【作者】 李静；

【导师】 王国云；

【作者基本信息】 山东大学 ， 妇产科学， 2022， 硕士

【摘要】 研究背景:子宫内膜异位症(EM)发病率逐年增高,复发率较高,对患者的身心健康产生了严重影响。近年来,手术切除治疗和激素类药物治疗仍是EM的主要治疗方式,但存在不良反应较多、复发率较高的问题。EM的发病机制尚不明确,但腹腔巨噬细胞在EM进展中发挥重要作用。EM患者的腹腔环境中巨噬细胞的数量和活力均有所增高,细胞因子的产生量也增加,但这些巨噬细胞的吞噬功能却显著减弱了,而巨噬细胞吞噬能力减弱的原因及其机制尚不明确。免疫检查点在巨噬细胞吞噬作用中发挥重要作用。巨噬细胞相关免疫检查点CD47-SIRPα通路是固有免疫中重要的自我保护机制,目标细胞表面的CD47与巨噬细胞表面的SIRPα相结合,导致巨噬细胞的吞噬功能减弱,从而实现了目标细胞的免疫逃逸。CD47阻断是肿瘤免疫治疗的新方向,并且已在非霍奇金淋巴瘤、乳腺癌、小细胞肺癌及非肿瘤性疾病—动脉粥样硬化等疾病的治疗中取得了实质性进展。PD-1-PD-L1途径通常被认为是癌细胞逃避适应性免疫应答的关键机制,但也有证据表明,PD-1在肿瘤相关的巨噬细胞表面表达,体内阻断PD-1-PD-L1信号通路可增加巨噬细胞吞噬肿瘤细胞的能力。在许多临床前肿瘤模型中,阻断CD47或PD-L1或两者同时阻断增加了巨噬细胞对肿瘤细胞的吞噬清除,表现出显著的抗肿瘤活性。研究目的:探讨基于巨噬细胞相关免疫检查点阻断治疗EM的可能性,阐明CD47-SIRPα及PD-1-PD-L1通路在EM中的表达情况,进而明确CD47及PD-L1在逃避巨噬细胞吞噬中的作用机理,并利用抗体阻断该通路研究其对EM进程的影响。研究方法:1.组织多重免疫荧光法定位CD47及PD-L1在EM患者在位及异位子宫内膜组织中的表达,并使用RT-PCR定量组织中CD47及PD-L1 mRNA水平的表达。进一步,使用流式细胞术定量原代子宫内膜间质细胞(ESCs)和HESCs细胞系中CD47及PD-L1蛋白水平的表达。2.使用小干扰RNA构建CD47低表达的子宫内膜间质细胞(包括HESCs细胞系及原代ESCs),并将其与人外周血单核细胞诱导来源的巨噬细胞或THP-1细胞系诱导来源的巨噬细胞按1:2的比例直接共培养,共培养24h后利用流式细胞术检测巨噬细胞吞噬ESCs的能力,并分析共培养后ESCs的凋亡改变。3.体外构建子宫内膜间质细胞与巨噬细胞共培养体系,并使用CD47或PD-L1单抗治疗。体外HESCs与巨噬细胞直接共培养时加入CD47中和抗体阻断CD47-SIRPα信号通路或加入PD-L1中和抗体阻断PD-1-PD-L1信号通路,共培养结束后利用流式细胞术检测巨噬细胞吞噬能力的改变。4.体内构建原代ESCs与巨噬细胞共培养体系,并使用CD47单抗治疗。体内共培养即使用CD47中和抗体封闭小鼠原代ESCs表面的CD47抗原,经PKH67染色后将其注射入受体小鼠腹腔形成ESCs异位的状态,12h后获取小鼠腹腔细胞悬液,利用腹腔巨噬细胞常用标记F4/80及CD11b区分大腹腔巨噬细胞(LPMs)及小腹腔巨噬细胞(SPMs),评估CD47阻断后两者吞噬能力的改变,并对异位ESCs的凋亡率进行评估。研究结果:1.CD47在EM异位病灶中表达上调,而PD-L1的表达并未上调。定位结果显示,异位子宫内膜组织中多个区域可以定位到CD47的表达,但仅有少数区域可观察到PD-L1的表达。RT-PCR结果表明,异位子宫内膜中CD47 mRNA的表达明显高于EM在位内膜及对照组在位内膜,且后两者CD47mRNA表达无统计学差异;PD-L1 mRNA水平在正常对照及EM异位及在位内膜组织中均无统计学差异。流式细胞术的结果表明:CD47在EM在位、异位以及正常对照组在位原代ESCs中均有所表达,但在异位ESCs中的表达明显高于后两者,且后两者中CD47表达无统计学差异;HESCs细胞系存在高水平CD47的表达。PD-L1在三组原代细胞中的表达无统计学差异。因此,通过多种方法、不同层面的分析,我们证实了 CD47在EM异位病灶中表达上调,而PD-L1的表达并未上调。2.CD47高表达使ESCs实现了吞噬逃逸及凋亡逃逸。构建CD47低表达的HESCs,并与巨噬细胞进行共培养,结果表明:巨噬细胞对HESCs的吞噬清除能力增强,且后者凋亡率增加。相较于CD47低表达的在位ESCs,CD47高表达的异位ESCs更容易逃脱巨噬细胞的吞噬清除,从而实现免疫逃逸。下调异位ESCs表面CD47的表达后,巨噬细胞对其吞噬清除增强。以上结果表明了 CD47表达量与吞噬逃逸以及凋亡逃逸之间的关系,即CD47高表达使ESCs实现了吞噬逃逸及凋亡逃逸。3.中和抗体阻断CD47增强了巨噬细胞的吞噬能力。体外共培养体系中,阻断CD47增强了巨噬细胞吞噬清除HESCs的能力;在共培养体系中加入PD-L1中和抗体并不能够改变巨噬细胞对HESCs的清除能力,这与EM异位病灶中PD-L1表达未上调的结果相对应。4.CD47阻断增强体内巨噬细胞的吞噬能力和ESCs的凋亡。体内ESCs与巨噬细胞共培养早期,主要是LPMs负责异位ESCs的清除,这与LPMs更早地募集至炎症发生部位相关。使用CD47单抗治疗能够增强LPMs的吞噬能力,同时增加了异位ESCs的凋亡率,从而延缓了 EM的发生。而SPMs的吞噬能力在共培养早期并不因为CD47抗体的治疗而改变。结论:1.CD47在EM异位病灶中表达上调,而PD-L1表达并未上调。2.异位ESCs表面CD47高表达使其实现了吞噬逃逸及凋亡逃逸。3.靶向CD47,增加了巨噬细胞对异位ESCs的吞噬清除并诱导后者凋亡。更多还原

【Abstract】 BackgroundEndometriosis(EM)has a high incidence and recurrence rate,which seriously affects the physical and mental health of patients.The current treatment modalities for EM are mainly limited to surgery and hormonal therapy,and both suffer from a high number of adverse effects and a high recurrence rate.The pathogenesis of EM is unclear,but abdominal macrophages play an important role in the progression of EM.The number and activity of macrophages in the abdominal environment of EM patients are increased,and the secretion of cytokines is also elevated,but the phagocytic function of these macrophages is significantly reduced,and the reasons for the reduced phagocytic capacity of macrophages have not been identified.Immune checkpoints play an important role in macrophage phagocytosis.Macrophage-associated immune checkpoint CD47-SIRPa pathway is an important self-protection mechanism in intrinsic immunity,where SIRPa on the surface of macrophages binds to CD47 on the surface of target cells,thereby attenuating the phagocytosis of target cells by macrophages and enabling immune escape.CD47 blockade is a new direction in tumour immunotherapy and has been used in non-Hodgkin’s lymphoma,breast cancer,small cell lung cancer.The PD-1-PD-L1 pathway is often thought to be a key mechanism by which cancer cells evade adaptive immune responses,but there is also evidence that tumour-associated macrophages also express PD-1 and that in vivo blockade of the PD-1-PD-L1 pathway increases macrophage phagocytosis of tumour cells.In many preclinical tumour models,blockade of CD47 or PD-L1 or both increased phagocytic clearance of tumour cells by macrophages and demonstrated significant anti-tumour activity.ObjectivesTo investigate the possibility of macrophage-based immune checkpoint blockade for the treatment of EM,to elucidate the expression of CD47-SIRPα and PD-1-PD-L1 pathways in EM,and then to clarify the mechanism of the role of CD47 and PD-L1 in evading macrophage phagocytosis,and to investigate their effects on the course of EM using antibody blockade of this pathway.Methods1.Tissue multiplex immunofluorescence was used to localize CD47 and PD-L1 expression in in situ and ectopic endometrial tissues of EM patients,and RT-PCR was used to quantify the expression of CD47 and PD-L1 mRNA levels in the tissues.Further,flow cytometry was used to quantify the expression of CD47 and PD-L1 protein levels in primary endometrial stromal cells(ESCs).2.CD47 low-expressing endometrial stromal cells(including HESCs cell line and primary ESCs)were constructed using small interfering RNA and co-cultured directly with human peripheral blood monocyte-induced macrophages or THP-1 cell line induced macrophages at a ratio of 1:2.After 24 h of co-culture,the ability of macrophages to phagocytose endometrial stromal cells was measured by flow cytometry and the apoptotic changes of endometrial mesenchymal cells were analyzed.3.In vitro co-culture system of endometrial stromal cells and macrophages was constructed and treated with CD47 or PD-L1 monoclonal antibody.In vitro HESCs were co-cultured directly with macrophages by adding CD47 neutralizing antibody to block the CD47-SIRPα signalling pathway or adding PD-L1 neutralizing antibody to block the PD-1-PD-L1 signalling pathway,and the altered phagocytic capacity of macrophages was detected by flow cytometry after co-culture.4.In vivo co-culture of primary ESCs with macrophages was constructed and treated with CD47 monoclonal antibody.In vivo co-culture was performed by using CD47-neutralizing antibody to block CD47 antigen on the surface of primary mouse ESCs,which was stained with PKH67 and then injected into the peritoneal cavity of recipient mice to form an ectopic state of endometrial stromal cells.The changes in phagocytic capacity of both were assessed after CD47 blockade and the apoptotic rate of ectopic endometrial stromal cells was evaluated.Results1.CD47 expression was upregulated in EM ectopic lesions,while PD-L1 expression was not upregulated.The localization results showed that CD47 expression could be localized in several regions of ectopic endometrial tissues,but PD-L1 expression could be observed in only a few regions.RT-PCR results showed that CD47 mRNA expression was significantly higher in ectopic endometrium than in EM eutopic endometrium and control eutopic endometrium,and there was no statistical difference in CD47 mRNA expression between the latter two;PD-L1 mRNA levels were not statistically different in control and EM ectopic and eutopic endometrial tissues.The results of flow cytometry showed that CD47 was expressed in EM eutopic and ectopic as well as in normal control eutopic primary ESCs,but the expression was significantly higher in ectopic ESCs than in the latter two,and there was no statistical difference in CD47 expression in the latter two;PD-L1 expression was not statistically different in the three groups of primary cells.Thus,through multiple methods and different levels of analysis,we confirmed that CD47 expression was up-regulated in EM ectopic lesions,while PD-L1 expression was not.2.High CD47 expression enabled endometrial stromal cells to achieve phagocytic escape and apoptotic escape.Constructs of endometrial stromal cells with low CD47 expression revealed enhanced phagocytic clearance of the former by macrophages and an increased rate of apoptosis in the former.Compared with eutopic ESCs with low CD47 expression,ectopic ESCs with high CD47 expression were more likely to escape phagocytic clearance by macrophages,thus achieving immune escape.The phagocytic clearance of ectopic ESCs by macrophages was enhanced after down-regulation of CD47 expression on their surface.The above results suggest a relationship between CD47 expression and phagocytic escape as well as apoptotic escape,high CD47 expression enabled endometrial stromal cells to achieve phagocytic escape and apoptotic escape.3.Blockade of CD47 with neutralizing antibodies enhanced the phagocytic capacity of macrophages.Blocking CD47 enhanced the phagocytic clearance of HESCs by macrophages in an in vitro co-culture system;the addition of PD-L1 neutralizing antibody to the co-culture system did not alter the clearance of endometrial cells by macrophages,which corresponds to the result that PD-L1 expression was not upregulated in EM ectopic lesions.4.CD47 blockade enhances phagocytosis of macrophages and apoptosis of ESCs in vivo.Early in vivo ESCs co-cultured with macrophages,mainly LPMs were responsible for the clearance of ectopic endothelial stromal cells,which correlated with earlier recruitment of LPMs to the site of inflammation.Treatment with CD47 monoclonal antibody enhanced the phagocytic capacity of LPMs while increasing the rate of apoptosis of ectopic endometrial stromal cells,thereby delaying the onset of EM.In contrast,the phagocytic capacity of SPMs was not altered by treatment with CD47 antibody early in co-culture.Conclusions1.Macrophage-associated immune checkpoint CD47 expression is upregulated in EM ectopic lesions,whereas PD-L1 expression is not upregulated.2.High CD47 expression on the surface of ectopic endometrial cells allowed them to evade macrophage phagocytosis and also apoptosis.3.CD47 mono-antibody treatment increased phagocytic clearance of ectopic endometrial cells by macrophages and induced apoptosis of ectopic endometrial cells.更多还原