乳腺癌细胞释放的EXO在机体肿瘤免疫反应中的初步研究
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摘要
研究背景
人体是由多种不同的细胞、组织、器官和系统构成的复杂有机体。但它们之间却能够在体内相互协调、共同维持整体生命活动机能的正常运行。这主要是与它们的基本组成单位---细胞之间的信息传递密切相关。而细胞之间的信息传递,要主要由细胞自己制造和释放的某些特定化学物质来实现。这就好比人要在复杂的社会中生存,就必须要通过人与人之间的对话来实现交流。目前认为,细胞间的信息传递分为直接和间接两种：1.间接的细胞交流,指细胞分泌一些化学物质(如激素)至细胞外,作为信号分子作用于靶细胞,调节其功能。根据化学信号分子可以作用的距离范围,可分为(1)内分泌：内分泌细胞(腺)分泌的激素随血液循环运输至全身,作用于相应的靶细胞；(2)旁分泌：内分泌细胞分泌的激素进入组织液,扩散至邻近的靶细胞,旁分泌细胞分泌的激素经组织液弥散至邻近的靶细胞,起局部调节作用,称为局部激素；(3)自分泌：信号发放细胞和靶细胞为同类或同一细胞,常见于癌变细胞。2.相邻细胞间直接信息交流：(1)通过与细胞膜结合的信号分子影响其他细胞。如精子和卵细胞之间的识别和结合；(2)相邻细胞间形成通道使细胞相互交流。即细胞←通道→细胞。如高等植物细胞之间通过胞间连丝相互连接,进行细胞间的信息交流。
同样的,包括癌症在内的多数疾病发生时,机体也会释放出一些分子信号物质如生长因子、细胞因子、小分子物质(如核苷酸、肽类)等来实现细胞间的信号传递,从而调节机体内环境以适应其自身所发生的变化。但除此之外,机体还能释放一些囊泡状物质到细胞外,以此来帮助完成细胞间的信号传递交流。这些物质按其大小、合成、及功能可分为微囊泡(microvesicles)、胞外体(exosome, EXO)、凋亡小体(apoptptic bodies)，脱落体(shed particles)等。
微囊泡(microvesicles， MV)是指当细胞遭受一系列刺激因素发生应激时,从细胞质膜上脱落的膜性小囊泡。直径在100nm-1000nm,密度在1.04-1.07g/m1。作为新发现的细胞间信息传递途径正逐渐引起科学界的关注。它主要含有与母细胞膜相似的脂类、蛋白质、mRNA、microRNA(miRNA)及包浆中的细胞器等。当含有多囊小体的细胞膜融合到质膜时,外核体便从细胞膜释放,MV可以通过介导配体-受体反应或传递胞浆成分及细胞器等方式使母细胞与靶细胞发生联系,其在免疫反应调节中发挥重要作用。除此之外,微囊泡参与动脉粥样硬化、心肌梗死.肝脏疾病等多种疾病的发生和发展。
凋亡小体是指程序性死亡细胞的核DNA在核小体处断裂成核小体片段,并向核膜下或中央异染色质区聚集形成浓缩的染色质块。随着染色质不断聚集,核纤层断裂消失,核膜在核孔处断裂,形成核碎片。同时在程序性死亡过程中,由于不断脱水,细胞质不断浓缩,细胞体积减小。凋亡细胞经核碎裂形成的染色质块(核碎片),然后整个细胞通过发芽(by budding)、起泡(byzelosls)等方式形成一个球形的突起,并在其根部绞窄而脱落形成一些大小不等,内含胞质、细胞器及核碎片的小体称为凋亡小体。最近研究发现肺癌细胞经放射线Co60y射线照射后可形成凋亡小体,经照射诱导的凋亡小体可作为抗原负载自体及异体树突细胞用于诱发特异性抗肿瘤免疫反应,为肿瘤的主动免疫治疗提供了新的途径。
EXO与微囊泡相似,是由包括肿瘤细胞在内的多种细胞分泌的一种囊泡状物质,其组分及功能根据所分泌的细胞种类不同而有所差异。EXO最早可追溯到1978年,由Johnstone等人率先提出,其后由Pan等人在研究网织红细胞成熟过程中证实。在体外培养羊网织红细胞的上清中经过10000g离心90min后,可以收集到直径60nm的囊性小泡,小泡中含有网织红细胞成熟过程中放出的胞膜结构。随后的研究相继表明,EXO是一种形态相似,大小相近,有双层脂膜包被,呈杯状,直径大约在30-100nnm之间,密度在1.13-1.21g/mL的膜性小囊泡。它主要形成于细胞内的多囊泡胞内体(multivesicular endosome, MVE)的区室中,当MVE与细胞膜融合时,这些内囊泡就作为EXO被释放到胞外,这些EXO能够运行到距离较远的组织从而影响细胞行为。多种真核细胞如T淋巴细胞、B淋巴细胞、上皮细胞、皮质神经原细胞、树突细胞、肿瘤细胞、胎儿细胞等的上清液和一些体液如羊水、胸水、母乳、尿液等都存在EXO。由于EXO的来源不同,EXO的组分及功能也有所差异。不同细胞来源的EXO参与了机体的多种不同的生理及病理过程。
对EXO功能的最初研究发现,EXO主要是清除细胞内多余的蛋白质,但后期发现,它具有更多潜在的生物功能。很多实验证实了EXO作为信息复合体能够转运胞质,并在胞间信号转导中起关键性的作用,这种信号模式与经典的模式不同,EXO不仅是一个配体,还是一个具有多种生物活性的纳米复合体。
在肿瘤方面,EXO具有双重作用。有学者发现,肿瘤细胞来源的EXO对肿瘤生长具有促进作用,若给予预先种植卵巢癌细胞的小鼠腹腔内注射从卵巢癌患者腹腔积液中分离出的EXO,可以明显促进小鼠腹腔内肿瘤的生长[34],来源于卵巢癌的MicroRNA可作为诊断卵巢癌的生物标记物。但也有研究发现,成熟的树突状细胞来源的EXO可以阻止肿瘤的生长作用,也能通过转运pMHC到成熟的树突状细胞上,间接递呈给T细胞,从而激活CD4+、CD8+特异性T细胞,起到免疫放大的作用。抗原递呈细胞(antigen-presenting cell, APC)释放的外泌体可以刺激T细胞的体外增殖及诱导体内的抗肿瘤免疫反应。从肿瘤细胞中分泌出的包含肿瘤抗原的外泌体则可以通过APC交叉呈递给细胞毒性T淋巴细胞(cytotoxic T lymphocyte， CTL),使其产生肿瘤杀伤作用。EXO携带有多种蛋白分子(CD54、CD80、CD3、D81等),被认为在细胞之间具有传递信息的功能。研究还表明恶性黑色素瘤来源的EXO其具有促进肿瘤血管新生的作用且与肿瘤诱导的血管生成,肿瘤的侵袭、转移及免疫抑制等密切相关。来源于结直肠癌细胞的富含与细胞周期相关mRNA,这些mRNA可以促进内皮细胞增殖,提示这些EXO可能促进肿瘤血管新生。在成胶质细胞瘤细胞来源的EXO中,富含促进血管新生的蛋白和mRNA,且这些EXO中的mRNA可以被正常宿主细胞摄入并表达。这些肿瘤细胞来源的EXO可以影响肿瘤微环境,促进肿瘤血管新生,进而促进肿瘤的侵袭和转移。肿瘤细胞来源的有膜结构的EXO,通过其携带的神经鞘磷脂,可以在体外实验中促进内皮细胞迁徙、侵袭和形成管腔,并在体内实验中促进血管新生。
乳腺癌是全球妇女恶性肿瘤中的主要杀手。同其它肿瘤一样,乳腺癌细胞也能分泌EXO。有实验研究表明,乳腺癌来源的EXO在肿瘤微环境中可促进肿瘤的免疫逃逸、血栓的形成、肌纤维母细胞的分化,从而为肿瘤的转移提供有力的条件。间充质干细胞释放的EXO可通过下调表达在乳腺癌细胞上的血管内皮生长因子(VEGF)从而抑制血管的生成。人乳腺癌细胞株释放的EXO中RNA的特征可作为子代的测序。另外,epigallocatechin gallate(EGCG),绿茶中的一种成分,所释放的EXO可用作乳腺癌的治疗。其机制主要是由于EGCG上调了肿瘤细胞中的miR-16,而乳腺癌细胞释放的EXO可将miR-16转移到肿瘤相关巨噬细胞(TAM)并抑制TAM的浸润及M2巨噬细胞的扩增从而减轻肿瘤的浸润。ER,PR,Her-2免疫组化三者阴性的乳腺癌患者释放的EXO参与了细胞与细胞之间的信号转导,且可根据其表型特性确定这些细胞的起源。在乳腺癌的转移中,EXO可介导自分泌Wnt-PCP信号通路的基质松解从而促进肿瘤的转移及浸润。从以上研究我们发现,EXO是实现细胞与细胞之间对话的一类物质,尤其在肿瘤微环境及肿瘤的免疫逃逸中起着重要的作用。
缺氧(Hypoxia)是实体肿瘤特征性的表现,随着实体瘤的增大,肿瘤内部不能得到足够血液供应,新近研究发现实体瘤中都存在一定程度或一定区域的缺氧[54-56]。肿瘤必须适应缺氧的微环境,因而缺氧对肿瘤的生物学特性有着重要影响。同时研究发现,缺氧也促进肿瘤的转移、血管生成及多数肿瘤细胞在缺氧条件下EXO分泌增加[57]。基于以上背景,我们采取了体外立体缺氧模型设计了本课题。
目的
使用购买于美国ATCC菌种保藏中心的人乳腺癌来源的细胞株BT-474,MDA-MB-231为研究对象,采取体外立体缺氧模型培养,比较正常氧含量和缺氧条件下培养乳腺癌细胞释放EXO的含量差异,并探索人乳腺癌细胞来源EXO中的TGF-β在肿瘤免疫抑制中的作用机制。研究缺氧培养的乳腺癌来源的EXO对小鼠脾淋巴细胞的增殖和活性抑制情况,进而应用蛋白组学方法,筛选淋巴细胞受到缺氧培养的乳腺癌细胞释放EXO刺激后的蛋白质谱变化,探讨其分子机制。
方法
本课题分为两部分：
第一部分：将置于37℃、5%C02饱和湿度的孵箱中含有10%灭活FBS的DEME培养基中的BT-474, MDA-MB-231人乳腺癌细胞培养条件改为02体积分数为1%,C02体积分数为5%,温度37-C,以制备肿瘤缺氧模型。运用梯度离心法制备EXO,用Q-PCR法和western blot法检测CD63、TGF-β、IL-10、PGE2的表达量。将EXO与T细胞共培养,运用3H-胸腺嘧啶核苷掺入法观察EXO抑制淋巴细胞情况。用RNA干扰策略,抑制EXO的TGF-β含量,比较其与对照EXO对淋巴细胞抑制的效应。
第二部分：应用蛋白质双向凝胶电泳技术比较缺氧培养的乳腺癌细胞释放EXO刺激小鼠脾来源淋巴细胞与对照细胞的蛋白质谱差异。用质谱鉴定差异蛋白并用Q-PCR和western blot技术选择4种差异蛋白进行验证。用RNA干扰技术抑制其中2种表达上调的蛋白质,观察其表达抑制后,对缺氧培养的乳腺癌细胞释放EXO刺激小鼠脾来源淋巴细胞抑制作用的影响。用生物信息学方法(STRING)对蛋白质组学技术获得的差异蛋白进行相互作用分析,以探讨其分子机制。
结果
第一部分结果：正常氧浓度(含氧量为21%)和缺氧条件下(含氧量1%)培养BT-47448h后细胞蛋白浓度比较显示差异有显著统计学意义,t=15.698,P<0.001:MDA-MB-231细胞蛋白浓度比较显示差异有显著统计学意义,t=10.844,P<0.001:BT-474和MDA.MB.231细胞中蛋白在正常氧浓度的总量高于缺氧条件下。
正常氧浓度(含氧量为21%)和缺氧条件下(含氧量1%)培养BT-47448h后同等数量细胞来源EXO蛋白浓度比较显示差异有显著统计学意义,t=-22.312,P<0.001；同等数量MDA-MB-231细胞来源EXO蛋白浓度比较显示差异有显著统计学意义,t=-10.209,P<0.00l；BT-474和MDA-MB-231细胞中EXO蛋白浓度在正常氧浓度的总量低于缺氧条件下。
正常氧浓度(含氧量为21%)和缺氧条件下(含氧量1%)培养BT-47448h后相同数量细胞获得EXO的CD63表达检测比较显示差异有显著统计学意义,t=-15.735,P<0.001;MDA-MB-231同数量细胞获得EXO的CD63表达检测比较显示差异有显著统计学意义,t=-24.441,P<0.001；BT-474和MDA-MB-231细胞中EXO的CD63表达量在正常氧浓度状态下低于缺氧条件下。表明,缺氧可通过肿瘤微环境促使来源于乳腺癌细胞的EXO分泌增多。
缺氧条件下来源于BT-474与MDA-MB-231人乳腺癌细胞株的EXO作用2天后的淋巴细胞cpm比较对照组淋巴细胞的cpm,显示差异有显著统计学意义,t=25.521,P<0.001； t=9.700,P=0.001；BT-474和MDA-MB-231细胞中EXO作用后的淋巴细胞cpm低于对照组淋巴细胞的cpm。说明缺氧条件下培养乳腺癌细胞获得的EXO小鼠脾来源淋巴细胞共同培养,发现EXO明显抑制淋巴细胞的增殖。
细胞释放的EXO的PGE2不论在正常条件还是缺氧条件培养的两种细胞释放的EXO中差异无统计学意义,f=-0.324, P=0.776; t=-0.659, P=0.546。细胞间与不同环境下,这两个因素也不存在交互效应(F=0.025,P=0.080)。细胞释放的EXO的IL-10不论在正常条件还是缺氧条件培养的两种细胞释放的EXO中差异也无统计学意义,t=-1.080,P=0.341; t=-0.629, P=0.563。细胞间与不同环境下,这两个因素也不存在交互效应(F=1.181,P=0.080)。在缺氧条件下,BT-474及MDA-MB-231细胞株释放的EXO TGF-p含量比正常氧浓度条件下差异有统计学意义,t=-9.047, P=0.001; t=-14.639, P<0.001。缺氧条件下,TGF-β含量均显著增高。不同细胞间与不同环境下,这两个因素也不存在交互效应(F=1.453,P=0.263)。说明检测缺氧培养乳腺癌细胞来源EXO中的TGF-β, IL-10、PGE2的含量,发现TGF-β明显增高,而IL-10与PGE2差异并不明显。
我们进而采用3H-胸腺嘧啶核苷掺入法检测了TGF-β对小鼠脾来源淋巴细胞增殖的影响。结果表明：TGF-β组和对照组比较显示淋巴细胞cpm差异有显著统计学意义,t=5.586,P=0.005；相对于对照组,TGF-β能够显著抑制细胞增殖。结果发现淋巴细胞受到的抑制减轻,即3H-胸腺嘧啶核苷的值增高。
进一步采用析因分析发现RNA干扰结果：使用TGF-β的干扰剂组与对照组的EXO TGF-β的差异有统计学意义(F=418.228,P<0.001),使用TGF-β的干扰剂组的EXO TGF-β低于对照组。正常氧培养与缺氧培养的EXO TGF-β差异有统计学意义(F=178.824,P<0.001),正常氧培养的EXO TGF-β低于缺氧的EXOTGF-β。干扰与未干扰组与环境处理这两个因素存在交互效应(F=147.260,P<0.001),其它交互效应不显著。经析因分析发现：EXO作用于脾来源淋巴细胞的平均cpm干扰剂组与对照组之间差异有统计学意义(F=65.887, P<0.001), EXO作用于脾来源淋巴细胞的平均cpm干扰剂组高于对照组。正常氧培养与缺氧培养EXO作用于脾来源淋巴细胞的平均cpm差异有统计学意义(F=22.682,P<0.001),正常氧培养的EXO作用于脾来源淋巴细胞的平均cpm高于缺氧组。各组间交互效应不显著当体外干扰EXO中的TGF-β时,这些结果表明：减少肿瘤细胞来源的EXO中TGF-β后,EXO对脾来源淋巴细胞增殖的抑制作用减弱。
第二部分结果：经过质谱鉴定,筛选得到20个表达量具有显著差异的蛋白质斑点。与对照组相比,EXO刺激组表达水平上调2倍以上的蛋白质点10个：Probable histidine--tRNA ligase, mitochondrial; DEAD (Asp-Glu-Ala-Asp) box polypeptide47; Heat shock protein beta-1; Class II histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; programmed cell death6; Doublesex-and mab-3-related transcription factor1; ferritin light chain; Protein Disulfide Isomerase Family A, Member3。相对于对照组,EXO刺激组表达水平下调2倍以上的蛋白质点10个:vimentin; MAGUK p55subfamily member2; ubiquitin-conjugating enzyme E2N; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; Nuclear factor of activated T-cells, cytoplasmic4; SS18-like protein2; Peroxiredoxin6; Peroxiredoxin-2; Dual specificity phosphatase28。
选择上调蛋白2个(programmed cell death6即PDCD6; Protein Disulfide Isomerase Family A, Member3即PDIA3、Erp57)、下调蛋白2个(Vimentin; Nuclear factor of activated T-cells, cytoplasmic4即NFATC4)采用Q-PCR方法和western-blot方法进行了验证。
Q-PCR检测结果表明EXO刺激组淋巴细胞的vimentin mRNA表达水平与对照组比较显示差异有统计学意义t=4.339, P=0.012, EXO刺激组vimentin表达量低于对照组；EXO刺激组淋巴细胞的NFATC4表达量与对照组比较显示差异有统计学意义t=7.425,P=0.002, EXO刺激组NFATC4表达量低于对照组；EXO刺激组淋巴细胞的PDCD6表达量与对照组比较显示差异有统计学意义t=-14.449, P<0.001, EXO刺激组PDCD6表达量高于对照组；EXO刺激组淋巴细胞的PDIA3表达量与对照组比较显示差异有统计学意义t=-14.260,P<0.001,EXO刺激组PDIA3表达量高于对照组。
用western-blot检测结果发现： EXO刺激组淋巴细胞的vimentin蛋白表达水平与对照组比较显示差异有统计学意义t=10.286, P=0.001, EXO刺激组vimentin表达量低于对照组；EXO刺激组淋巴细胞的NFATC4表达量与对照组比较显示差异有统计学意义t=3.734, P=0.020, EXO刺激组NFATC4表达量低于对照组；EXO刺激组淋巴细胞的PDCD6表达量与对照组比较显示差异有统计学意义t=-13.598, P<0.001, EXO刺激组PDCD6表达量高于对照组：EXO刺激组淋巴细胞的PDIA3表达量与对照组比较显示差异有统计学意义t=-12.763,P<0.001, EXO刺激组PDIA3表达量高于对照组。这些蛋白在EXO刺激组和对照组之间表达变化趋势与蛋白质组学结果一致。
采用RNAi方法抑制淋巴细胞的PDCD6和PDIA3表达,然后用EXO刺激细胞。结果表明转染PDCD6siRNA组与对照组比较,PDCD6表达量差异有统计学意义t=20.647,P<0.001；转染PDIA3siRNA组与对照组比较,差异有统计学意义t=11.002,P<0.001。说明淋巴细胞的PDCD6和PDIA3的表达受到明显抑制。
我们用3H-胸腺嘧啶核苷掺入法检测淋巴细胞PDCD6和PDIA3表达抑制后MDA-MB-231来源EXO对小鼠脾来源淋巴细胞增殖的作用,结果显示：PDCD6表达抑制后,缺氧培养MDA-MB-231来源EXO作用于脾来源淋巴细胞的平均cpm与对照组的平均cpm比较差异有统计学意义,t=-8.110, P=0.001; PDIA3表达抑制后,缺氧培养MDA-MB-231来源EXO作用于脾来源淋巴细胞的平均cpm与对照组的平均cpm比较差异有统计学意义t=-7.775,P=0.001。说明PDCD6和PDIA3表达抑制后,给予缺氧培养MDA-MB-231细胞来源EXO刺激5d后,细胞的增殖抑制减轻(相对于两种蛋白表达未抑制组增殖能力增强)。
对蛋白质组获得的差异蛋白进行生物信息学分析发现：20种差异蛋白中,14种蛋白存在直接或者间接的相互作用,包括vimentin; DEAD(Asp-Glu-Ala-Asp) box polypeptide47; ubiquitin-conjugating enzyme E2N; Protein Disulfide Isomerase Family A, Member3; Nuclear factor of activated T-cells, cytoplasmic4; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; Dual specificity phosphatase28; Probable histidine--tRNA ligase, mitochondrial; Peroxiredoxin6; Peroxiredoxin-2; Class II histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; ferritin light chain; Heat shock protein beta-1。这14种差异蛋白都直接或者间接通过ubiquitin C (UBC)联系起来。另外,这14种蛋白所形成的相互作用网络主要涉及的细胞信号转到通路包括MAPK通路和Akt通路。
结论
缺氧条件可诱导乳腺癌释放的EXO表达量增加,该EXO可抑制小鼠脾淋巴细胞,体外干扰EXO的中TGF-β可减轻对淋巴细胞的抑制。蛋白质组学提示乳腺癌来源的EXO可通过上调淋巴细胞PDCD6和PDIA3等蛋白而发挥抑制淋巴细胞的免疫功能,从而促进肿瘤的发生。我们的结果为探讨肿瘤免疫抑制的机制提供了新的线索,且对乳腺癌的生物治疗具有一定的潜在性意义。
BACKGROUND
Cells, the basic unit of human body, just like a human in the world, must communicate if they are to organize themselves into a complex society, cells do. And just as human communication involves more than the passage of noises from mouth to ear, so cell-cell communication involves more than the transmission of chemical signals across the space between one cell and another. At present, there are two ways of cells interaction, one is cell-matrix interaction, long range signals, electrical signal and via extracellular chemical molecules such as proteins, lipids etc. We called it indirect cell-cell communication. Cells communicate can be classified into endocrine, paracrine, and autocrine. Endocrine target distant cells, cells produce hormones that travel through the blood to reach all parts of the body. Paracrine is a form of cell-cell communication in which a cell produces a signal to induce changes in nearby cells, altering the behavior or differentiation of those cells. Neurotransmitters represent an example. Autocrine are produced by the target cell, are secreted, and effect the target cell itself via receptors. Sometimes autocrine cells can target cells close by if they are the same type of cell as the emitting cell, an example of this are immune cells. Another is direct cell-cell contact, it refers to some cells can form gap junctions that connect their cytoplasm to the cytoplasm of adjacent cells, such as plant direct cell-cell communication, and the combination of sperm and ovum.
The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes. And at the same time, some signal molecules like grow factors, cytokines, small molecules (such as nucleotide, active lipid, peptides) are involves the pathological process of cell interaction. In addition to the extracellular molecules that cells can use to influence their environment, more and more attention is focused on the release of extracellular membrane vesicles. The main populations of vesicles released are usually classified by their size, synthesis, and function and include microvesicles, exosome(EXO), apoptptic bodies, shed particles.
Microvesicles are small vesicles are released by virtually cells under a series of stimulates. It has the size between100-1,000nm in diameter, and a density of1.04-1.07g/ml. It main contain lipids, protein, RNAs, microRNA(miRNA),and some cytoplasmic organoids which are similar with their parent cells. Now it is act as vectors of information that regulate the function of target cells. MVs are derives cell types and released by outward bud from the plasma membrane of variouding and fission of the plasma membrane. After fusion with the plasma membrane, MVs are shed from the cell surface of activated cells. It is reported microvesicles involves diseases such as atherosclerosis and heart infarction.
Apoptotic bodies, it refers to the process of programmed cell death (PCD), lead to characteristic cell changes and death. These changes include cell shrinkage, nuclear fragment, and chromatin condensation. Apoptotic bodies, or vesicles, are1-5μm in diameter (approximately the size range of platelets) and released from the plasma membrane as blebs when cells undergo apoptosis. They are characterized by phosphatidylserine exteralization, which contain several intracellular fragments and cellular organelles, including histones and fragmented DNA.
EXO, similar with microvesicles, are kind of vesicles released by many types of cells in our body, and it has different functions in different cells. It was first discovered by Taylor in1987, and Pan confirmed it in the reticulocyte becomes a mature red blood cell. Cell culture in vitro found vesicles with a diameter60nm in the serum of goat reticulocytes after centrifugation with10000g/min for90mins. The vesicles contains membrane structure which are released by reticulocyte becomes a mature red blood cell. It is reported EXO was enclosed by double lipid membrane, with a cup-shaped morphology and similar size, the diameter of EXO is between30and100nm, the density of EXO is between1.13-1.21g/ml. it was formed by the inward budding of celluar compartment knows as muticles endosome(MV). When MV fused with the plasma membrane, these internal vesicles are released as EXO, which can travel to distant tissues to influence various aspects of cell behavior and physiology. EXO are released by multiples eukaryotic cells, including T cells, epithelial cell, reticulocytes, dendritic cell, B cells, mast cells, platelet and some of tumor cells. In addition, EXO were found in biological fluids, such as urine, breast milk, pleural effusion and amniotic fluid. EXO from different cell derived with different function and they involves different pathological process of diseases.
Earlier study on EXO function discover it was a tool to remove abundant protein are produced by cells. But later research found, it has more potential function. It is reported EXO can be a information complex and be transferred to cytoplasm, meanwhile, it play a key role in signal transduction of cells. It different from traditional model of signal transduction, it more than a ligand, but a multibiological complex.
EXO has double function on tumors. Some study found EXO from tumors can promote growth, if mice were injected with EXO isolated from ovarian cancer which were planted by ovary cells, it can promote tumor growth in mice. But other study discovered EXO from mature dendritic cell can inhibit tumor growth, and it help transfer pMHC to mature dendritic cells, then present to T cell and activated CD4+,CD8+lead to signal expansion. EXO released by antigen-presenting cell (APC)promote T cell proliferation in vitro and induce anti-tumor immune reaction in vivo. EXO with tumor antigen secreted by tumor cells can kill tumors cells through APC cross present to cytotoxic T lymphocyte(CTL). EXO contains proteins like CD54, CD80, CD3, D81etc and it was consider to pass information. It was also reported EXO involves the angiogenesis, tumor invasive and metastasis. The example of this EXO derived from colorectal cancer has mRNA which related cell cycle, and these mRNA promotes endothelial proliferation, it suggested those EXO might induce the tumor angiogenesis. EXO released from glioblastoma rich in protein promotes angiogenesis and mRNA which was expressed by host cells. Study also support these EXO accelerate tumor invasive, invasive and angiogenesis. EXO from tumor cells promotes endothelial immigration, invasive and the form of lumen of blood cell through the effect of sphingomyelin.
Breast cancer is the leading killer of women in malignant tumor in the world. Study show breast cell also released its EXO and it involves immune escape of tumor, the form of thrombi, the differentiate of myofibroblast to provide a proper environment promotes tumor metastasis in tumor microenvironment. EXO secreted by mesenchymal stem cell suppress angiogenesis through down-regulating vascular endothelial grow factor(VEGF) expression in breast cancer. Characterization of RNA in EXO from breast cancer cell line was consider as the sequencing for next generation. In addition, EXO derived from eipgallocatechin gallate(EGCC) was used to treat breast cancer. The mechanism is EGCG up-regulates miR-16in tumor cells, which can be transferred to TAM via exosomes and inhibits tumor-associated microphage(TAM) via EXO and inhibits TAM infiltration and M2polarization. EXO derived from triple-negative(her-2, PR, ER) breast cancer maybe involved in cancer cell-to cell communication, cinferring phenotypic traits to secondary cells that reflect those of their cells origin. EXO mediate autocrine Wnt-PCP signal pathway promotes tumor metastasis and infiltrate by lose matrix. These study showed EXO is a messenger between cells interaction, especially in tumor microenvironment and the immune escape of tumors.
Hypoxia is a character of solid tumor, and study showed hypoxia become more serious as the volume of solid tumor because lack of enough blood. But tumor cell will build its special environment for suit hypoxia. Another study has suggested EXO derived from breast cancer was increased in hypoxia. We designed our study using this model.
OBJECTIVE
To find the role of TGFp in EXO derived from breast tumor in tumor immune suppression and the changes of relative protein mass spectrometry by using breast cancer cell lines BT-1474, MDA-MB-231, which purchased from ATCC of America. It will be helpful to understand the molecular basis of breast cancer, and to explore a new way to treat breast cancer.
METHODS
1. BT-474, MDA-MB-231cell were cultured in DMEM medium supplemented with10%FBS, and to established the model of hypoxia, the cells were cultured in Sanyo O2/CO2incubator at1%02at37℃in a5%CO2humidified condition. EXO were harvested through serial centrifugation of supernatants. The expression of CD63, TGF-β, IL-10, PGE2were detected by Q-PCR and western blot. EXO and T cell were co-cultured and measument the inhibition of T lymphocyte by [3H] thymidine.
2. Compared with the change of protein from T lymphocyte derived from spleen with EXO stimulate and the control group in hypoxia using two-dimensional electrophoresis (2DE) technology. Get the purpose protein with mass spectrometry and choose4target protein using Q-PCR and western bolt. Two of up-regulate protein were inhibited by RNA interference (RNAi), then observed the effect of lymphocyte inhibition using EXO from breast cancer cells stimulate under hypoxia. Differential protein were get from proteomics technology were analysed by STRING for explore its possible mechanism.
RESULTS
1.The main results and findings are as follows:EXO derived from BT-474, MDA-MB-231human breast cell lines were increased in hypoxia with l%02and5%CO2humidity; Lymphocyte proliferation were inhibited when lymphocyte from the spleen of mice were cultured with EXO released by breast cancer cell in hypoxia; TGF-β, IL-10, PGE2were measured in EXO derived from breast cancer cell in hypoxia, and the results showed TGFβwere obviously increased, IL-10and PGE2has no significance. The inhibition of lymphocyte were alleviated when TGF-pin EXO were interfered in vitro,[3H] thymidine were increased.
2. We got20proteins spots with differentiated after mass mass spectrometry identification. Compared with control group, there are10spots with the expression of EXO-stimulated were up-regulated more than2times. They are Probable histidine-tRNA ligase, mitochondrial; DEAD (Asp-Glu-Ala-Asp) box polypeptide47; Nuclear factor of activated T-cells, cytoplasmic4; Class II histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; programmed cell death6; Doublesex-and mab-3-related transcription factor1; ferritin light chain; Protein Disulfide Isomerase Family A, Member3。 Compared with control group, there are10spots with the expression of EXO-stimulated were down-regulated more than2times. They are vimentin; MAGUK p55subfamily member2; ubiquitin-conjugating enzyme E2N; Heat shock protein beta-1; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; SS18-like protein2; Peroxiredoxin6; Peroxiredoxin-2; Dual specificity phosphatase28.
3. Two of up-regulated protein (programmed cell death6, PDCD6; Protein Disulfide Isomerase Family A, Member3, PDIA3、Erp57)and two of down-regulated protein (Vimentin; Nuclear factor of activated T-cells, cytoplasmic4,NFATC)were selected randomly, we get the same results in EXO-stimulated group and control group using the method of Q-PCR and western blot. The expression of PDCD6and PDIA3of lymphocyte were inhibited by RNAi and cells were stimulated with EXO. Results showed lymphocyte proliferation were alleviate when given EXO from MDA-MB-231which cultured5days in hypoxia.
4. Protein come from proteomics technology were analysed by STRING found14proteins has direct or indirect interaction in20differentiated protein. They including vimentin; DEAD (Asp-Glu-Ala-Asp) box polypeptide47; ubiquitin-conjugating enzyme E2N; Protein Disulfide Isomerase Family A, Member3; Nuclear factor of activated T-cells, cytoplasmic4; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; Dual specificity phosphatase28; Probable histidine-tRNA ligase, mitochondrial; Peroxiredoxin6; Peroxiredoxin-2; Class Ⅱ histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; ferritin light chain; Heat shock protein beta-1, and they are cross together by contact ubiquitin C (UBC).In addition, these14proteins formed a net mainly involves the MAPK and Akt signal pathway.
CONCLUSIONS
The expression of EXO derived from human breast cells were increased in hypoxia and Lymphocytes from spleen of mice were suppressed by it. TGF-β in EXO were interfered in vitro can alleviate the suppression of lymphocyte. Proteomics suggested EXO from human breast cancer inhibited the function of lymphocyte by up-regulated PDCD6and PDIA3of lymphocyte to promote tumor formed. Our results provide new clue for explore the mechanism of immune suppression in tumor microenvironment, meanwhile, it might be a potential tumor biological treatment for breast cancer.
人体是由多种不同的细胞、组织、器官和系统构成的复杂有机体。但它们之间却能够在体内相互协调、共同维持整体生命活动机能的正常运行。这主要是与它们的基本组成单位---细胞之间的信息传递密切相关。而细胞之间的信息传递,要主要由细胞自己制造和释放的某些特定化学物质来实现。这就好比人要在复杂的社会中生存,就必须要通过人与人之间的对话来实现交流。目前认为,细胞间的信息传递分为直接和间接两种：1.间接的细胞交流,指细胞分泌一些化学物质(如激素)至细胞外,作为信号分子作用于靶细胞,调节其功能。根据化学信号分子可以作用的距离范围,可分为(1)内分泌：内分泌细胞(腺)分泌的激素随血液循环运输至全身,作用于相应的靶细胞；(2)旁分泌：内分泌细胞分泌的激素进入组织液,扩散至邻近的靶细胞,旁分泌细胞分泌的激素经组织液弥散至邻近的靶细胞,起局部调节作用,称为局部激素；(3)自分泌：信号发放细胞和靶细胞为同类或同一细胞,常见于癌变细胞。2.相邻细胞间直接信息交流：(1)通过与细胞膜结合的信号分子影响其他细胞。如精子和卵细胞之间的识别和结合；(2)相邻细胞间形成通道使细胞相互交流。即细胞←通道→细胞。如高等植物细胞之间通过胞间连丝相互连接,进行细胞间的信息交流。
同样的,包括癌症在内的多数疾病发生时,机体也会释放出一些分子信号物质如生长因子、细胞因子、小分子物质(如核苷酸、肽类)等来实现细胞间的信号传递,从而调节机体内环境以适应其自身所发生的变化。但除此之外,机体还能释放一些囊泡状物质到细胞外,以此来帮助完成细胞间的信号传递交流。这些物质按其大小、合成、及功能可分为微囊泡(microvesicles)、胞外体(exosome, EXO)、凋亡小体(apoptptic bodies)，脱落体(shed particles)等。
微囊泡(microvesicles， MV)是指当细胞遭受一系列刺激因素发生应激时,从细胞质膜上脱落的膜性小囊泡。直径在100nm-1000nm,密度在1.04-1.07g/m1。作为新发现的细胞间信息传递途径正逐渐引起科学界的关注。它主要含有与母细胞膜相似的脂类、蛋白质、mRNA、microRNA(miRNA)及包浆中的细胞器等。当含有多囊小体的细胞膜融合到质膜时,外核体便从细胞膜释放,MV可以通过介导配体-受体反应或传递胞浆成分及细胞器等方式使母细胞与靶细胞发生联系,其在免疫反应调节中发挥重要作用。除此之外,微囊泡参与动脉粥样硬化、心肌梗死.肝脏疾病等多种疾病的发生和发展。
凋亡小体是指程序性死亡细胞的核DNA在核小体处断裂成核小体片段,并向核膜下或中央异染色质区聚集形成浓缩的染色质块。随着染色质不断聚集,核纤层断裂消失,核膜在核孔处断裂,形成核碎片。同时在程序性死亡过程中,由于不断脱水,细胞质不断浓缩,细胞体积减小。凋亡细胞经核碎裂形成的染色质块(核碎片),然后整个细胞通过发芽(by budding)、起泡(byzelosls)等方式形成一个球形的突起,并在其根部绞窄而脱落形成一些大小不等,内含胞质、细胞器及核碎片的小体称为凋亡小体。最近研究发现肺癌细胞经放射线Co60y射线照射后可形成凋亡小体,经照射诱导的凋亡小体可作为抗原负载自体及异体树突细胞用于诱发特异性抗肿瘤免疫反应,为肿瘤的主动免疫治疗提供了新的途径。
EXO与微囊泡相似,是由包括肿瘤细胞在内的多种细胞分泌的一种囊泡状物质,其组分及功能根据所分泌的细胞种类不同而有所差异。EXO最早可追溯到1978年,由Johnstone等人率先提出,其后由Pan等人在研究网织红细胞成熟过程中证实。在体外培养羊网织红细胞的上清中经过10000g离心90min后,可以收集到直径60nm的囊性小泡,小泡中含有网织红细胞成熟过程中放出的胞膜结构。随后的研究相继表明,EXO是一种形态相似,大小相近,有双层脂膜包被,呈杯状,直径大约在30-100nnm之间,密度在1.13-1.21g/mL的膜性小囊泡。它主要形成于细胞内的多囊泡胞内体(multivesicular endosome, MVE)的区室中,当MVE与细胞膜融合时,这些内囊泡就作为EXO被释放到胞外,这些EXO能够运行到距离较远的组织从而影响细胞行为。多种真核细胞如T淋巴细胞、B淋巴细胞、上皮细胞、皮质神经原细胞、树突细胞、肿瘤细胞、胎儿细胞等的上清液和一些体液如羊水、胸水、母乳、尿液等都存在EXO。由于EXO的来源不同,EXO的组分及功能也有所差异。不同细胞来源的EXO参与了机体的多种不同的生理及病理过程。
对EXO功能的最初研究发现,EXO主要是清除细胞内多余的蛋白质,但后期发现,它具有更多潜在的生物功能。很多实验证实了EXO作为信息复合体能够转运胞质,并在胞间信号转导中起关键性的作用,这种信号模式与经典的模式不同,EXO不仅是一个配体,还是一个具有多种生物活性的纳米复合体。
在肿瘤方面,EXO具有双重作用。有学者发现,肿瘤细胞来源的EXO对肿瘤生长具有促进作用,若给予预先种植卵巢癌细胞的小鼠腹腔内注射从卵巢癌患者腹腔积液中分离出的EXO,可以明显促进小鼠腹腔内肿瘤的生长[34],来源于卵巢癌的MicroRNA可作为诊断卵巢癌的生物标记物。但也有研究发现,成熟的树突状细胞来源的EXO可以阻止肿瘤的生长作用,也能通过转运pMHC到成熟的树突状细胞上,间接递呈给T细胞,从而激活CD4+、CD8+特异性T细胞,起到免疫放大的作用。抗原递呈细胞(antigen-presenting cell, APC)释放的外泌体可以刺激T细胞的体外增殖及诱导体内的抗肿瘤免疫反应。从肿瘤细胞中分泌出的包含肿瘤抗原的外泌体则可以通过APC交叉呈递给细胞毒性T淋巴细胞(cytotoxic T lymphocyte， CTL),使其产生肿瘤杀伤作用。EXO携带有多种蛋白分子(CD54、CD80、CD3、D81等),被认为在细胞之间具有传递信息的功能。研究还表明恶性黑色素瘤来源的EXO其具有促进肿瘤血管新生的作用且与肿瘤诱导的血管生成,肿瘤的侵袭、转移及免疫抑制等密切相关。来源于结直肠癌细胞的富含与细胞周期相关mRNA,这些mRNA可以促进内皮细胞增殖,提示这些EXO可能促进肿瘤血管新生。在成胶质细胞瘤细胞来源的EXO中,富含促进血管新生的蛋白和mRNA,且这些EXO中的mRNA可以被正常宿主细胞摄入并表达。这些肿瘤细胞来源的EXO可以影响肿瘤微环境,促进肿瘤血管新生,进而促进肿瘤的侵袭和转移。肿瘤细胞来源的有膜结构的EXO,通过其携带的神经鞘磷脂,可以在体外实验中促进内皮细胞迁徙、侵袭和形成管腔,并在体内实验中促进血管新生。
乳腺癌是全球妇女恶性肿瘤中的主要杀手。同其它肿瘤一样,乳腺癌细胞也能分泌EXO。有实验研究表明,乳腺癌来源的EXO在肿瘤微环境中可促进肿瘤的免疫逃逸、血栓的形成、肌纤维母细胞的分化,从而为肿瘤的转移提供有力的条件。间充质干细胞释放的EXO可通过下调表达在乳腺癌细胞上的血管内皮生长因子(VEGF)从而抑制血管的生成。人乳腺癌细胞株释放的EXO中RNA的特征可作为子代的测序。另外,epigallocatechin gallate(EGCG),绿茶中的一种成分,所释放的EXO可用作乳腺癌的治疗。其机制主要是由于EGCG上调了肿瘤细胞中的miR-16,而乳腺癌细胞释放的EXO可将miR-16转移到肿瘤相关巨噬细胞(TAM)并抑制TAM的浸润及M2巨噬细胞的扩增从而减轻肿瘤的浸润。ER,PR,Her-2免疫组化三者阴性的乳腺癌患者释放的EXO参与了细胞与细胞之间的信号转导,且可根据其表型特性确定这些细胞的起源。在乳腺癌的转移中,EXO可介导自分泌Wnt-PCP信号通路的基质松解从而促进肿瘤的转移及浸润。从以上研究我们发现,EXO是实现细胞与细胞之间对话的一类物质,尤其在肿瘤微环境及肿瘤的免疫逃逸中起着重要的作用。
缺氧(Hypoxia)是实体肿瘤特征性的表现,随着实体瘤的增大,肿瘤内部不能得到足够血液供应,新近研究发现实体瘤中都存在一定程度或一定区域的缺氧[54-56]。肿瘤必须适应缺氧的微环境,因而缺氧对肿瘤的生物学特性有着重要影响。同时研究发现,缺氧也促进肿瘤的转移、血管生成及多数肿瘤细胞在缺氧条件下EXO分泌增加[57]。基于以上背景,我们采取了体外立体缺氧模型设计了本课题。
目的
使用购买于美国ATCC菌种保藏中心的人乳腺癌来源的细胞株BT-474,MDA-MB-231为研究对象,采取体外立体缺氧模型培养,比较正常氧含量和缺氧条件下培养乳腺癌细胞释放EXO的含量差异,并探索人乳腺癌细胞来源EXO中的TGF-β在肿瘤免疫抑制中的作用机制。研究缺氧培养的乳腺癌来源的EXO对小鼠脾淋巴细胞的增殖和活性抑制情况,进而应用蛋白组学方法,筛选淋巴细胞受到缺氧培养的乳腺癌细胞释放EXO刺激后的蛋白质谱变化,探讨其分子机制。
方法
本课题分为两部分：
第一部分：将置于37℃、5%C02饱和湿度的孵箱中含有10%灭活FBS的DEME培养基中的BT-474, MDA-MB-231人乳腺癌细胞培养条件改为02体积分数为1%,C02体积分数为5%,温度37-C,以制备肿瘤缺氧模型。运用梯度离心法制备EXO,用Q-PCR法和western blot法检测CD63、TGF-β、IL-10、PGE2的表达量。将EXO与T细胞共培养,运用3H-胸腺嘧啶核苷掺入法观察EXO抑制淋巴细胞情况。用RNA干扰策略,抑制EXO的TGF-β含量,比较其与对照EXO对淋巴细胞抑制的效应。
第二部分：应用蛋白质双向凝胶电泳技术比较缺氧培养的乳腺癌细胞释放EXO刺激小鼠脾来源淋巴细胞与对照细胞的蛋白质谱差异。用质谱鉴定差异蛋白并用Q-PCR和western blot技术选择4种差异蛋白进行验证。用RNA干扰技术抑制其中2种表达上调的蛋白质,观察其表达抑制后,对缺氧培养的乳腺癌细胞释放EXO刺激小鼠脾来源淋巴细胞抑制作用的影响。用生物信息学方法(STRING)对蛋白质组学技术获得的差异蛋白进行相互作用分析,以探讨其分子机制。
结果
第一部分结果：正常氧浓度(含氧量为21%)和缺氧条件下(含氧量1%)培养BT-47448h后细胞蛋白浓度比较显示差异有显著统计学意义,t=15.698,P<0.001:MDA-MB-231细胞蛋白浓度比较显示差异有显著统计学意义,t=10.844,P<0.001:BT-474和MDA.MB.231细胞中蛋白在正常氧浓度的总量高于缺氧条件下。
正常氧浓度(含氧量为21%)和缺氧条件下(含氧量1%)培养BT-47448h后同等数量细胞来源EXO蛋白浓度比较显示差异有显著统计学意义,t=-22.312,P<0.001；同等数量MDA-MB-231细胞来源EXO蛋白浓度比较显示差异有显著统计学意义,t=-10.209,P<0.00l；BT-474和MDA-MB-231细胞中EXO蛋白浓度在正常氧浓度的总量低于缺氧条件下。
正常氧浓度(含氧量为21%)和缺氧条件下(含氧量1%)培养BT-47448h后相同数量细胞获得EXO的CD63表达检测比较显示差异有显著统计学意义,t=-15.735,P<0.001;MDA-MB-231同数量细胞获得EXO的CD63表达检测比较显示差异有显著统计学意义,t=-24.441,P<0.001；BT-474和MDA-MB-231细胞中EXO的CD63表达量在正常氧浓度状态下低于缺氧条件下。表明,缺氧可通过肿瘤微环境促使来源于乳腺癌细胞的EXO分泌增多。
缺氧条件下来源于BT-474与MDA-MB-231人乳腺癌细胞株的EXO作用2天后的淋巴细胞cpm比较对照组淋巴细胞的cpm,显示差异有显著统计学意义,t=25.521,P<0.001； t=9.700,P=0.001；BT-474和MDA-MB-231细胞中EXO作用后的淋巴细胞cpm低于对照组淋巴细胞的cpm。说明缺氧条件下培养乳腺癌细胞获得的EXO小鼠脾来源淋巴细胞共同培养,发现EXO明显抑制淋巴细胞的增殖。
细胞释放的EXO的PGE2不论在正常条件还是缺氧条件培养的两种细胞释放的EXO中差异无统计学意义,f=-0.324, P=0.776; t=-0.659, P=0.546。细胞间与不同环境下,这两个因素也不存在交互效应(F=0.025,P=0.080)。细胞释放的EXO的IL-10不论在正常条件还是缺氧条件培养的两种细胞释放的EXO中差异也无统计学意义,t=-1.080,P=0.341; t=-0.629, P=0.563。细胞间与不同环境下,这两个因素也不存在交互效应(F=1.181,P=0.080)。在缺氧条件下,BT-474及MDA-MB-231细胞株释放的EXO TGF-p含量比正常氧浓度条件下差异有统计学意义,t=-9.047, P=0.001; t=-14.639, P<0.001。缺氧条件下,TGF-β含量均显著增高。不同细胞间与不同环境下,这两个因素也不存在交互效应(F=1.453,P=0.263)。说明检测缺氧培养乳腺癌细胞来源EXO中的TGF-β, IL-10、PGE2的含量,发现TGF-β明显增高,而IL-10与PGE2差异并不明显。
我们进而采用3H-胸腺嘧啶核苷掺入法检测了TGF-β对小鼠脾来源淋巴细胞增殖的影响。结果表明：TGF-β组和对照组比较显示淋巴细胞cpm差异有显著统计学意义,t=5.586,P=0.005；相对于对照组,TGF-β能够显著抑制细胞增殖。结果发现淋巴细胞受到的抑制减轻,即3H-胸腺嘧啶核苷的值增高。
进一步采用析因分析发现RNA干扰结果：使用TGF-β的干扰剂组与对照组的EXO TGF-β的差异有统计学意义(F=418.228,P<0.001),使用TGF-β的干扰剂组的EXO TGF-β低于对照组。正常氧培养与缺氧培养的EXO TGF-β差异有统计学意义(F=178.824,P<0.001),正常氧培养的EXO TGF-β低于缺氧的EXOTGF-β。干扰与未干扰组与环境处理这两个因素存在交互效应(F=147.260,P<0.001),其它交互效应不显著。经析因分析发现：EXO作用于脾来源淋巴细胞的平均cpm干扰剂组与对照组之间差异有统计学意义(F=65.887, P<0.001), EXO作用于脾来源淋巴细胞的平均cpm干扰剂组高于对照组。正常氧培养与缺氧培养EXO作用于脾来源淋巴细胞的平均cpm差异有统计学意义(F=22.682,P<0.001),正常氧培养的EXO作用于脾来源淋巴细胞的平均cpm高于缺氧组。各组间交互效应不显著当体外干扰EXO中的TGF-β时,这些结果表明：减少肿瘤细胞来源的EXO中TGF-β后,EXO对脾来源淋巴细胞增殖的抑制作用减弱。
第二部分结果：经过质谱鉴定,筛选得到20个表达量具有显著差异的蛋白质斑点。与对照组相比,EXO刺激组表达水平上调2倍以上的蛋白质点10个：Probable histidine--tRNA ligase, mitochondrial; DEAD (Asp-Glu-Ala-Asp) box polypeptide47; Heat shock protein beta-1; Class II histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; programmed cell death6; Doublesex-and mab-3-related transcription factor1; ferritin light chain; Protein Disulfide Isomerase Family A, Member3。相对于对照组,EXO刺激组表达水平下调2倍以上的蛋白质点10个:vimentin; MAGUK p55subfamily member2; ubiquitin-conjugating enzyme E2N; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; Nuclear factor of activated T-cells, cytoplasmic4; SS18-like protein2; Peroxiredoxin6; Peroxiredoxin-2; Dual specificity phosphatase28。
选择上调蛋白2个(programmed cell death6即PDCD6; Protein Disulfide Isomerase Family A, Member3即PDIA3、Erp57)、下调蛋白2个(Vimentin; Nuclear factor of activated T-cells, cytoplasmic4即NFATC4)采用Q-PCR方法和western-blot方法进行了验证。
Q-PCR检测结果表明EXO刺激组淋巴细胞的vimentin mRNA表达水平与对照组比较显示差异有统计学意义t=4.339, P=0.012, EXO刺激组vimentin表达量低于对照组；EXO刺激组淋巴细胞的NFATC4表达量与对照组比较显示差异有统计学意义t=7.425,P=0.002, EXO刺激组NFATC4表达量低于对照组；EXO刺激组淋巴细胞的PDCD6表达量与对照组比较显示差异有统计学意义t=-14.449, P<0.001, EXO刺激组PDCD6表达量高于对照组；EXO刺激组淋巴细胞的PDIA3表达量与对照组比较显示差异有统计学意义t=-14.260,P<0.001,EXO刺激组PDIA3表达量高于对照组。
用western-blot检测结果发现： EXO刺激组淋巴细胞的vimentin蛋白表达水平与对照组比较显示差异有统计学意义t=10.286, P=0.001, EXO刺激组vimentin表达量低于对照组；EXO刺激组淋巴细胞的NFATC4表达量与对照组比较显示差异有统计学意义t=3.734, P=0.020, EXO刺激组NFATC4表达量低于对照组；EXO刺激组淋巴细胞的PDCD6表达量与对照组比较显示差异有统计学意义t=-13.598, P<0.001, EXO刺激组PDCD6表达量高于对照组：EXO刺激组淋巴细胞的PDIA3表达量与对照组比较显示差异有统计学意义t=-12.763,P<0.001, EXO刺激组PDIA3表达量高于对照组。这些蛋白在EXO刺激组和对照组之间表达变化趋势与蛋白质组学结果一致。
采用RNAi方法抑制淋巴细胞的PDCD6和PDIA3表达,然后用EXO刺激细胞。结果表明转染PDCD6siRNA组与对照组比较,PDCD6表达量差异有统计学意义t=20.647,P<0.001；转染PDIA3siRNA组与对照组比较,差异有统计学意义t=11.002,P<0.001。说明淋巴细胞的PDCD6和PDIA3的表达受到明显抑制。
我们用3H-胸腺嘧啶核苷掺入法检测淋巴细胞PDCD6和PDIA3表达抑制后MDA-MB-231来源EXO对小鼠脾来源淋巴细胞增殖的作用,结果显示：PDCD6表达抑制后,缺氧培养MDA-MB-231来源EXO作用于脾来源淋巴细胞的平均cpm与对照组的平均cpm比较差异有统计学意义,t=-8.110, P=0.001; PDIA3表达抑制后,缺氧培养MDA-MB-231来源EXO作用于脾来源淋巴细胞的平均cpm与对照组的平均cpm比较差异有统计学意义t=-7.775,P=0.001。说明PDCD6和PDIA3表达抑制后,给予缺氧培养MDA-MB-231细胞来源EXO刺激5d后,细胞的增殖抑制减轻(相对于两种蛋白表达未抑制组增殖能力增强)。
对蛋白质组获得的差异蛋白进行生物信息学分析发现：20种差异蛋白中,14种蛋白存在直接或者间接的相互作用,包括vimentin; DEAD(Asp-Glu-Ala-Asp) box polypeptide47; ubiquitin-conjugating enzyme E2N; Protein Disulfide Isomerase Family A, Member3; Nuclear factor of activated T-cells, cytoplasmic4; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; Dual specificity phosphatase28; Probable histidine--tRNA ligase, mitochondrial; Peroxiredoxin6; Peroxiredoxin-2; Class II histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; ferritin light chain; Heat shock protein beta-1。这14种差异蛋白都直接或者间接通过ubiquitin C (UBC)联系起来。另外,这14种蛋白所形成的相互作用网络主要涉及的细胞信号转到通路包括MAPK通路和Akt通路。
结论
缺氧条件可诱导乳腺癌释放的EXO表达量增加,该EXO可抑制小鼠脾淋巴细胞,体外干扰EXO的中TGF-β可减轻对淋巴细胞的抑制。蛋白质组学提示乳腺癌来源的EXO可通过上调淋巴细胞PDCD6和PDIA3等蛋白而发挥抑制淋巴细胞的免疫功能,从而促进肿瘤的发生。我们的结果为探讨肿瘤免疫抑制的机制提供了新的线索,且对乳腺癌的生物治疗具有一定的潜在性意义。
BACKGROUND
Cells, the basic unit of human body, just like a human in the world, must communicate if they are to organize themselves into a complex society, cells do. And just as human communication involves more than the passage of noises from mouth to ear, so cell-cell communication involves more than the transmission of chemical signals across the space between one cell and another. At present, there are two ways of cells interaction, one is cell-matrix interaction, long range signals, electrical signal and via extracellular chemical molecules such as proteins, lipids etc. We called it indirect cell-cell communication. Cells communicate can be classified into endocrine, paracrine, and autocrine. Endocrine target distant cells, cells produce hormones that travel through the blood to reach all parts of the body. Paracrine is a form of cell-cell communication in which a cell produces a signal to induce changes in nearby cells, altering the behavior or differentiation of those cells. Neurotransmitters represent an example. Autocrine are produced by the target cell, are secreted, and effect the target cell itself via receptors. Sometimes autocrine cells can target cells close by if they are the same type of cell as the emitting cell, an example of this are immune cells. Another is direct cell-cell contact, it refers to some cells can form gap junctions that connect their cytoplasm to the cytoplasm of adjacent cells, such as plant direct cell-cell communication, and the combination of sperm and ovum.
The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes. And at the same time, some signal molecules like grow factors, cytokines, small molecules (such as nucleotide, active lipid, peptides) are involves the pathological process of cell interaction. In addition to the extracellular molecules that cells can use to influence their environment, more and more attention is focused on the release of extracellular membrane vesicles. The main populations of vesicles released are usually classified by their size, synthesis, and function and include microvesicles, exosome(EXO), apoptptic bodies, shed particles.
Microvesicles are small vesicles are released by virtually cells under a series of stimulates. It has the size between100-1,000nm in diameter, and a density of1.04-1.07g/ml. It main contain lipids, protein, RNAs, microRNA(miRNA),and some cytoplasmic organoids which are similar with their parent cells. Now it is act as vectors of information that regulate the function of target cells. MVs are derives cell types and released by outward bud from the plasma membrane of variouding and fission of the plasma membrane. After fusion with the plasma membrane, MVs are shed from the cell surface of activated cells. It is reported microvesicles involves diseases such as atherosclerosis and heart infarction.
Apoptotic bodies, it refers to the process of programmed cell death (PCD), lead to characteristic cell changes and death. These changes include cell shrinkage, nuclear fragment, and chromatin condensation. Apoptotic bodies, or vesicles, are1-5μm in diameter (approximately the size range of platelets) and released from the plasma membrane as blebs when cells undergo apoptosis. They are characterized by phosphatidylserine exteralization, which contain several intracellular fragments and cellular organelles, including histones and fragmented DNA.
EXO, similar with microvesicles, are kind of vesicles released by many types of cells in our body, and it has different functions in different cells. It was first discovered by Taylor in1987, and Pan confirmed it in the reticulocyte becomes a mature red blood cell. Cell culture in vitro found vesicles with a diameter60nm in the serum of goat reticulocytes after centrifugation with10000g/min for90mins. The vesicles contains membrane structure which are released by reticulocyte becomes a mature red blood cell. It is reported EXO was enclosed by double lipid membrane, with a cup-shaped morphology and similar size, the diameter of EXO is between30and100nm, the density of EXO is between1.13-1.21g/ml. it was formed by the inward budding of celluar compartment knows as muticles endosome(MV). When MV fused with the plasma membrane, these internal vesicles are released as EXO, which can travel to distant tissues to influence various aspects of cell behavior and physiology. EXO are released by multiples eukaryotic cells, including T cells, epithelial cell, reticulocytes, dendritic cell, B cells, mast cells, platelet and some of tumor cells. In addition, EXO were found in biological fluids, such as urine, breast milk, pleural effusion and amniotic fluid. EXO from different cell derived with different function and they involves different pathological process of diseases.
Earlier study on EXO function discover it was a tool to remove abundant protein are produced by cells. But later research found, it has more potential function. It is reported EXO can be a information complex and be transferred to cytoplasm, meanwhile, it play a key role in signal transduction of cells. It different from traditional model of signal transduction, it more than a ligand, but a multibiological complex.
EXO has double function on tumors. Some study found EXO from tumors can promote growth, if mice were injected with EXO isolated from ovarian cancer which were planted by ovary cells, it can promote tumor growth in mice. But other study discovered EXO from mature dendritic cell can inhibit tumor growth, and it help transfer pMHC to mature dendritic cells, then present to T cell and activated CD4+,CD8+lead to signal expansion. EXO released by antigen-presenting cell (APC)promote T cell proliferation in vitro and induce anti-tumor immune reaction in vivo. EXO with tumor antigen secreted by tumor cells can kill tumors cells through APC cross present to cytotoxic T lymphocyte(CTL). EXO contains proteins like CD54, CD80, CD3, D81etc and it was consider to pass information. It was also reported EXO involves the angiogenesis, tumor invasive and metastasis. The example of this EXO derived from colorectal cancer has mRNA which related cell cycle, and these mRNA promotes endothelial proliferation, it suggested those EXO might induce the tumor angiogenesis. EXO released from glioblastoma rich in protein promotes angiogenesis and mRNA which was expressed by host cells. Study also support these EXO accelerate tumor invasive, invasive and angiogenesis. EXO from tumor cells promotes endothelial immigration, invasive and the form of lumen of blood cell through the effect of sphingomyelin.
Breast cancer is the leading killer of women in malignant tumor in the world. Study show breast cell also released its EXO and it involves immune escape of tumor, the form of thrombi, the differentiate of myofibroblast to provide a proper environment promotes tumor metastasis in tumor microenvironment. EXO secreted by mesenchymal stem cell suppress angiogenesis through down-regulating vascular endothelial grow factor(VEGF) expression in breast cancer. Characterization of RNA in EXO from breast cancer cell line was consider as the sequencing for next generation. In addition, EXO derived from eipgallocatechin gallate(EGCC) was used to treat breast cancer. The mechanism is EGCG up-regulates miR-16in tumor cells, which can be transferred to TAM via exosomes and inhibits tumor-associated microphage(TAM) via EXO and inhibits TAM infiltration and M2polarization. EXO derived from triple-negative(her-2, PR, ER) breast cancer maybe involved in cancer cell-to cell communication, cinferring phenotypic traits to secondary cells that reflect those of their cells origin. EXO mediate autocrine Wnt-PCP signal pathway promotes tumor metastasis and infiltrate by lose matrix. These study showed EXO is a messenger between cells interaction, especially in tumor microenvironment and the immune escape of tumors.
Hypoxia is a character of solid tumor, and study showed hypoxia become more serious as the volume of solid tumor because lack of enough blood. But tumor cell will build its special environment for suit hypoxia. Another study has suggested EXO derived from breast cancer was increased in hypoxia. We designed our study using this model.
OBJECTIVE
To find the role of TGFp in EXO derived from breast tumor in tumor immune suppression and the changes of relative protein mass spectrometry by using breast cancer cell lines BT-1474, MDA-MB-231, which purchased from ATCC of America. It will be helpful to understand the molecular basis of breast cancer, and to explore a new way to treat breast cancer.
METHODS
1. BT-474, MDA-MB-231cell were cultured in DMEM medium supplemented with10%FBS, and to established the model of hypoxia, the cells were cultured in Sanyo O2/CO2incubator at1%02at37℃in a5%CO2humidified condition. EXO were harvested through serial centrifugation of supernatants. The expression of CD63, TGF-β, IL-10, PGE2were detected by Q-PCR and western blot. EXO and T cell were co-cultured and measument the inhibition of T lymphocyte by [3H] thymidine.
2. Compared with the change of protein from T lymphocyte derived from spleen with EXO stimulate and the control group in hypoxia using two-dimensional electrophoresis (2DE) technology. Get the purpose protein with mass spectrometry and choose4target protein using Q-PCR and western bolt. Two of up-regulate protein were inhibited by RNA interference (RNAi), then observed the effect of lymphocyte inhibition using EXO from breast cancer cells stimulate under hypoxia. Differential protein were get from proteomics technology were analysed by STRING for explore its possible mechanism.
RESULTS
1.The main results and findings are as follows:EXO derived from BT-474, MDA-MB-231human breast cell lines were increased in hypoxia with l%02and5%CO2humidity; Lymphocyte proliferation were inhibited when lymphocyte from the spleen of mice were cultured with EXO released by breast cancer cell in hypoxia; TGF-β, IL-10, PGE2were measured in EXO derived from breast cancer cell in hypoxia, and the results showed TGFβwere obviously increased, IL-10and PGE2has no significance. The inhibition of lymphocyte were alleviated when TGF-pin EXO were interfered in vitro,[3H] thymidine were increased.
2. We got20proteins spots with differentiated after mass mass spectrometry identification. Compared with control group, there are10spots with the expression of EXO-stimulated were up-regulated more than2times. They are Probable histidine-tRNA ligase, mitochondrial; DEAD (Asp-Glu-Ala-Asp) box polypeptide47; Nuclear factor of activated T-cells, cytoplasmic4; Class II histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; programmed cell death6; Doublesex-and mab-3-related transcription factor1; ferritin light chain; Protein Disulfide Isomerase Family A, Member3。 Compared with control group, there are10spots with the expression of EXO-stimulated were down-regulated more than2times. They are vimentin; MAGUK p55subfamily member2; ubiquitin-conjugating enzyme E2N; Heat shock protein beta-1; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; SS18-like protein2; Peroxiredoxin6; Peroxiredoxin-2; Dual specificity phosphatase28.
3. Two of up-regulated protein (programmed cell death6, PDCD6; Protein Disulfide Isomerase Family A, Member3, PDIA3、Erp57)and two of down-regulated protein (Vimentin; Nuclear factor of activated T-cells, cytoplasmic4,NFATC)were selected randomly, we get the same results in EXO-stimulated group and control group using the method of Q-PCR and western blot. The expression of PDCD6and PDIA3of lymphocyte were inhibited by RNAi and cells were stimulated with EXO. Results showed lymphocyte proliferation were alleviate when given EXO from MDA-MB-231which cultured5days in hypoxia.
4. Protein come from proteomics technology were analysed by STRING found14proteins has direct or indirect interaction in20differentiated protein. They including vimentin; DEAD (Asp-Glu-Ala-Asp) box polypeptide47; ubiquitin-conjugating enzyme E2N; Protein Disulfide Isomerase Family A, Member3; Nuclear factor of activated T-cells, cytoplasmic4; Phosphoglycerate mutase1; E3ubiquitin-protein ligase RNF169; Dual specificity phosphatase28; Probable histidine-tRNA ligase, mitochondrial; Peroxiredoxin6; Peroxiredoxin-2; Class Ⅱ histocompatibility antigen, M alpha chain; MHC class I RT1.C-type protein; RAD23homolog B; ferritin light chain; Heat shock protein beta-1, and they are cross together by contact ubiquitin C (UBC).In addition, these14proteins formed a net mainly involves the MAPK and Akt signal pathway.
CONCLUSIONS
The expression of EXO derived from human breast cells were increased in hypoxia and Lymphocytes from spleen of mice were suppressed by it. TGF-β in EXO were interfered in vitro can alleviate the suppression of lymphocyte. Proteomics suggested EXO from human breast cancer inhibited the function of lymphocyte by up-regulated PDCD6and PDIA3of lymphocyte to promote tumor formed. Our results provide new clue for explore the mechanism of immune suppression in tumor microenvironment, meanwhile, it might be a potential tumor biological treatment for breast cancer.
引文
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