CN111601890A - 用于治疗肾癌的包含miRNA的药物载体 - Google Patents
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- C12N2320/32—Special delivery means, e.g. tissue-specific
Abstract
本发明涉及包含选自miR‑15a、miR‑181b、miR‑320c、miR‑874及其任意组合的微RNA的可药用载体,其用于治疗肾癌。所述可药用载体优选地为来源于选自以下的成体干细胞的细胞外囊泡(EV):间充质干细胞(MSC)、非卵圆人肝祖细胞(HLSC)和脂肪干细胞(ASC)。
Description
本发明涉及肾癌的新治疗性处理。更具体地,本发明涉及携带在肾癌的治疗中有效的一组特定miRNA的药物载体的用途。重点在于来源于干细胞的细胞外囊泡(extracellular vesicle,EV),其作为用于治疗肾癌的药物载体。
肿瘤血管形成是肿瘤生长和转移中的基本步骤。在缺乏氧和营养物的血管供应的情况下,实体瘤事实上无法生长超过数平方毫米。此外,转移的数目与原发性肿瘤的血管密度相关。
肿瘤内皮细胞(tumor endothelial cell,TEC)与正常内皮细胞不同,并且显示促血管生成表型。例如,TEC表现出更高的运动性和增殖,在体外不依赖于血清,以及通过Akt信号传导增强存活。在表型上,TEC可增强生长因子受体表达,包括VEGF和EGF受体。TEC还对某些化学治疗药物具有抗性并且对靶向VEGF的抗血管生成药物不那么可感知。此外,TEC在遗传学上与正常内皮细胞不同。
细胞外囊泡(EV)表现为细胞间通信的重要机制,并且其活性载物(cargo)可通过改变接受体细胞的功能和表型对其进行重编程。事实上,EV的活性看起来依赖于许多不同因子的转移,这些因子包括蛋白质、RNA、DNA和脂质,其中微RNA显示出具有主要作用。已知干细胞来源的EV并且特别是人骨髓来源的间充质基质细胞(bone-marrow derivedmesenchymal stromal cell,BM-MSC)显示出促肿瘤发生活性和抗肿瘤发生活性二者,其取决于肿瘤类型和发展阶段。类似地,MSC-EV也可正向或负向调节肿瘤血管形成(Zhu W,Huang L,Li Y,Zhang X,Gu J,Yan Y,等Exosomes derived from human bone marrowmesenchymal stem cells promote tumor growth in vivo.Cancer letters.2012;315(1):28-37)。据报道,MSC-EV在施用于荷瘤(tumor-bearing)小鼠之后在体内促进血管生成(Zhu W,Xu W,Jiang R,Qian H,Chen M,Hu J,等Mesenchymal stem cells derived frombone marrow favor tumor cell growth in vivo.Exp Mol Pathol.2006;80(3):267-74)。另一些研究观察到MSC-EV对通过肿瘤细胞的VEGF分泌具有间接作用。
最近,本发明人已经表明,另一种来源的表达分离自肝的肝细胞标志物的人驻留非卵圆多能祖细胞(即人肝干细胞(human liver stem cell,HLSC))可显示出抗肿瘤作用(Herrera MB,Bruno S,Buttiglieri S,Tetta C,Gatti S,Deregibus MC,等Isolationand characterization of a stem cell population from adult human liver.Stemcells.2006;24(12):2840-50)。特别地,HLSC-EV显示出通过涉及特定抗肿瘤miRNA的递送的机制降低肝癌生长(Fonsato V,Collino F,Herrera MB,Cavallari C,Deregibus MC,Cistema B,等Human liver stem cell-derived microvesicles inhibit hepatomagrowth in SCID mice by delivering antitumor microRNAs.Stem cells.2012;30(9):1985-98)。然而,未研究HLSC-EV是否也影响肿瘤血管生成。
在WO2006/126236中已对HLSC进行了描述。
此外,据本发明人所知,从未报道过干细胞来源的EV通过特定miRNA的递送针对肾癌具有抗肿瘤作用。
WO 2011/107437公开了来源于成体干细胞(例如BM-MSC、G1-MSC和HLSC)的微泡在治疗肿瘤疾病例如肝、上皮、肺、前列腺、卵巢、乳房、胃和结肠肿瘤中有效。WO 2011/070001公开了HLSC来源的条件培养基用于治疗肝癌、卡波西肉瘤(Kaposi’s sarcoma)和乳腺癌的用途。在这些专利中既没有讨论肾癌也没有讨论miRNA的作用。
如将在本说明书的实验部分中更详细地举例说明的,现在本发明人已出人意料地发现,来源于成体干细胞的细胞外囊泡(EV)例如HLSC-EV和MSC-EV能够在体外抑制肾肿瘤内皮细胞的迁移,并且HLSC-EV还显著降低血管样形成(vessel-like formation)。体内实验还显示HLSC-EV能够抑制肿瘤血管生成。非常有趣的是,在这些实验中,HLSC-EV对正常内皮细胞未显示出任何作用。因此,本发明解决了提供在肾癌的治疗性处理中高效的产品的问题。
EV具有复杂的组成,并且包含许多功能性蛋白质、脂质和核酸。本发明人已经确定了由HLSC-EV携带的四种微RNA,即miR-15a、miR-181b、miR-320c和miR-874,其能够在肾癌-TEC转染之后体外抑制肿瘤血管生成。用这些miRNA转染分别导致其靶基因FGF1、PLAU、ITGB3和EPHB4的表达降低。当用携带以上确定的miRNA的EV刺激TEC时,观察到与FGF1和PLAU表达的抑制相关的miRNA的表达显著增强,然而未观察到ITGB3和EPHB4的抑制。FGF1是参与肿瘤血管生成的最重要的促血管生成因子之一并且能够不依赖于VEGF地调节血管生成。PLAU是编码尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,uPA)(激活来自纤溶酶原的纤溶酶的酶)的基因。纤溶酶参与胞外基质降解的蛋白水解过程,这对血管生成和癌症进展是重要的。FGF1和PLAU通过FGF1受体(FGFR)相联系(colligate),FGF1受体(FGFR)可激活uPA并增强其受体uPAR的表达。FGF1、uPA和uPAR,其全部通过FGFR在正反馈回路(positive feedback loop)中连接。
鉴于这些结果,包含miR-15a、miR-181b、miR-320c、miR-874或其混合物的药物载体对于肾癌的治疗是非常有前景的治疗剂。
因此,本发明的第一方面是包含选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合的微RNA的可药用载体,其用于治疗肾癌。根据本发明的一个优选实施方案,可药用载体包含miR-15a、miR-181b、miR-320c和miR-874中的至少两种的组合。甚至更优选地,可药用载体包含所有miR-15a、miR-181b、miR-320c和miR-874。
miR-15a、miR-181b、miR-320c和miR-874是本身已知的微RNA(miRNA);其特征和序列可在例如定名为miRBase的数据库中根据登录号MI0000069、MI0000269、MI0003778、MIPF0000401找到。
药物效果可归因于药物载体中包含的miRNA。设想用miRNA对靶细胞的任何高效转染有效的用于治疗肾癌。miRNA的高效转染需要合适的药物载体,优选地以微米粒或纳米粒的形式。这样的载体可商购获得,包括基于藻酸盐的微载体(GEM,Global CellSolutions)、基于右旋糖酐的微载体(Cytodex,GE Healthcare)、基于胶原的微载体(Cultispher,Percell)和基于聚苯乙烯的微载体(SoloHill Engineering)。
作为替代,miRNA的药物载体可以是病毒载体。基于病毒的系统通常使用逆转录病毒、慢病毒、腺病毒或腺相关病毒(adeno-associated virus,AVV)作为递送载体,如例如在Ningning Yang.An overview of viral and nonviral delivery systems formicroRNA.Int J Pharm Investig.2015年10月至12月;5(4):179-181中所公开的。因此,miRNA的合适载体的选择和使用完全在本领域技术人员的能力范围内。
miRNA的甚至更优选的药物载体是囊泡,例如脂质体或细胞外囊泡(EV)。细胞外囊泡(例如细胞来源的微泡或外排体(exosome)是最优选的药物载体。
因此,根据本发明的另一个优选实施方案,可药用载体是细胞外囊泡(EV),其来源于干细胞,优选地来源于成体干细胞,更优选地来源于间充质干细胞(mesenchymal stemcell,MSC)(例如如骨髓基质干细胞)、或来源于脂肪干细胞(adipose stem cell,ADS)、或来源于非卵圆人肝祖细胞(human liver progenitor cell,HLSC)。HLSC及其获得方法在作为WO2006126219公开的国际专利申请中公开。
本发明的另一方面是分离自干细胞条件培养基的细胞外囊泡的组合物,所述条件培养基分离自优选成体干细胞的条件培养基,更优选分离自间充质干细胞(MSC)或人肝干细胞(HLSC)或脂肪干细胞(ADS)的条件培养基,以用于治疗肾癌。这样的细胞外囊泡是初始的,即分离自干细胞条件培养基的非经改造的EV,其凭借其生物学活性分子的内在载物(特别是miRNA)在肾癌的治疗性处理中有效。
因此,根据本发明所述应用的细胞外囊泡(EV)是分离的、天然存在的EV,或者作为替代,是已改造成包含选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合中的一种或更多种微RNA。
作为EP 2010663公开的欧洲专利申请向本领域技术人员提供了关于如何用特定miRNA改造EV的说明。技术人员已知的用于将RNA引入到囊泡或外排体的技术是转染或共孵育。已知的转染方法是例如电穿孔、脂质体转染、显微注射、通过病毒和非病毒载体转染、磁辅助转染和声致穿孔(sonoporation)。因此,将已离体引入选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合的微RNA的经改造的EV是本发明的另一方面。
根据本发明所述应用的经改造的细胞外囊泡(EV)包含的上述微RNA的量显著高于在天然存在的细胞外囊泡(EV)中所包含的量。
根据本发明所述应用的细胞外囊泡(EV)是天然存在的EV,或者作为替代,是与天然存在的细胞外囊泡(EV)相比,改造成包含显著更高量的一种或更多种微RNA的EV,所述微RNA选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合。所述经改造的EV可通过离体地将选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合的一种或更多种微RNA负载到分离的细胞外囊泡中获得。作为替代,可通过在如上限定的干细胞中转染miRNA,并随后从经转染干细胞的条件培养基中分离EV并进行纯化来获得经改造的EV。
评估与天然存在的细胞外囊泡(EV)相比显著更高量的miRNA的合适方法是qPCR数据分析的ΔΔCT方法。
负载效率(即存在于本发明的经改造的EV中的靶miRNA的量)表示为相对值,其与天然量相比为至少2倍。或者,负载效率可以以绝对值表示为每个EV负载的靶分子数。设想该值可比天然量高约1×103至约1×105个靶分子/EV。
公开了本发明人用来源于MSC和HLSC的EV进行的实验的以下实验部分仅以举例说明的方式提供,并且不旨在限制由所附权利要求书确定的本发明的范围。
实验部分
材料和方法
细胞培养
先前已在本发明人的实验室中从患有肾癌4的患者的手术标本中分离TEC并培养。TEC是通过磁性细胞分选(magnetic cell sorting)(MACS系统,Miltenyi Biotech,Auburn,CA)使用抗CD105阳性选择从消化的组织中分离,并在EndoGro完全培养基(Millipore)中进行培养,如所描述的那样(Bussolati B,Deambrosis I,Russo S,Deregibus MC,Camussi G.Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB journal,official publication of theFederation of American Societies for Experimental Biology.2003;17(9):1159-61)。
HLSC是在本发明人的实验室中从获自Lonza的人冷冻保存的正常肝细胞中分离的,如先前所描述的那样(Herrera MB,Bruno S,Buttiglieri S,Tetta C,Gatti S,Deregibus MC,等Isolation and characterization of a stem cell population fromadult human liver.Stem cells.2006;24(12):2840-50)。简言之,将细胞在肝细胞无血清培养基(Gibco Hepatozyme-SFM;Invitrogen)中以1.0至1.5×105个活细胞/cm2的密度平板接种在胶原包被的培养板上2周。2周的培养之后,肝细胞死亡,并随后将培养基用补充有L-谷氨酰胺(5mM)、Hepes(12mM,pH7.4)、青霉素(50IU/ml)、链霉素(50μg/ml)(均来自Sigma)和10%FBS(Lonza)的α-极限必需培养基/内皮细胞基础培养基-1(α-minimum essentialmedium/endothelial cell basal medium-1,α-MEM/EBM)(3∶1)(Gibco/Euroclone)替换。3周之后克隆单独附着细胞并扩增。HLSC对CD73、CD90、CD29和CD44呈阳性,并且对CD45、CD34、CD117(c-kit)和CD133呈阴性。
MSC购自Lonza并在MSCBM完全培养基(Lonza)中培养。
EV分离和表征
如先前所述(Herrera MB,Fonsato V,Gatti S,Deregibus MC,Sordi A,Cantarella D,等Human liver stem cell-derived microvesicles accelerate hepaticregeneration in hepatectomized rats.Journal of cellular and molecularmedicine.2010;14(6B):1605-18)(做了较小的修改)进行EV分离。简言之,将汇合的HLSC或MSC的培养基对无FBS的RPMI进行更换经18小时。第二天,将该培养基在3000g下离心30分钟以去除细胞碎片和凋亡小体。在那之后,使用带有转子类型为45Ti 45000RPM的BeckmanCoulter Optima L-100K Ultracetrifuge将上清液在100.000g,4℃下超速离心2小时。EV沉淀物重悬在补充有10%DMSO的RPMI中。然后将HLSC-EV的悬浮液在-80℃下进行冷冻直至使用。使用NTA分析和电子显微术对EV进行分析。EV的平均尺寸为90nm(±20)。对于一些体内成像实验,EV通过1μM Vybrant Cell Tracers DiD(Ex:640nm;Em:700nm)或不含血清的Dil溶液(Ex:530nm;Em:580nm)(Molecular Probes,Oregon,USA)进行标记,然后在PBS 1×40中通过超速离心洗涤两次。
生存力和迁移测试
对于增殖的测试,将TEC以2×103个/孔的密度接种在96孔板中。第二天,将细胞在EndoGro完全培养基(Lonza)中以1×1010或5×1010或10×1010个EV/TEC的浓度用HLSC-EV或MSC-EV进行处理。在EV刺激之后24、48和72小时,使用Cell Proliferation ELISA,BrdU(比色)试剂盒(Roche,11647229001)根据制造商的说明书通过BrdU掺入来测量增殖。在TEC上进行迁移测试,接种在24孔板上并生长直至汇合。就在刮擦完成之后,以1×1015或5×1015或10×1015个EV/孔的浓度添加EV。在刮擦之后0、3、7和24小时的时间时,在放大率为10×的显微镜上采集图像。该距离通过LAS软件(Leica)测量。结果表示为平均辐射率±SD。
体外血管样结构形成
将TEC以25×103个细胞/孔的密度接种到基质胶(Matrigel)包被的24孔板上,并在存在1×1010、5×1010、10×1010或20×1010个EV/TEC的情况下在EndoGro完全培养基中培养。没有EV的TEC用作对照。孵育24小时之后,记录相衬图像(phase-contrast image)(放大率,×10),并使用LAS软件(Leica)测量网络结构的总长度。在五个随机视场中计算每个视场的总长度,并表示为与相应对照的比率。数据表示为平均辐射率±SD。
体内血管生成模型
动物研究根据国家准则和法规进行,并经都灵大学伦理委员会(EthicsCommittee of the University of Torino)批准(协议号:338/2016-PR)。将通过在基质胶内的TEC注射获得的体内肿瘤血管生成的模型用于评估干细胞来源的EV的作用,如前所述(参考FASEB 2003)。为了防止肿瘤血管生成的发生,在注射之前对TEC进行预处理。为此,对SCID小鼠(6至8周龄)(Charles River Laboratories,Lyon,France)在基质胶内用1×106TEC进行皮下注射,所述TEC用或未用HLSC-EV/MSC-EV(10×103个EV/细胞)进行预处理:(对于对照和每个EV处理,n=8)。7天之后,切除基质胶栓(plug),并通过Masson三色反应(Masson’s trichromic reaction)对血管密度进行分析。为了评价EV对已建立的肿瘤血管的影响,在SCID小鼠中在基质胶内皮下注射1×106个TEC。在TEC注射之后三天和第七天,将HLSC-EV(10×103个EV/细胞)两次注射到基质胶栓中。对照小鼠用载剂(PBS)进行注射。在实验的第10天,处死小鼠并切除基质胶栓用于组织化学分析(对于对照和HLSC-EV处理,n=8)
生物分布分析
为了通过TEC的EV摄取的体内成像,对25只SCID小鼠在基质胶内用1×106个TEC进行皮下注射。肿瘤血管形成(1周)之后,将小鼠分成5组(每组n=5):接收载剂(PBS)的对照、DiL MSC-EV、DiD MSC-EV、DiL HLSC-EV和DiD HLSC-EV。静脉内注射经DiD或DiL标记的EV(1.3×1010个EV/小鼠),并在5小时之后处死小鼠。回收获自用DiL EV处理的小鼠的器官(基质胶栓、皮肤、肾、脾、肝和肺)以用于免疫荧光。将每个器官的冷冻切片用Dapi进行染色以用于核复染色,并通过共聚焦显微术进行分析以检测经DiL标记的EV。
经DiD标记的EV的生物分布通过光学成像进行评价。所有研究均用使用640nm的激发滤光片和700nm的发射滤光片的IVIS 200小动物成像系统(PerkinElmer,Waltham,MA)进行。使用相同的照明设置(例如曝光时间、像素合并因子(binning factor)、f/光圈(f/stop)和视场(Grange 2014))来获取所有图像,并将荧光发射相对于光子每秒每平方厘米每球面度(p/sec/cm2/sr)归一化。在EV注射之后5小时收集在器官上获取的图像。为了对背景光子发射进行控制,使用在535nm激发下捕获的数据对获得的数据进行平均背景减除。使用Living Image 4.0软件(PerkinElmer)(Grange 2014)采集和分析图像。在徒手绘制的目的区域(region of interest,ROI)中量化荧光(p/sec/cm2/sr)。数据表示为平均辐射率±SD。
基因表达研究和实时PCR
使用Applied BiosystemsArray Human MicroRNA A/B Cards(Applied Biosystems,Foster City,CA)对HLSC-EV或MSC-EV中的miRNA表达水平进行分析,以通过qRT-PCR描述754个成熟miRNA。该试剂盒使用微RNA特异性茎环逆转录引物和TaqMan探针以在两步骤实时逆转录PCR测定中检测成熟的miRNA转录物。简言之,使用环状引物的混合物(Multiplex RT试剂盒,Applied Biosystems)按照制造商的方案通过逆转录从总RNA样品(80ng)中产生单链cDNA。然后将RT反应物稀释并与Taqman universal masterMix(Applied)以1∶1的比例混合,加载到TaqMan微流体卡中,并进行qRT-PCR实验。所有反应均使用配备有384孔反应板的Applied Biosystems 7900HT实时PCR仪进行。使用自动基线和阈值,使用SDS软件2.3版计算原始Ct值。我们分析了3个HLSC-EV样品中的miR表达。在35次PCR循环之后扩增的所有微RNA均被归类为未表达。只将检测到的或在多于两个样品中未检测到的微RNA考虑在内。
将qRT-PCR用于确定TEC中的miRNA或靶基因表达。简言之,将来自所有样品的200ng输入RNA用miScript逆转录试剂盒进行逆转录,并随后使用miScript SYBR GreenPCR试剂盒(全部来自Qiagen,Valencia,CA,USA)通过qRT-PCR将cDNA用于检测和量化目的miRNA或基因。如制造商的方案(Qiagen)所述,对于每个反应,所有样品均使用3ng的cDNA,以一式三份运行。然后,根据35个PCR循环的Ct检测截止值,使用对每个阵列中的总体miRNA表达计算的平均表达值对相对表达数据进行归一化,如在MestdaghP,Van VlierbergheP,De Weer A,Muth D,WestermannF,SpelemanF,等A novel and universal method formicroRNA RT-qPCR data normalization.Genome biology.2009;10(6):R64)中所述。使用人血管生成PCR阵列(RT2 Profiler PCR阵列,96/孔格式,Qiagen),按照制造商的说明书以一式三份进行TEC(用或未用HLSC-EV进行处理)中促血管生成基因表达的PCR分析。数据使用SaBioscience(Qiagen)在线软件进行分析并且表示为相对定量值±CI(置信区间)。
细胞转染
使用HiPerfect试剂(Qiagen)进行TEC的转染。为了找到最佳转染浓度,用所有推荐浓度的模拟miR-FITC和HiPerfect试剂转染TEC。还进行了HiPerfect最大剂量(9μlHiPerfect每50×103TEC)的双倍提高。转染之后那天进行的FACS分析揭示,在不损害其生存力和增殖的情况下两倍最大剂量的HiPerfect允许转染超过60%的TEC。将该剂量用于所有转染实验。
使用以下模拟miRNA进行TEC的转染:miR-15a、miR-20b、miR-23a、miR-93、miR-181b、miR-320c、miR-424和miR-874(均来自Qiagen)。转染之后那天更换新鲜生长培养基,并在第2天将细胞用于体外实验(增殖、凋亡测试、体外血管生成测定)或基因表达分析(实时PCR、Western印迹、FACS分析)。
荧光激活细胞分选(FACS)分析
使用CytoFLEX流式细胞仪(Beckman Coulter)进行HLSC-EV和MSC-EV的FACS分析。使用的抗体是经FITC缀合的抗体抗CD63(Abnova),抗CD105(Dako Cytomation,Copenhagen,Denmark),抗CD90(BD Pharmigen),抗CD44(Miltenyi Biotech),CD45(BDPharmigen),抗ICAM和抗VCAM(Serotec),CD31(BioLegend),整合素亚基α4、α5、α6(来自BDPharmigen);经PE缀合的抗体抗CD73(BD Pharmigen)、抗整合素亚基α4、α5(均来自BDPharmigen)和VE-钙黏着蛋白(BioLegend)。FITC或PE小鼠非免疫同型IgG(DakoCytomation)用作对照。
Western印迹
蛋白质样品通过4%至15%梯度十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecyl sulfate-polyacrylamide gel electrophoresis,SDS PAGE)进行分离,并用针对PLAU(Abcam,ab131433)或FGF1(Abcam ab9588)的抗体对其进行免疫印迹。用增强化学发光(enhanced chemiluminescence,ECL)检测试剂盒和ChemiDocTM XRS+System(BioRad)使蛋白质带显现。加载20μg/孔的细胞裂解物。
统计
使用Kolmogorov-Smirnov测试对数据的分布正态性进行评估。使用SigmaPlot11.0软件进行统计分析。然后,当分布为正态时,使用Student t检验对处理组与对照组之间的差异进行分析。数据表示为平均±SEM。当p<0.05时,差异被认为是显著的。
结果
HLSC-EV在体外抑制血管生成潜能和肾TEC的迁移
通过在体外评价增殖、凋亡和血管样结构形成来对MSCEV和HLSC-EV对TEC的影响进行评估。HLSC-EV刺激在体外以剂量依赖性方式显著抑制TEC血管生成:10×103个EV/TEC的添加使血管样结构形成降低了37%,并且20×103个EV/TEC的剂量使血管样结构形成降低了44%(图1,A、B、D)。MSCEV和HLSC-EV二者均未改变TEC的生存力(数据未示出)。还通过创伤愈合测定评价了MSC-EV和HLSC-EV对TEC运动性的作用。两种EV均显著抑制了TEC的迁移,并且HLSC-EV在1×103/TEC的剂量下已经是有效的(图1,E)。
在对照实验中,评价了MSC-EV和HLSC-EV对正常内皮细胞的作用:MSC-EV能够增强人微血管内皮细胞(microvascular endothelial cell,HMEC)的血管生成特性,而HLSC-EV没有显示出任何作用(图1,F)。这表明来自MSC和HLSC的EV对正常血管生成和肿瘤血管生成具有不同的作用。
图1.HLSC-EV或MSC-EV在体外对TEC的血管生成特性的作用。通过对照TEC(A),通过用HLSC-EV处理的TEC(B)以及通过用MSC-EV处理的TEC(C)的血管样结构的形成;通过对照TEC或用EV处理的TEC形成的每个视场血管样结构的总长度的图(D);创伤愈合测定期间TEC迁移的图(E);通过用EV处理的HMEC形成的每个视场血管样结构的总长度的图(F)。
HLSC-EV在体内防止肿瘤血管生成
通过使用由在SCID小鼠中皮下植入时的基质胶内的TEC组织诱导的人肿瘤血管生成模型,在体内对MSC-EV和HLSC-EV的作用进行评价。在此模型中,TEC在7天内组织了与小鼠循环连接的结构。在预处理环境中,将TEC用HLSC-EV或MSC-EV处理24小时,并皮下植入到SCID小鼠中。植入之后7天,切除基质胶栓并且对血管密度进行免疫组织化学分析。如预期的那样,对对照栓的分析表明存在与鼠脉管连接的血管(图2,A)。植入之前用HLSC-EV处理24小时的TEC的栓未呈现包含血管的红细胞(图2,B、D),而用MSC-EV进行的预处理未显示出任何作用(图2,C、D)。
由于HLSC-EV能够防止肿瘤血管生成,因此还评价了其对已建立的肿瘤血管的作用。为此,在TEC植入之后的3天和7天将HLSC-EV注射到基质胶栓中,并在10天之后回收栓。HLSC-EV处理显著降低了几乎50%的血管密度(图2,E)。
图2.体内肿瘤血管生成。用Masson三色反应染色(细胞外基质染成蓝色、细胞染成红色以及红细胞染成黄色)的基质胶切片的代表性图像:A-包含对照TEC的基质胶栓;B-包含用HLSC-EV处理的TEC的基质胶栓;C-包含用MSC-EV处理的TEC的基质胶栓。包含血管的红细胞由箭头指示;D-在包含对照或用EV预处理的TEC的基质胶中血管密度的图(n=8,对5个视场/实验进行分析,***-p<0.001,相对于对照TEC)。E-在注射之后第3天和第7天时,包含用或未用HLSCEV处理的TEC的基质胶中血管密度的图(n=8,对5个视场/实验进行分析,*-p<0.05,相对于对照基质胶)。
在TEC中HLSC-EV的体内摄取
为了评价已组织成血管的TEC在体内摄取EV的能力,静脉内注射经标记的MSC-EV和HLSC-EV(1.3×1010个EV/小鼠)。将EV用荧光染料标记,近红色(DiD)用于光学成像以及红色(DiL)用于免疫荧光(见方法)。5小时之后,处死小鼠并回收器官。基于初步实验(未示出)选择EV的剂量和时间。通过光学成像,我们证明了在基质胶内体内注射的TEC可摄取两种类型的经DiD标记的EV(MSC-EV和HLSC-EV),如图3A和C中所示。相反,在栓附近分离的真皮组织产生非常低的荧光信号(图3B和C)。关于所有器官内的生物分布,EV主要在肝内积聚。
图3.经标记的EV的生物分布。A和B.在EV注射之后5小时收集的通过对基质胶栓(A)和移植器官(B)进行光学成像的代表性图像。CTL:未经处理;MSC-EV:用DiD MSC-EV处理;HLSC-EV:用DiD HLSC-EV处理。C.处死用DiD MSC-EV和DiD HLSC-EV处理的小鼠之后5小时,表示为平均辐射率±SD的基质胶栓和邻近皮肤的荧光强度的量化。减去来源于未经处理小鼠的背景(N=5)。D.处死用DiD MSC-EV和DiD HLSC-EV处理的小鼠之后5小时,表示为平均辐射率±SD的器官(肺、肝、脾和肾)的荧光强度的量化。减去来源于未经处理小鼠的背景(N=5)。
通过移植器官上的免疫荧光获得相似的结果。在基质胶栓中,通过共聚焦分析在人TEC内可检测到由MSC和HLSC二者脱落的经DiL标记的EV(图4,A)。当与皮肤中的正常内皮细胞相比时,这种积聚对TEC相当具有特异性(图4,A)。这确定了TEC能够摄取基质胶内的EV。通过免疫荧光,EV在所有样品的肝实质和脾内也是可见的(图4,B)。几乎没有阳性细胞存在于肺和肾中。与HLSC相比,由MSC脱落的EV的生物分布没有观察到差异。
图4.组织中经DiL标记的EV的检测。在静脉内注射经MSC或HLSC标记的EV之后5小时,小鼠的基质胶栓和皮肤(A)以及其他器官(肝、脾、肾和肺)的代表性荧光图像。在基质胶栓和排泄器官中的细胞内可检测到的DiL EV(红色斑点)。细胞核与DAPI共同染色(蓝色)(N=5)。
HLSC-EV对TEC的分子作用
基于这些结果,通过使用血管生成阵列对体外血管样结构组织期间在HLSC-EV刺激之后TEC中发生的变化进行分子分析。用HLSC-EV(10×103个EV/TEC)处理TEC,并且从血管生成测定收获扩散细胞。在测试的84个基因中,我们鉴定了在体外血管生成过程中通过HLSC-EV在TEC中显著下调的11种促血管生成因子(图5,A)。特别地,TEC使以下下调:促血管生成表面受体,其包括Tie-1、β3整合素(beta 3integrin,ITGB3)、肝配蛋白受体B4(ephrinreceptor B4,EPHB4)和内皮联蛋白(或CD105));以及生长因子,例如FGF1、TGF家族成员、尿激酶型纤溶酶原激活物(PLAU)和组织因子(F3)。最后,已知参与TEC的促血管生成作用的Akt1也被下调。
图5.造成对TEC的抗血管生成作用的HLSC-EV特异性miRNA的选择。A-用HLSC-EV处理之后TEC中下调的基因的列表(n=3,数据表示为平均倍数变化±CI(置信区间);这些基因可被136个miRNA(B)靶向,其中42个由HLSC-EV携带。在这42个miRNA中有26个还由MSC-EV携带,其对TEC未显示出任何抗肿瘤作用,因此,这26个miRNA被排除在研究之外(C)。16个HLSC-EV特异性miRNA可以与EV对TEC的生物作用有关。选择用于研究的miRNA是粗体的。
由HLSC-EV携带的抗血管生成微RNA的确定
随后,为了剖析所观察的基因调控的可能效应物,研究了可能涉及的HLSC的微RNA含量。为此,使用了生物信息学分析的策略,随后进行了体外功能验证。
使用Funrich V3软件,本发明人预测了靶向11种下调基因的miRNA。确定了136个miRNA,并将其与由HLSC-EV携带的miRNA匹配(在囊泡(vesiclepedia)上证实的数据库)。其中,确定了由HLSC-EV表达的42个miRNA(图5,B)。考虑到缺少对TEC的作用,随后的分析在由HLSC-EV携带的42个miRNA中排除了也存在于MSC-EV中的那些(图5,C)。靶向确定的基因的16个miRNA仅存在于HLSC-EV中并且用于功能研究(图5,D)。其中,3个被描述为促肿瘤发生的(hasmiR-30e-5p、has-miR-301a-3p、has-miR-212-3D),并且3个(miR-23、miR-181、miR-320)与多于一个的miRNA家族成员一起存在。因此,发明人考虑了8种miRNA:miR-15a、miR-20b、miR-23a、miR-93、miR-181b、miR-320c、miR-424和miR-874(图5,D,粗体)。有趣的是,这些选定的miRNA在对照TEC中下调(Ct>33)。
HLSC-EV miRNA对TEC血管生成的作用
为了证明这些单独的miRNA对TEC血管生成特性的特异性作用,将TEC用选定的模拟物进行转染。转染之后两天,进行血管样结构形成体外测定。4种miRNA显著抑制体外血管样结构形成:miR-15a、miR-181b、miR-320c和miR-874(图6,A)。没有观察到模拟物对增殖或凋亡的作用(图6,B和C)。
图6.选定的HLSC-EV特异性miRNA对TEC的促血管生成特性和生存力的影响。A-通过用选定的模拟RNA或乱序(scramble)RNA转染的TEC的体外血管样结构形成的图。(n=3,一式两份,每孔10张图像,*-p<0.05,相对于乱序RNA);B-经转染TEC的凋亡率;C-经转染TEC的增殖率。
因此,本发明人调查了用4个活性miRNA模拟物的转染如何改变预测的靶标(EPHB4、ITGB3、FGF1和PLAU)的表达。TEC转染显著下调了其靶标(表1,图7)。
表1.相对于对照TEC,用选定的miRNA转染的TEC中促血管生成基因的相对表达。
图7.在用选定的模拟miRNA转染的TEC中miRNA及其靶标的表达。A-miR-15a及其靶基因FGF1、EPHB4的表达;B-miR-181b及其靶基因PLAU、ITGB3、FGF1、EPHB4的表达;C-miR-320c及其靶基因PLAU、ITGB4、FGF1的表达;D-miR-874及其靶基因EPHB4、PLAU、ITGB3、FGF1的表达;(n=5,*-p<0.05,相对于乱序RNA)。
发现这些miRNA的表达在用HLSC-EV处理的TEC中显著增强(图8,A),表明经由电脑模拟(insilico)数据的有效性。与此同时,在RNA和蛋白质二者的水平上评价了HLSC-EV对模拟物的4个靶基因(EPHB4、ITGB3、FGF1和PLAU)的作用。
图8.在用HLSC-EV处理的TEC中miRNA及其靶标的表达。A-在对照TEC和用HLSC-EV处理的TEC中的miRNA的相对表达(n=7,*-p<0.05,相对于对照);B-在对照TEC和用HLSC-Ev处理的TEC中的靶基因的相对表达(n=7,*-p<0.05,相对于对照);C-在对照TEC、用miR-15a转染或用HLSC-EV刺激的TEC中FGFl表达的Western印迹分析的代表性图像。D-在对照TEC、用miR-181b转染或用HLSC-EV刺激的TEC中PLAU表达的Western印迹分析的代表性图像。
确定了在正常培养条件下用HLSC-EV处理的细胞中两个靶标(即FGF1和PLAU)显著降低(图8,B)。通过Western印迹确定了在经转染TEC或用HLSC-EV处理的TEC中FGF1和PLAU表达的下调(分别为图8的C和D)。
肿瘤内皮细胞与miRNA组合的转染以及抗血管生成作用的评价
使用HiPerfect试剂(Qiagen),用以下miRNA的组合转染TEC:miR-874/miR-15a;miR-874/miR-181b;miR-847/miR-320c;和miR181c/miR-320。转染之后两天,进行血管生成体外测定。所有miRNA组合均显著降低TEC的促血管生成特性。此外,rniRNA的组合比单独的miRNA更高效。
结果在图9中进行了报告,图9为条形图,其示出了由用单独或组合的miRNA转染的TEC形成的血管样结构的相对数量。(*=p<0.01,相对于乱序RNA)。
Claims (19)
1.包含选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合的微RNA的可药用载体,其用于治疗肾癌。
2.根据权利要求1所述应用的可药用载体,其包含由miR-15a、miR-181b、miR-320c和miR-874组成的组中至少两种的组合。
3.根据权利要求1或2所述应用的可药用载体,其中所述可药用载体是微米粒或纳米粒,并且其中所述微RNA包含在所述微米粒或纳米粒内或者附着在所述微米粒或纳米粒的表面上。
4.根据权利要求1至3中任一项所述应用的可药用载体,其中所述可药用载体是细胞外囊泡(EV)。
5.根据权利要求4所述应用的可药用载体,其中所述细胞外囊泡(EV)来源于干细胞。
6.根据权利要求5所述应用的可药用载体,其中所述细胞外囊泡(EV)来源于成体干细胞。
7.根据权利要求6所述应用的可药用载体,其中所述细胞外囊泡(EV)来源于选自以下的成体干细胞:间充质干细胞(MSC)、非卵圆人肝祖细胞(HLSC)和脂肪干细胞(ASC)。
8.根据权利要求4至7中任一项所述应用的可药用载体,其中所述细胞外囊泡(EV)改造成包含选自以下的微RNA:miR-15a、miR-181b、miR-320c、miR-874及其任意组合。
9.经改造的细胞外囊泡(EV),其与天然存在的细胞外囊泡(EV)相比包含显著更高量的选自以下的微RNA:miR-15a、miR-181b、miR-320c和miR-874,或及其任意组合,所述经改造的细胞外囊泡(EV)可通过离体地将选自miR-15a、miR-181b、miR-320c和miR-874及其任意组合的微RNA负载到分离的细胞外囊泡中获得。
10.根据权利要求9所述的经改造的细胞外囊泡(EV),其包含的miR-15a、miR-181b、miR-320c、miR-874或其任意组合的量比存在于所述天然存在的细胞外囊泡(EV)中的量超出1×103至×105个分子/EV。
11.根据权利要求9所述的经改造的细胞外囊泡(EV),其包含的miR-15a、miR-181b、miR-320c、miR-874或其任意组合的量为存在于所述天然存在的细胞外囊泡(EV)中的量的至少2倍,如通过qPCR数据分析的ΔΔCT方法所测得的。
12.根据权利要求9至11中任一项所述的经改造的细胞外囊泡(EV),其中所述细胞外囊泡是从干细胞或体液中分离的。
13.根据权利要求12所述的经改造的细胞外囊泡(EV),其中所述干细胞是成体干细胞。
14.根据权利要求9至13中任一项所述的经改造的细胞外囊泡(EV),其中已通过选自以下的转染方法将所述微RNA引入到所述EV中:电穿孔、脂质体转染、显微注射、通过病毒和非病毒载体转染、磁辅助转染、共孵育和声致穿孔。
15.制备经改造的细胞外囊泡(EV)的方法,其包括通过选自以下的转染方法将选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合的微RNA引入到所述细胞外囊泡中的步骤:电穿孔、脂质体转染、显微注射、通过病毒和非病毒载体转染、磁辅助转染、共孵育和声致穿孔。
16.制备经改造的细胞外囊泡(EV)的方法,其包括从细胞的条件培养基中分离并纯化所述细胞外囊泡的步骤,所述细胞已通过选自以下的转染方法用选自miR-15a、miR-181b、miR-320c、miR-874及其任意组合的微RNA进行转染:电穿孔、脂质体转染、显微注射、通过病毒和非病毒载体转染、磁辅助转染、共孵育和声致穿孔。
17.组合物,其包含分离自干细胞条件培养基的细胞外囊泡(EV)的混合物,所述组合物用于治疗肾癌。
18.根据权利要求17所述的组合物,其中所述干细胞是成体干细胞。
19.根据权利要求18所述的组合物,其中所述成体干细胞选自间充质干细胞(MSC)、非卵圆人肝祖细胞(HLSC)和脂肪干细胞(ASC)。
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