【作者】 刘佳；

【导师】 毛海婷；

【作者基本信息】 山东大学 ， 肿瘤学， 2020， 博士

【摘要】 研究背景白细胞介素-35（IL-35）是IL-12家族的新成员,是由EBI3（Epstein-Barr virus-induced gene 3）和p35两个亚基组成的异源二聚体细胞因子。IL-35与IL-10、TGF-p同为免疫抑制性细胞因子。其生物学功能主要包括:（1）抑制T淋巴细胞或B淋巴细胞的增殖;（2）促进Treg细胞的增殖:（3）抑制Thl7细胞的极化和IL-17的产生;（4）诱导初始T淋巴细胞或B淋巴细胞转化为分泌IL-35的Treg（IL-35 induced regulatory Tcell,iTR35）或Breg（1L₃5+Breg）。基于IL-35的生物学功能,其在免疫抑制的调节及免疫耐受微环境的形成中可能发挥着重要作用。为深入探讨IL-35在免疫耐受形成中的作用,本研宂分别选取了两个典型-的免疫耐受微环境:病理性耐受微环境（乳腺肿瘤局部微环境）和生理性耐受环境（妊娠环境）作为研究对象,分别研究IL₃5在机体的病理和生理不同状态下对微环境内细胞的诱导及免疫耐受状态的维持作用。肿瘤微环境（Tumormicroenvironment,TME）是由肿瘤基质细胞（包括肿瘤浸润淋巴细胞、成纤维细胞和血管内皮细胞等）、细胞外基质和可溶性分子等共同构成的肿瘤细胞生活的特殊环境。在肿瘤发展的过程中,TME中的免疫细胞受到周围环境的训导,逐渐转化为抑制性免疫细胞,从而促进了局部免疫耐受状态的形成,这对于肿瘤的进展和远处转移起着不可或缺的作用。我们的前期研究发现,乳腺肿瘤细胞及TME中的调节性T细胞（Regulatory Tcell,Treg）均可表达和分泌免疫抑制性细胞因子IL-35。乳腺肿瘤组织中1L-35水平升高被认为是预后不良的独立因素。然而,肿瘤局部高水平的IL-35通过何种方式促进了肿瘤的发展和演进?IL-35是否可通过影响乳腺TME中的基质细胞,如肿瘤浸润淋巴细胞和血管内皮细胞,对肿瘤的进展发挥调节作用,尚不清楚。作为细胞间交流的重要媒介,外泌体（Exosomes）是细胞分泌的一种胞外膜泡,具有脂质双分子层膜结构,起着向特定靶细胞传递基因信息和进一步调节其表型和生物学过程的作用。肿瘤细胞能够通过外泌体影响肿瘤细胞的增殖代谢,免疫逃逸和远处转移。在这一过程中,血管生成对于肿瘤细胞的局部增殖和逃逸到血流进而建立远处转移有着重要意义。在TME中,由肿瘤细胞分泌的、富含蛋白和核酸的外泌体是否充当了肿瘤细胞和内皮细胞之间交流的关键介质,帮助肿瘤细胞调节血管生成?另一方面,乳腺TME中富含大量的肿瘤浸润淋巴细胞,包括T淋巴细胞和B淋巴细胞。肿瘤局部微环境中具有免疫抑制作用的、高浓度的IL-35是否调节了这些淋巴细胞向抑制性细胞的转化（如转化为iTR35和IL-35+Breg）和局部免疫耐受的形成?均有待于进一步的研究证明。与肿瘤局部的免疫耐受微环境相似,妊娠早期的母胎界面也存在着大量具有抑制功能的免疫细胞和抑制性细胞因子,形成特殊的“免疫赦免”状态,避免母体对胎儿的排斥。例如,处于母体蜕膜的Treg细胞可抑制局部免疫活性、支持母体血管增殖,从而促进滋养层的侵袭和胎盘血液的供应。另一方面,妊娠期间,母体外周免疫系统也存在着大量抑制性免疫细胞,如Treg细胞和调节性B细胞（Regulatory B cell,Breg）。作为妊娠外周免疫耐受的重要组成部分,Breg细胞可以促进Treg细胞扩增、抑制Thl分化和树突状细胞成熟,同时降低炎性因子,如TNF-a和IL-6的产生。在妊娠期间的外周免疫耐受中发挥着重要作用。我们的前期研究发现,除Treg细胞和乳腺肿瘤细胞外,人早孕滋养细胞也可表达和分泌IL₃5,妊娠早期的孕妇外周血中IL-35水平显著升高。因此,我们推测由滋养细胞分泌的IL-35可能诱导了T和B淋巴细胞向iTR35或IL-35+Breg的转化,从而在母胎局部和母体外周发挥免疫调节作用,确保妊娠的顺利进行。在本研究中,我们分别构建了乳腺肿瘤小鼠模型和自然流产小鼠模型:并通过体内、体外实验分别探索IL-35在病理和生理状态下促进免疫抑制、诱导免疫耐受微环境形成的“双刃剑”作用及其机制。本研究不仅丰富和补充了IL-35的研究范围,也为乳腺肿瘤和习惯性流产的治疗提供了新的方向和治疗靶点。第一0分IL-35通过调节乳腺肿瘤细胞来源外泌体的mRNA表达谱促进肿瘤血管生成目的:在肿瘤发展演进和转移的过程中,血管生成对于肿瘤细胞逃逸到血流中和建立远处转移是必不可少的。现有的研究证明,肿瘤细胞可通过分泌各种生物活性介质促进肿瘤血管的生成。外泌体（exosomes）是近年来发现的具有双层磷脂分子膜的细胞外囊泡,能够由大多数细胞分泌并被受体细跑吞噬。对乳腺TME的研宂表明,肿瘤细胞来源的外泌体可以通过与周围的基质细胞交流向受体细胞传递特定信息,从而改善肿瘤细胞的生长环境,促进肿瘤的生长。我们之前的研究表明,乳腺肿瘤细胞和肿瘤局部浸润淋巴细胞均可表达抑制性细胞因子IL-35。在乳腺肿瘤微环境中,肿瘤局部高浓度的IL-35对乳腺肿瘤细胞自身的活性和功能有何影响?经IL-35诱导后的乳腺肿瘤细胞分泌的外泌体是否可对TME中的血管内皮细胞起到调节作用?为回答以上问题,本部分研究将着重探讨乳腺肿瘤细胞分泌的外泌体在TME中的血管生成作用,以及IL-35对这一过程的影响。方法:1.采用超速离心法分离乳腺肿瘤细胞外泌体;通过透射电子显微镜和纳米颗粒跟踪分析技术鉴定外泌体的形态和直径;通过Western blotting的方法检测外泌体表面标志蛋白CD9和TSG10]的表达情况。2.应用P1CH67荧光染料标记分离得到的外泌体,鬼笔环肽标记人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells,HUVECs）,结合激光共聚焦显微镜观察HUVECs对外泌体的摄取。3.用乳腺肿瘤细胞来源的外泌体（con-Exo）和IL-35刺激后的肿瘤细胞分泌的外泌体（IL-35-Exo）分别诱导HUVECs,利用CCK8及流式细胞术检测HUVECs的增殖活性及细胞周期变化。4.con-Exo和IL-35-Exo分别诱导HUVECs,通过成管试验及鸡胚绒毛尿囊膜血管试验分析HUVECs的成管能力变化。5.采用基因芯片技术分析con-Exo和IL-35-Exo内mRNA表达谱,并筛选出显著差异的基因。6.通过生物信息学手段对差异表达的基因进行聚类分析和功能富集,得到参与外泌体调控血管生成的相关基因和信号通路。并通过构建蛋白互作（Protein-protein interaction,PPI）网络,筛选出关键中枢基因。结果:1.从乳腺肿瘤细胞中分离得到的外泌体为典型双层囊泡结构,直径主要分布在lOOnm左右,可表达外泌体表面标志蛋白CD9和TSGI01。2.乳腺肿瘤细胞来源的外泌体可被HUVECs内吞并且主要富集在细胞质中。3.CCK-8结果显示,乳腺肿瘤细胞来源外泌体显著促进HUVECs增殖;流式细胞术分析结果显示乳腺肿瘤细胞来源外泌体显著增加了处于S期和G2/M期的HUVECs细胞数量。与阴性对照组的外泌体（con-Exo）相比,IL-35-Exo对细胞的增殖活性和周期具有更加明显的促进作用。4.成管试验结果显示,来自乳腺肿瘤细胞的外泌体显著增加了HUVECs网络结构中的总分支长度。在鸡胚绒毛尿囊膜血管试验中,con-Exo和IL-35-Exo均能促进鸡胚绒毛尿囊膜分支血管的形成。与con-Exo组相比,IL-35-Exo刺激组形成更多的微血管。5.通过比较Exo和IL-35-Exo内mRNA表达谱,确定了3508个显著差异表达基因。聚类分析和功能富集结果显示,这些差异基因参与血管生成和细胞增殖等GO项目,并在RAS和PPAR信号通路中发挥作用。PPI网络分析进一步筛选出R-RAS,INSR和KDR等关键中枢基因。结论:本部分的研究表明,乳腺肿瘤细胞来源的外泌体可以被HUVECs内吞,并促进肿瘤微环境中的血管生成。IL-35通过改变乳腺肿瘤细胞来源外泌体的mRNA增强其血管生成作用。本研究证实,IL-35可改变肿瘤细胞的核酸信息,并通过其分泌的外泌体间接促进肿瘤血管的形成和肿瘤的发展。第二部分IL-35通过诱导乳腺肿瘠撖环境中淋巴细胞的转化促进肿瘤进展目的:乳腺肿瘤的复发和转移是造成不良预后的重要原因,其主要机制包括肿瘤细胞耐药、肿瘤免疫耐受微环境的形成和肿瘤细胞逃脱免疫监视等。在肿瘤免疫学中,免疫效应细胞和免疫抑制细胞之间的平衡在肿瘤发生和进展中发挥着至关重要的作用。肿瘤细胞可驯化TME中的免疫细胞,诱导产生免疫抑制性细胞亚群,从而加速免疫逃逸,导致肿瘤恶性进展。最近的研究已经确定了一系列的免疫细胞,如调节性T细胞（Treg）和调节性B细胞（Breg）等,它们是抑制抗肿瘤免疫反应和促进肿瘤进展的关键免疫调节因素。乳腺肿瘤细胞及乳腺TME中的Treg均可表达和分泌免疫抑制性细胞因子IL-35。肿瘤组织内IL-35水平升高被认为是预后不良的因素。我们的体外研究已证实,乳腺肿瘤细胞分泌的IL-35可通过介导T细胞向iTR35细胞的转化。另一方面,IL-35也可抑制B细胞的增殖,并介导B细胞转化为可分泌IL-10和IL-35的Breg细胞亚群,即丨L-35+Breg。乳腺TME中高水平的IL-35是否会通过诱导TME的浸润淋巴细胞转化为抑制性淋巴细胞,进而促进免疫抑制的形成?本研宂通过建立小鼠荷瘤模型,模拟高IL-35的肿瘤微环境,通过体内实验探索IL-35对乳腺TME中浸润淋巴细胞分化的诱导作用,以及IL-35对TME血管生成、肿瘤局部进展及远处转移的影响。方法:1.将小鼠乳腺癌4T1细胞悬液接种到BALB/c雌鼠胸壁皮下,构建小鼠荷乳腺肿瘤模型。2.将成瘤的BALB/c小鼠随机分为两组,对照组肿瘤给予PBS注射,实验组肿瘤给予IL-35注射,观察并记录肿瘤的生长曲线。3.利用磁珠分离CD19+B和CD4+T细胞,流式细胞术检测两组小鼠肿瘤内淋巴细胞分化情况。4.免疫组织化学方法检测不同组别中与细胞增殖相关的核抗原Ki67和血管内皮细胞标志物CD31表达,并对其在肿瘤组织中的表达强度和范围进行评分用以评估肿瘤增殖情况及血管密度。同时分离小鼠肺脏,免疫组织化学方法检测肺转移瘤情况。结果:1.与对照组相比,IL-35处理组的小鼠肿瘤生长速度较快且肿瘤体积显著增大。免疫组化结果显示,IL-35处理组肿瘤组织内Ki67阳性细胞的比例明显升高。2.与对照组相比,IL-35处理组小鼠肺脏乳腺肿瘤转移灶明显增多。3.流式细胞术检测结果显示,IL-35可诱导乳腺肿瘤TME中的淋巴细胞转化为iTR35及IL-35+Breg。4.免疫组化结果显示,IL-35处理组小鼠乳腺肿瘤内CD3]阳性细胞比例显著高于对照组。结论:本部分研究证实,IL-35可通过直接刺激肿瘤细胞增殖、诱导肿瘤局部免疫抑制性淋巴细胞的转化,以及间接促进肿瘤血管的生成,形成有利于肿瘤生长的微环境,为肿瘤细胞的发展和转移创造有利条件。第三部分IL-35通过诱导淋巴细胞转化维持麵期免疫耐受目的:在第二部分的研究中,我们主要探讨了IL-35在病理性环境（肿瘤微环境）中促进局部免疫耐受的作用和机制。本部分研究则主要聚焦于IL-35在生理性环境（妊娠）中促进免疫耐受的作用。妊娠早期的母体中存在大量免疫耐受淋巴细胞与抑制性细胞因子,与肿瘤的免疫耐受特性相似。妊娠期的免疫耐受主要包括母胎界面免疫耐受及外周免疫耐受。母胎界面中富含大量的T细胞。滋养层细胞可通过分泌IL-10、胸腺基质淋巴细胞生成素等细胞因子,调节ThlATi2平衡,增加Th2细胞含量,并抑制母胎界面Thl7细胞的免疫效应。我们先前的研究表明,妊娠早期滋养细胞可表达并分泌免疫抑制细胞因子IL-35,并且早孕妇女外周血中IL-35水平显著升高。考虑到IL-35的免疫抑制作用及其在滋养层细胞中的表达,我们推测母胎界面中IL-35可能通过诱导iTR35细胞分化参与维持母胎耐受。此外,作为抑制性免疫细胞的重要组成部分,Breg细胞可通过调节外周免疫应答在自身免疫性疾病和感染中发挥调节作用。作为Breg细胞的重要亚群,IL-10+Breg细胞（也被称为B10）通过分泌IL-10来发挥免疫抑制功能。BIO细胞在小鼠脾脏中呈现CD19+CD5+CDldhi表型。此外,IL-35可抑制B细胞的增殖,并介导B细胞转化为一种新的Breg细胞亚群:IL-35+Breg,该亚群通过分泌IL-35发挥免疫抑制作用。在前一部分的研究中我们证实,IL-35可通过促进乳腺肿瘤浸润的iTR35及IL-35+Breg细胞扩增促进肿瘤进展及肿瘤局部免疫耐受的形成。本部分研究将通过妊娠小鼠模型进一步探讨IL-35在促进T和B淋巴细胞转化,维持妊娠期免疫耐受中的作用。由于B淋巴细胞很少存在于母胎界面,因此我们主要研究了Breg细胞在妊娠期间外周免疫耐受中的调节作用。此外,妊娠期间,母体外周血中雌二醇（E2）及人绒毛膜促性腺激素（hCG）水平发生剧烈变化,其在免疫反应调控中的作用也在以往的研究中得到了证实。在本部分研究中,我们也探讨了这些妊娠相关激素在抑制性淋巴细胞转化中的作用和相关机制。方法:1.构建自然流产小鼠模型CBA/JXDBA/2J和正常妊娠小鼠模型CBA/JxBALB/c。2.通过Westen blotting的方法检测孕鼠母胎界面IL-35含量。流式细胞术检测孕鼠脾脏内BI0细胞的比例以及母胎界面iTR35含量。3.将小鼠重组IL-35细胞因子及IL-35中和性抗体分别对不同组孕鼠进行腹腔注射治疗,观察其对流产的影响以及母胎界面iTR35含量变化。4.提取未孕鼠脾脏原代CD19+B细胞,在体外用妊娠相关激素（人绒毛膜促性腺激素hCG,雌激素E2）及IL-35进行培养及诱导。采用CCK8检测诱导后的B细胞增殖情况。流式细胞术分析诱导后B10和IL-35+Breg的比例变化。5.通过Westen blotting的方法检测妊娠相关激素（人绒毛膜促性腺激素hCG,雌激素E2）及IL-35诱导产生B10和IL-35+Breg的相关信号通路和转录因子。结果:1.自然流产孕鼠胚胎丢失率明显高于正常妊娠孕鼠。2.自然流产孕鼠母胎界面IL-35水平和iTR35细胞的比例显著低于正常妊娠孕鼠。3.IL-35治疗显著提髙了母胎界面iTR35含量并抑制流产的发生;而IL-35中和性抗体则降低了母胎界面iTR35含量并增加了胎盘丢失率。4.正常妊娠孕鼠外周B10细胞明显扩增,而自然流产孕鼠的外周B10细胞比例则显著低于正常妊娠孕鼠。5.IL-35和hCG均能抑制小鼠脾脏B细胞的增殖。IL-35可激活B细胞内的STAT1和STAT3蛋白,诱导其转化生成B10及IL-35+Breg。hCG可促使小鼠B细胞内的STAT3磷酸化,诱导其转化生成BIO。E2在小鼠脾脏B细胞增殖及诱导转化方面无显著作用。结论:本部分研究证实,IL-35可分别在母胎界面和母体外周诱导抑制性iTR35细胞和IL-35+Breg、B10细胞产生,从而促进了母胎局部及妊娠期外周的免疫耐受形成。妊娠相关激素hCG也在外周B10细胞的诱导中发挥着重要作用。我们的研究为IL-35在治疗复发性流产中的应用提供了新的依据,具有重要的临床应用价值。更多还原

【Abstract】 BackgroundInterleukin-35（IL-35）is a new member of the IL-12 family which consists of two subunits: EBI3（Epstein Barr virus induced gene 3）and p35.It was found that IL-35 can act as immunosuppressive cytokines and participate in the formation of immune tolerance environment.In this study,the typical pathological（breast cancer）and physiological（pregnancy）immune environments were selected as the research objects to observe the induction of IL-35 on cells’ 5 transformation in the microcirculation and the maintenance of immune tolerance in different states.Tumor microenvironment（TME）is a special environment for tumor cells,which is composed of tumor stromal cells（including tumor infiltrating lymphocytes,fibroblasts,vascular endothelial cells,etc.）,extracellular matrix and soluble molecules.TME plays an indispensable role in the local progression and distant metastasis of tumor,and participates in the regulation of tumor occurrence,development and drug response.Breast cancer cells and regulatory T cells（Treg）in TME can express and secrete immunosuppressive cytokine IL-35.The high level of IL-35 in breast cancer is considered as an independent factor of poor prognosis.However,it is not clear whether IL-35 can regulate tumor progression by inducing stromal cells（such as tumor infiltrating lymphocytes and vascular endothelial cells）in breast TME.As an important medium of cell communication,exosomes are extracellular vesicles secreted by cells,which have the structure of lipid bimolecular membrane,and participate in the transport of specific gene information to target cells and the regulation of their phenotype and biological process.New research shows that tumor cells can affect infiltrating lymphocytes and vascular endothelial cells in TME by secreting cytokines and exosomes,and then regulate the metabolism,immune escape and distant metastasis of tumor cells.Therefore,the exploration of breast cancer TME provides a new clue for the search of tumor molecular markers and therapeutic targets.At the same time,there are a lot of regulatory B cells（Breg）and Treg cells in breast TME.Breg and Treg can inhibit inflammation,maintain immune tolerance,and regulate immune response in inflammation,autoimmune diseases and transplantation rejection.Previous studies have confirmed that IL-35 inhibits the proliferation of B cells and T cells and induces them into special Breg cell subsets（IL-35 + Breg）and Treg cell subsets（iTR35）that play an immunomodulatory role by seCTeting IL-35.Therefore,in order to clarify the induction effect of IL-35 in breast TME,this study explore whether IL-35 can change the mRNA expression profile of tumor cell-derived exosomes,promote the angiogenesis in breast TME;or induce the transformation of T and B lymphocytes in breast TME,maintain immune tolerance microenvironment,help tumor cells escape immune surveillance and attack,accelerate the tumor progression and lung metastasis by simulating the tumor microenvironment witii high IL-35.Similar to tumor immune tolerance,there are large numbers of immune tolerant lymphocytes and suppressive cytokines in early pregnancy,which form a special"immune tolerant55 state.As an important part of peripheral immune tolerance in early pregnancy,Breg cells can promote Treg cell expansion,inhibit Thl differentiation and dendritic cells maturation while reducing the production of pro-inflammatory factors,such as TNF-a and IL-6.Treg cells inhibit effector immunity and support maternal vascular adaptations,thereby facilitating trophoblast invasion and placental access to the maternal blood supply.Therefore,Treg and Breg cells are considered to be highly related to pregnancy immune tolerance.Previous studies have found that the level of IL-35 in pregnant women’s peripheral blood is increased,and IL-35 is expressed by human trophoblasts in early pregnancy.IL-35 can induce B and T cell transformation into IL-35+Breg and iTR35.We supposed that IL-35 may play an immune-modulatory role by inducing B and T lymphocytes conversion during pregnancy.In this study,we explored the role of BIO and IL-35+Breg,in maintaining maternal-fetal and peripheral immune tolerance,and further investigated the effect of pregnancy related hormones（hCG and E2）and IL-35 on BIO and IL-35+Breg cells.In the present study,we selected breast cancer microenvironment and pregnancy immunity,two typical immune tolerance environments,as research objects to explore the regulation of immunosuppressive cytokine IL-35 on angiogenesis and the induction of B cells in pathological or physiological microenvironment.Our study provides a new clue to further exploring the interaction between tumor cells and stromal cells in breast cancer TME,and is of great significance for elucidating the unique mechanisms of tumor cell immune escape and distant metastasis.Part I EL-35 promotes tumor angiogenesis by regulating the mRNA expression profile of exosomes derived from breast cancer cellsObjective:In the process of tumor development and metastasis,angiogenesis is essential for the escape of tumor cells into blood stream and distant metastasis establishment.It has been proved that tumor cells can promote tumor angiogenesis by secreting a variety of bioactive mediators.Exosomes is an extracellular vesicle with bilayer phospholipid membrane,which can be secreted by most cells and internalized by recipient cells.The study of breast TME shows that exosomes from tumor cells can communicate with the surrounding stromal cells to transmit specific information to the recipient cells,so as to improve the growth environment of tumor cells and promote tumor growth.Our previous studies showed that both breast cancer cells and tumor infiltrating lymphocytes can express suppressor cytokine IL-35.In the breast tumor microenvironment,IL-35 secreted by tumor cells and infiltrating lymphocytes mediates the transformation of tumor local T lymphocytes into suppressive iTr35 cells,further promoting and expanding the positive feedback regulation of tumor local immunosuppression.In addition to the regulation of lymphocyte transformation,whether the high concentration of IL-35 has an effect on the activity and function of breast cancer cells? Can exosomes secreted by IL-35-stimulated breast tumor cells regulate vascular endothelial cells in TME? In order to answer the above questions,this part of the study will focus on the angiogenesis of exosomes secreted by breast cancer cells in TME and the effect of IL-35 on this process.In order to answer the above questions,this part of the study will focus on the angiogenesis of exosomes secreted by breast cancer cells in TME and the effect of IL-35 on this process.Methods:1.The exosomes of breast cancer cells were isolated by differential centrifugation;the morphology and diameter of exosomes were identified by transmission electron microscopy and nanoparticle tracking analysis;the expression of CD9 and TSG101 were detected by Western blotting.2.Exosomes were dyed with PKH67,human umbilical vein endothelial cells（HUVECs）were labeled by phalloidin.Internalization of exosomes by HUVECs was observed by laser confocal microscopy.3.HUVECs were cultured with exosomes from breast cancer cells（con-exo）or exosomes stimulated by IL-35（IL-35-exo）.The proliferation activity and cell cycle of HUVECs were detected by CCK8 and flow cytometry.4.HUVECs were induced by con-35-exo and IL-35-exo respectively.Tube form abilities of HUVECs were analyzed by tube formation test and chick chorioallantoic membrane model.5.Gene chip technology was used to analyze the mRNA expression profiles in Con-exo and IL-35-exo,and the significant different genes were screened out.6.By using bioinformatics to cluster and enrich the differentially expressed genes,the related genes and signal pathways involved in angiogenesis were obtained.By constructing protein protein interaction（PPI）network,key central genes were screened out_Results:1.The exosomes were typical double-layer vesicles with diameter of about 100 nm and can express CD9 and TSG101.2.Exosomes derived from breast tumor cells can be internalized by HUVECs and mainly enriched in cytoplasm.3.The results of CCK-8 showed that the exosomes from breast cancer cells significantly promoted the proliferation of HUVECs;the results of flow cytometry showed that the exosomes from breast cancer cells significantly increased the number of HUVECs cells in S and G2 / M stages.Compared with the negative control group,IL-35-exo has a more significant effect on cell proliferation.4.Tube formation results showed that exosomes from breast cancer cells significantly increased the total branch length of HUVECs network.Both con-exo and IL-35-exo can promote the formation of chorioallantoic branching vessels.Compared with con-exo,IL-35-exo has more obvious effect on angiogenesis.5.By comparing the mRNA expression profiles of Con-exo and IL-35-exo,3508 significantly different expressed genes were identified.Cluster analysis and functional enrichment showed that these genes were significantly enriched in RAS and PPAR signaling pathways.PP1 network analysis further identified hub genes such as R-Ras,INSR and KDR.Conclusion:Our study demonstrates that exosomes derived from breast cancer cells can be internalized by HUVECs and promote angiogenesis in tumor microenvironment.IL-35 can enhance the angiogenesis by changing the mRNA of exosomes.This study provides new clues for better understanding the role of tumor secreting exosomes and IL-35 in tumor microenvironment and breast tumor progression.Part II IL-35 promotes tumor progression by inducing lymphocyte transformation in breast tumor microenvironmentObjective:Recurrence and metastasis of breast cancer are the important reasons for its poor prognosis.The main mechanisms include drug resistance of tumor cells,tumor immune tolerance microenvironment formation and tumor cells escaping immune surveillance.In tumor immunology,the balance between immune effector cells and immunosuppressive cells plays an important role in tumor development.Tumor cells can acclimate the immune cells in TME,induce the generation of immunosuppressive cell subsets,thus accelerate the immune escape and lead to tumor malignant progress.Recently,a series of immune cells have been identified,such as Treg and Breg,which are the key immune modulatory factors to inhibit anti-tumor immune response and promote tumor progression.Breast tumor cells and Treg in TME can express and secrete IL-35.The increased level of IL-35 in tumor tissue is considered to be a poor prognostic factor.Our in vitro study has confirmed that IL-35 secreted by breast cancer cells can induce the formation of local immune tolerance microenvironment by mediating the transformation of T cells into 1Tr35 cells.On the other hand,IL-35 can also inhibit the proliferation of B cells,and mediate the transformation of B cells into Breg cell subsets that can secrete IL-10 and IL-35,which is IL-35+Breg.Whether the high level of IL-35 in breast TME affects tumor progression by inducing lymphocyte differentiation in TME? The purpose of this study was to investigate the effect of IL-35 on the differentiation of infiltrating lymphocytes in breast TME by establishing tumor bearing mice model and simulating tumor microenvironment with high IL-35.At the same time,the effects of IL-35 on angiogenesis,local tumor progression and distant metastasis were studied by in vivo experiments.Methods:1.The breast cancer model of BALB/c mice was established by injecting the breast cancer 4T1 cell suspension into the subcutaneous chest of female BALB/c mice.2.After tumor formation,BALB/c mice were randomly divided into two groups.The tumor in the control group was injected with PBS,and the tumor in the experimental group was injected with IL-35.The growth curve was detected and recorded.3.CD19+B and CD4+ were separated by magnetic beads,and lymphocyte differentiation was detected by flow cytometry.4.Immunohistochemical method was used to detect the expression of Ki67 and CD31 in different groups of tumor cells,and the expression intensity and range of Ki67 and CD31 in tumor tissues were scored to evaluate the tumor proliferation and vascular density.At the same time,the mouse lung was separated and the metastasis was detected by immunohistochemistry.Results:1.Compared with the control group,the tumor growth rate of IL-35 treated group was faster and the tumor volume increased significantly.The results of immunohistochemistry showed that the proportion of Ki67 positive cells in tumor tissue of IL-35 treatment group increased significantly.2.Compared with the control group,the number of lung metastases in IL-35 treatment group was significantly increased3.The results of flow cytometry showed that IL-35 could induce the transformation of lymphocytes in breast tumor TME into JTr35 and IL-35+Breg.4.The results of immunohistochemistry showed that the proportion of CD31 positive cells in breast tumor from IL-35 treated group was significantly higher than that of the control group.Conclusion:This study confirmed that IL-35 not only promote the development of breast cancer by directly stimulating the proliferation of tumor cells,but also indirectly create favorable conditions for the growth and metastasis of tumor cells by increasing the micro-vessel density in tumor microenvironment and inducing immune tolerance.Therefore,IL-35 plays an important role in the development and prognosis of breast cancer and may become a new target of treatment.Part III IL-35 maintains immune tolerance during pregnancy by inducing lymphocyte transformationObjective:In the second part,we mainly discussed the role and mechanism of IL-35 in promoting local immune tolerance in pathological environment（tumor microenvironment）.Here we focused on the role of IL-35 in promoting immune tolerance in physiological environment（maternal fetal tolerance microenvironment）.There are a large number of immunotolerant lymphocytes and suppressive cytokines in early pregnancy,which are similar to the characteristics of tumor immunotolerance.The immune tolerance during pregnancy mainly includes maternal fetal interface and peripheral immune tolerance.There are a lot of T cells in the maternal fetal interface.Trophoblast cells can regulate Th] / Th2 balance,increase Th2 ce】】 content and inhibit the immune effect of Thl7 cells at the maternal fetal interface by secreting IL-10,thymic stromal lymphopoietin and other cytokines.Our previous studies have shown that trophoblasts in early pregnancy can express and secrete immunosuppressive cytokine IL-35,and the level of IL-35 in peripheral blood of early pregnant women is significantly increased.Considering the immunosuppressive effect of IL-35 and its expression in trophoblast cells,we speculated that IL-35 might be involved in the maintenance of maternal fetal tolerance by inducing the differentiation of iTR35 cells.In addition,as an important part of inhibitory immune cells,Breg cells can play a regulatory role in autoimmune diseases and infection by regulating the peripheral immune response.As an important subgroup of Breg cells,IL-10+ Breg cells（also known as BIO）exert the immunosuppressive effects through secreting IL-10.BIO cells present CD]9+CD5+CDldh,phenotype in mouse spleen.In addition,IL-35 can inhibit the proliferation of B cells,and mediate the transformation of B cells into a new subgroup of Breg cells: IL-35+ Breg,which plays an immunosuppressive role by secreting IL-35.In the previous study,we confirmed that IL-35 can promote the tumor progression and the formation of local immune tolerance by promoting the expansion of tumor infiltrating Treg and Breg cells.This study will further explore the role of IL-35 in promoting the transformation of T and B lymphocytes and maintaining the immune tolerance during pregnancy.Because B lymphocytes rarely presente at the maternal fetal interface,we mainly studied the regulatory role of Breg cells in peripheral immune tolerance during pregnancy.In addition,during pregnancy,the expression levels of estradiol（E2）and human chorionic gonadotropin（hCG）in peripheral blood change dramatically.In autoimmune encephalomyelitis（EAE）,exogenous estradiol（E2）can increase the production of IL₁0 by spleen cells.Human chorionic gonadotropin（hCG）is also considered to be an important factor in the regulation of B cell phenotype and antibody production.Therefore,we also discussed the role and mechanism of these hormones in the transformation of Breg cellsMethod:1. CBA / J x DBA / 2J model of spontaneous abortion and CBA / J x BALB / C model of normal pregnancy were constructed.2.The expression level of IL-35 at the maternal fetal interface was detected by Western blotting.Flow cytometry was used to detect the proportion of BIO cells in the spleen and the iTR35 at the maternal fetal interface.3.Intraperitoneal injection of recombinant IL-35 cytokines and IL-35 neutralizing antibody into pregnant mice was carried out to observe the effect of IL-35 on abortion and the change of 1Tr35 proportion at the maternal fetal interface.4.The primary CD19+B cells in the spleen of non-pregnant mice were isolated and cultured with pregnancy related hormones（hCG,E2）and IL-35.CCK8 was used to detect the B cell proliferation.The ratio of BIO and IL-35+Breg was analyzed by flow cytometry.5.Western blotting was used to detect the signaling pathways that hCG or IL-35 induced BregResults:1.The embryo loss rate of spontaneous abortion mice was significantly higher than that of normal pregnant mice.2.The expression level of IL-35 and the proportion of iTR35 cells at the maternal fetal interface of spontaneous abortion mice were significantly lower than that of normal pregnant mice.3.IL-35 significantly increased the iTR35 content at the maternal fetal interface and inhibited abortion;IL-35 neutralizing antibody decreased the iTr35 propotion at the matemalfetalinterfaceandincreasedtherateofplacentaloss.4.The peripheral BIO cells in normal pregnant mice were significantly expanded,while the proportion of peripheral BIO cells in spontaneous abortion mice was significantly lower than that of normal pregnant mice.5.IL-35 and hCG inhibited the proliferation of B cells in mice spleen.IL-35 activated STAT1 and STAT3 proteins in B cells and mediated their transform into BIO and IL-35+Breg.hCG activated the phosphorylation of STAT3 in B cells and induced its transformation into BIO.E2 had no significant effect on the proliferation and transformation of spleen B cells.Conclusion:This study confirmed that decidual iTR35 cells and spleen BIO cells play an important role in maintaining maternal fetal interface and peripheral immune tolerance during pregnancy.IL-35 can maintain normal pregnancy by inducing the differentiation of iTr35 cells at the maternal fetal interface.In addition,hCG and IL*35 can inhibit the proliferation of B cells and mediate their transformation into BIO and IL-35+Breg cells.Our study provides a new basis for the application of IL-35 in the treatment of recurrent abortion.At the same time,it also provides a new way to study the mechanism of IL-35 maintaining immune tolerance in tumor microenvironment.更多还原