes.With the rapid advancement of molecular biology techniques,such as spatial transcriptomics and single-cell sequencing,the mechanisms underlying the reciprocal interactions between the genetic expression programs of glioma cells and the tumor microenvironment are gradually being uncovered.Glioma-associated macrophages(GAMs)are the most widely distributed immune cells in the tumor microenvironment and are also one of the main cell groups exerting immune function.The recruitment and activation of monocytes and macrophages mainly depend on specific cytokines,and their sources are mainly divided into two cell types:resident macrophages in brain tissue and activated macrophages derived from bone marrow.GAMs mainly contain two activated types with different functions and morphologies,namely classically activated M1 macrophages and alternatively activated M2 macrophages.The two types have distinct functions.M1 macrophages mainly exert anti-cancer and immune activation effects,while M2 macrophages mainly exert cancer-promoting and immune suppressive effects.Studies have shown that GAMs have been demonstrated to have significant effects on various biological functions of glioma,including but not limited to promoting tumor growth and invasion,angiogenesis,energy metabolism,and drug resistance.Both M1 and M2 macrophages have high plasticity and can polarize or transform into each other in various malignant environments.With deep research and technological innovation,advanced diagnostic techniques and newly discovered biomarkers will provide more precise paths for the detection and treatment of glioma.Currently,with the rapid progress of cell vesicle-based liquid biopsy technology,researchers are actively exploring the mechanism of interaction between glioma cells and macrophages,aiming to clarify tumor burden and treatment effects.It is worth mentioning that extracellular vesicles(EVs)are emerging as a potential source of cancer biomarkers due to their low invasiveness.EVs are a heterogeneous group of membrane-enclosed small vesicles that exist in various body fluids.They are released by various cell types,including cancer cells,and carry cargoes including nucleic acids,proteins,and metabolites,which vary according to the biopathological state of the secreting cells.Therefore,EVs are considered as one of the reliable sources of cancer biomarkers.Due to the possibility of minimally invasive sampling,EVs are gradually becoming a widely used method in liquid biopsy,which can not only compensate for the deficiencies of existing cancer diagnostic techniques but also enable continuous monitoring and evaluation of the disease.Non-coding RNAs(ncRNAs),including microRNAs(miRNAs),IncRNAs,and circRNAs,play crucial roles in extracellular vesicles and have key regulatory effects on the progression of glioma.These molecules can regulate protein expression levels involved in tumor cell proliferation,invasion,and migration,thereby promoting angiogenesis and cell differentiation.Meanwhile,some non-coding RNA molecules also function as tumor suppressors.Therefore,accurately identifying changes in the expression levels of these molecules in tumor tissue,blood,or cerebrospinal fluid will provide us with a precise and powerful means for accurate diagnosis and personalized treatment of glioma.This study aims to further explore the association between the malignant progression of glioma and the tumor microenvironment.Through advanced sequencing analysis techniques,we found that the expression of miR-3591-3p in glioma tissue shows a significant downward trend compared to normal brain tissue,but its expression level in cerebrospinal fluid exosomes is relatively high.This finding indicates that miR-3591-3p could potentially have a significant impact on the advancement of glioma.Our study investigated this phenomenon from two aspects.First,miR-3591-3p in exosomes can be phagocytosed by macrophages,inducing the transformation of macrophages into an immunosuppressive phenotype through the activation of JAK2/PI3K/AKT/mTOR and STAT3 signaling pathways,which promotes the invasion and migration abilities of glioma.Second,we found that miR-3591-3p has a regulatory effect on the glioma cell cycle,able to induce cell cycle arrest and promote apoptosis,which is achieved by inhibiting the MAPK pathway,thereby helping to inhibit the malignant progression of glioma.This study reveals a novel immune evasion mechanism of glioma cells.Tumor cells selectively secrete specific tumor suppressor molecules into exosomes and then transfer them to macrophages,inducing their transformation into an immunosuppressive phenotype.This process helps create an inhibitory immune microenvironment conducive to the malignant progression of glioma,providing new perspectives and ideas for our understanding and treatment of glioma.ObjectivesTo investigate the potential mechanism of glioma-derived exosomal miRNAs in tumor malignancy progression and immune microenvironment formation through deep sequencing analysis of gene expression levels in glioma tissue,cerebrospinal fluid,and normal brain tissue.Methods1.miRNA sequencing analysis was performed on glioma tumor tissue,preoperative and postoperative cerebrospinal fluid exosomes,and normal brain tissue from craniotomy patients with traumatic brain injury or cerebral hemorrhage to identify differentially expressed miRNA molecules.2.qRT-PCR,flow cytometry,and enzyme-linked immunosorbent assay(ELISA)were used to detect the effect of miR-3591-3p on the expression of M1 and M2 markers in macrophages.3.Through sequencing analysis and database prediction,the target genes and downstream signaling pathways of miR-3591-3p in macrophages were determined.Knockdown and overexpression experiments were performed in macrophages,and their validation was conducted using Western Blot,qRT-PCR,flow cytometry,and ELISA.4.The effect of miR-3591-3p on the malignant biological behavior of glioma cells was explored using flow cytometry to detect cell cycle,apoptosis,and ELISA and Transwell experiments to assess the role of macrophages in glioma cell malignancy.5.miR-3591-3p target genes and downstream signaling pathways in glioma cells were identified through sequencing and database prediction.Knockdown and overexpression experiments were conducted in glioma cells,and their function was validated using flow cytometry and Western Blot.6.The in vivo effect of miR-3591-3p on macrophages and glioma cells was validated using a nude mouse orthotopic tumor model.Tumor growth was detected using an animal luminescence imaging system and HE staining.Immunohistochemical methods were used to detect glioma cell proliferation and M2 macrophage marker expression levels.Results1.The expression of miR-3 591-3p was significantly reduced in postoperative cerebrospinal fluid exosomes and its expression level was lower in tumor tissue compared to normal brain tissue.2.Glioma cells promote macrophage polarization towards the M2 phenotype by releasing exosomes containing miR-3591-3p.3.miR-3591-3p can induce M2 polarization of macrophages and promote glioma invasion and migration both in vitro and in vivo.4.miR-3591-3p induces macrophage M2 polarization by targeting CBLB and regulating JAK2/PI3K/AKT/mTOR and JAK2/STAT3 signaling pathways.5.miR-3591-3p can induce glioma cell cycle arrest,promote apoptosis,and inhibit tumor growth.6.miR-3591-3p inhibits glioma growth by targeting MAPK1 and regulating the MAPK signaling pathway.Conclusion(1)miR-3591-3p in glioma cells can be encapsulated in exosomes and phagocytosed by macrophages,inducing M2 macrophage polarization and promoting the malignant progression of glioma.(2)Exosomal miR-3591-3p promotes macrophage M2 polarization by inhibiting the expression of CBLB in macrophages and activating JAK2/PI3K/AKT/mTOR and JAK2/STAT3 signaling pathways.(3)miR-3591-3p has the effect of inhibiting glioma cell cycle arrest and promoting apoptosis,and its mechanism is achieved by targeting MAPK1,thereby inhibiting the MAPK molecular pathway.更多还原