CN105473710B - 同源盒转录因子VentX调控人树突状细胞的分化和成熟 - Google Patents
同源盒转录因子VentX调控人树突状细胞的分化和成熟 Download PDFInfo
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Abstract
本发明涉及人同源盒转录因子VentX及其对人树突状细胞的分化和成熟的调控,并且涉及相关的治疗和诊断组合物及其使用方法。
Description
优先权及相关申请
本申请要求2013年4月10日提交的美国临时申请号61/810,610的权益,其全部内容通过引用明确地并入本文。
技术领域
本发明大体上涉及人类生物学发现,以及基于此的治疗和诊断组合物。更具体地,本发明涉及人同源盒(homeobox)转录因子VentX及其对人树突状细胞的分化和成熟的调控,并且涉及相关的治疗和诊断组合物及其使用方法。
背景技术
树突状细胞(DC)是最有效力的专职抗原呈递细胞,其在启动免疫应答和维持免疫内稳态中起到关键作用。源自骨髓造血干/多能祖细胞的DC在外周非淋巴组织中以未成熟状态存在。(Geissmann等,2010Science 327(5966):p.656-61;Liu等,2010 Immunol Rev234(1):p.45-54.)这些未成熟的DC能够有效地摄入并处理各种抗原。在被诸如细菌脂多糖(LPS)的炎性细胞因子或微生物组分刺激时,未成熟的DC经历成熟过程并且开始迁移至局部淋巴结,在局部淋巴结中与通过CD40与抗原特异性T细胞上的CD40配体相互作用,并且成熟为有效力的免疫刺激性或免疫耐受性细胞(移除(remove))。(Geissmann等,2010Science 327(5966):p.656-61;Randolph等.2005 Nat Rev Immunol 5(8):p.617-28;Mellman等.2005 Adv Exp Med Biol 560:p.63-7.)DC的成熟与MHC II类和共刺激分子的上调表达、免疫刺激性细胞因子的生产、以及刺激幼稚型和抗原特异性T细胞能力的取得相关联。
肠粘膜富含DC,DC存在于粘膜固有层中并调控对于病原体入侵的免疫应答。肠DC的成熟和功能受宿主因子和微生物组分两者的调控。肠DC通过模式识别受体检测病原体的存在,模式识别受体诸如Toll样受体(TLR)家族的成员。已经假定肠DC的异常成熟和激活在诸如炎症性肠病(IBD)的自体免疫性/炎症性病症中起致病作用。已经发现TLR2、TLR4、CD40的表达在从IBD患者的发炎粘膜分离的DC中增强。另外,已经发现异常的TLR信号转导使患者倾向于克罗恩氏病(Crohn’s disease)。随着证据的累积,暗示了在自体免疫-炎症性病症中靶向DC的潜在作用。然而,目前对于能够操控以调节DC成熟和功能的固有DC因子所知甚少。
在炎症的发病机制中已经涉及了循环单核细胞衍生的DC,并且体外人单核细胞衍生的树突状细胞已经广泛用作探索DC分化的分子机制的模式系统。在炎症的发病机制中已经涉及了循环单核细胞衍生的DC。先前的研究表明粒细胞-巨噬细胞集落-刺激因子(GM-CSF)和IL4驱动外周血CD14+单核细胞向CD14-CD1a+DC的分化。已经表明诸如IL6、IL10和(-IFN的几个细胞因子负向调控单核细胞向DC的分化,然而其他细胞因子被报道促进DC的分化。(Chomarat等,2000 Nat Immunol 1(6):p.510-4;Mitani等,2000 Br J Haematol 109(2):p.288-95;Allavena等,1998 Eur J Immunol 28(1):p.359-69;Delneste等,2003Blood 101(1):p.143-50;Chomarat等,2003 J Immunol 171(5):p.2262-9;Iwamoto等,2007 J Immunol 179(3):p.1449-57;Gabriele等,2004 Blood 103(3):p.980-7.)最近的基因图谱分析揭示了在诱导人CD14+单核细胞向DC的分化期间大量差异表达的基因。(LeNaour等,2001 J Biol Chem 276(21):p.17920-31.)尽管如此,人单核细胞向DC的分化的潜在关键转录调控机制仍然知之甚少。
发明概述
本发明部分基于VentX是DC成熟和功能的关键调控子、并且VentX能够作为炎症性疾病和免疫疗法的干预目标的发现。
VentX是BMP4信号通路的非洲爪蟾蜍(Xenopus)同源盒基因Vent/Xom的人类同源物,并且最近被定义为控制造血和免疫细胞的增殖和分化的新型造血转录因子。起初在非洲爪蟾蜍胚胎形成中通过反向遗传建模鉴别为经典Wnt信号转导的新型LEF/TCF相关拮抗剂的VentX,被发现为p53/p21和p16ink4a/Rb肿瘤抑制通路的转录激活子。VentX在造血细胞发育中的关键作用,进一步表现为其在控制HSC/MPPs和单核细胞两者向巨噬细胞终末分化的增殖和分化中的作用。
如本文中所公开的,已经示出VentX在从外周血液以及肠粘膜两者分离的人原代DC中表达。利用功能缺失和功能获得两种方法,展示出VentX通过IL6介导机制调控DC的分化和成熟。在从IBD患者的发炎粘膜分离的DC中VentX表达升高,并且VentX的敲低减少了DC的成熟应答。而且,VentX是皮质类固醇的下游靶标和效应子。本文中公开的数据展示了VentX是DC成熟和功能的关键调控子,并且VentX能够作为炎症性疾病和免疫疗法的干预目标。
在一个方面,本发明大体上涉及一种调控树突状细胞的方法。该方法包括向需要给药的对象给药治疗上有效量的组合物,该组合物包含发挥人同源盒基因VentX的调节作用的生物或化学药剂。
在另一个方面,本发明大体上涉及一种筛选用于树突状细胞的调控功能的化合物的方法。该方法包括:(a)提供包含多核苷酸的细胞,该多核苷酸包含VentX启动子;(b)使细胞与候选化合物接触;以及(c)检测VentX表达的活性。
在又一个方面,本发明大体上涉及一种药物组合物,该药物组合物包含通过人同源盒基因VentX的调节作用在树突状细胞上发挥调控作用的生物或化学药剂。
在又一个方面,本发明大体上涉及一种诊断对象的与树突状细胞的成熟相关联的炎症性病症的方法,包括人同源盒基因VentX的表达谱的测定。在某些实施方式中,炎症性病症是自体免疫疾病。
在又一个方面,本发明大体上涉及一种抑制肿瘤的方法。该方法包括向需要给药的对象给药治疗上有效量的组合物,该组合物包含在树突状细胞功能上发挥调节作用的生物或化学药剂。
附图说明
图1:VentX调控原代单核细胞向树突状细胞分化。(A)用靶向VentX的吗啉代(MO)寡核苷酸或对照MO转染人原代单核细胞,如材料和方法中所述。转染后24小时,向培养基中加入GM-CSF和IL4,以诱导树突状细胞分化。在添加细胞因子后5天收集细胞,并且通过流式细胞术分析CD1a和CD14的表面表达。(B)示出由VentX MO或对照MO转染的单核细胞的CD14-CD1a+细胞的百分比的柱形图。结果为(A)中6个不同实验的平均值+标准差(SD)。(C-D)如上所述,用VentX MO或对照MO寡核苷酸转染单核细胞。在添加细胞因子后5天,通过流式细胞术分析CD1b和CD1c的表面表达。示出的结果至少是5个单独实验的代表。(E)通过电穿孔,用pcDNA-GFP或pcDNA-GFP.VentX质粒转染单核细胞。转染后2小时向培养基加入细胞因子,并且在添加细胞因子后3天收集细胞用于流式细胞术分析。GFP阳性种群被设门用于分析CD1a表达。(F)示出(E)中的4个单独实验的平均值+SD。
图2:VentX过度表达促使THP1细胞向树突状细胞的分化。(A)在四环素可诱导启动子的控制下表达GFP或GFP.VentX的THP1细胞系用1.0(g/mL强力霉素(DOX)处理10天。在指示的日期计数细胞数量并作图。(B)暴露于DOX 10天后,表达GFP或GFP.VentX的THP1细胞的细胞周期图谱。细胞用碘化丙啶染色,并且通过流式细胞术分析。(C-D)用DOX处理THP1细胞3天,并收集THP1细胞。分别通过实时PCR和蛋白印迹法(western blot)分析p21和c-myc的mRNA的RNA水平(C)和蛋白质水平(D)。(E-F)用DOX处理THP1细胞,并且在材料和方法中描述的条件下培养,以诱导树突状细胞分化。(E)在处理后2天,收集细胞,以分析由流式细胞术指示的抗原的表面表达。(F)使用相差显微镜对细胞拍照,以示出VentX表达细胞的形态变化。示出的结果是至少3个单独实验的代表。
图3:VentX抑制IL6的表达。(A)用VentX MO或对照MO转染单核细胞5天,并且在培养基中加入GM-CSF和IL4。通过流式细胞术判定细胞间IL6水平。(B)通过电穿孔,用pcDNA-GFP或pcDNA-GFP.VentX转染单核细胞。然后在GM-CSF和IL4的存在下培养细胞3天。在转染的细胞中进行IL6的细胞间染色,并且通过流式细胞术分析GFP阳性细胞的IL6水平。示出的结果是至少3个单独实验的一个代表。(C)如上所述转染并收集单核细胞。进行实时PCR以判定IL6的mRNA水平。(D)通过电穿孔,用pcDNA-GFP或pcDNA-VentX质粒将-592bp人IL6启动子报告基因转染至原代单核细胞。转染后2天,收集细胞以分析荧光素酶活性。(E)将各种IL6启动子报告基因转染至用强力霉素诱导时表达VentX的U2OS细胞。添加DOX后2天收集细胞,并且在DOX的存在或缺失下分析荧光素酶活性。(F)将具有突变的NF(B结合位点的-80bpIL6启动子报告基因转染至U2OS细胞。然后向介质中加入DOX或忽略此步,并且在转染后2天判定荧光素酶活性。(G)表达GFP或GFP.VentX的THP1细胞用DOX处理2天,并且收集并与抗NFκB/p65抗体一起用于染色质免疫沉淀(ChIP)分析。如材料和方法中所述,通过实时PCR放大含有NF(B结合位点的人IL6启动子区域。示出的结果是一个代表性实验的三次重复的M+SD,并且进行了三个独立实验。
图4:增强的自分泌IL6对VentX敲低的树突状细胞中的分化缺陷有作用。如所指示的,用VnetX MO或对照MO转染人原代单核细胞。第二天,向培养基中加入细胞因子GM-SCF和IL4,同时加入中和IL6抗体或对照免疫球蛋白(IgG)。在添加细胞因子后5天收集细胞,并且通过流式细胞术分析(左栏)。在右栏中示出4个单独实验的平均值+SD。
图5:过度表达VentX的树突状细胞的PCR阵列分析。(A-E)通过电穿孔,用pcDNA-GFP或pcDNA-GFP.VentX质粒转染单核细胞。2小时后,向培养基中加入GM-CSF和IL4,以诱导DC分化。转染后3天,加入LPS用于过夜处理并且收集细胞,通过流式细胞术用GFP进行分选。分离总RNA,并且通过PCR阵列分析指示的基因的mRNA水平。(F)通过实时PCR,以不同的引物集判定选择的基因的mRNA水平。示出的结果是两个独立实验中的一个的三次重复的平均值+SD,并且对于全部基因,两组之间的差异显著不同(p<0.05)。
图6:对于粘膜固有层(lamina propria)DC的成熟,需要VentX。(A)从5个IBD患者的发炎的和非发炎的肠粘膜分离LPDC。通过实施PCR判定VentX水平。(B)通过电穿孔,用靶向VentX的siRNA或对照GFP转染LPDC,随后在培养基中培养2天。然后用LPS刺激细胞过夜以诱导成熟,并且收集以用于表面标志物和细胞间细胞因子/趋化因子的流式细胞术分析。填充柱状图指示siGFP转染。(C)柱形图示出(B)中4个不同实验的平均荧光强度(MFI)。结果表示为按对照标准化的MFI百分比。除CD83之外,两组之间的差异显著不同(p<0.05),CD83的差异不显著(N.S.)。(D)对于指示的基因的mRNA表达水平,分析了用siGFP或siVentX转染的LPDC。除CCL19和CXCL1之外,两组之间的差异显著不同(p<0.05)。(E)VentX的敲低损害DC刺激异体T细胞在混合淋巴细胞反应中增殖的能力。(F)用强的松(prednisolone)(10(g/ml)处理DC 48小时或模拟处理,并且以实时PCR判定VentX水平。(G)用GFP或VentX转染DC,并且如指示用强的松处理。24小时后加入LPS,并且在转染后48小时收集细胞,以分析CCL3、CCL5、IL12A和TNF(的mRNA水平。示出的结果是两个独立实验中的一个的三次重复的平均值+SD。
图S1:VentX在单核细胞和树突状细胞中的表达。从外周血液分离的人原代单核细胞在GM-CSF和IL4的存在下培养5天,以诱导树突状细胞分化。(A)对从新鲜单核细胞和分化的树突状细胞分离的总RNA进行反转录和常规PCR。通过琼脂糖凝胶电泳分离VentX和GAPDH的PCR产物,并且通过溴化乙锭染色可见。(B)从新鲜单核细胞和分化的树突状细胞收集总细胞溶解产物,并且使用VentX特异性抗体,通过蛋白印迹分析判定VentX蛋白质水平。微管蛋白用作上样参照。
图S2:通过VentX吗啉代(MO)寡核苷酸敲低DC中VentX表达。通过电穿孔,用靶向VentX的MO寡核苷酸(VentX MO和VentX MO-2)或对照MO寡核苷酸转染原代人单核细胞(每个10M),如材料和方法中所述。24小时后加入细胞因子GM-CSF和IL4,并且在细胞因子添加5天后收集细胞用于VentX蛋白质水平的蛋白印迹分析。微管蛋白用作上样参照。通过密度计量学判定VentX敲低效率的定量。
图S3:VentX敲低树突状细胞标志物的表面表达上的效果。用靶向VentX的吗啉代(MO)寡核苷酸或对照MO转染人原代单核细胞,如材料和方法中所述。转染后24小时,向培养基中加入GM-CSF和IL4,以诱导树突状细胞分化。在添加细胞因子后5天收集细胞,并且通过流式细胞术分析CD11c、CD16、CD36、CD64的表面表达。实线:对照MO转染的细胞;虚线:VentXMO转染的细胞;着色直方图:同位素对照。
图S4:VentX过度表达在THP1细胞的分化标志物上的效果。用DOX处理THP1细胞,并且在材料和方法中描述的条件下培养,以诱导树突状细胞分化。处理后2天,收集细胞,以通过流式细胞术分析指示的抗原的表面表达。
图S5:VentX的敲低在单核细胞中下调GM-CSF受体(CD116)的表达。(A)通过电穿孔,用靶向VentX的MO寡核苷酸或对照MO寡核苷酸转染原代人单核细胞,如材料和方法中所述。24小时后加入细胞因子GM-CSF和IL4,并且在细胞因子添加5天后收集细胞。通过流式细胞术分析CD116的表面表达。实线:对照MO转染的细胞;虚线:VentX MO转染的细胞。(B)示出中的4个单独实验的平均值+SD。
定义
出于理解本文中公开的主题的目的,提供下列定义,作为本领域普通技术人员通常理解的概念的总结。该定义无意作为本文的发明和权利要求的限制。
如本文中所使用,术语“抗体”指代能够结合表位或抗原决定簇的分子。该术语意欲包括整个抗体及其抗原结合片段,包括单链抗体。抗体可以来自任意的动物来源。优选的是,抗体是哺乳动物类的,如人、鼠、兔、羊、豚鼠、骆驼和马等,或者其他合适的动物。抗体可以识别多肽或多核苷酸抗原。该术语包括活性片段,例如包括免疫球蛋白的抗原结合片段、重链的可变和/或恒定区、轻链的可变和/或恒定区、互补性决定区(cdr)和构架区。该术语包括多克隆和单克隆抗体制剂,以及包括下列的制剂:混合抗体、改造抗体、嵌合抗体、混合抗体分子、F(ab)2和F(ab)片段;Fv分子(例如非共价异质二聚体)、二聚和三聚的抗体片段构建;微抗体、人源化抗体分子、以及从这些分子获得的任何功能性片段,其中这些片段保留特异性结合。
在组合物或剂型中明显存在多于一个抗体时,单数术语“一(a)”或“一(an)”或“所述(the)”抗体的使用无意限制其为单一抗体。另外,除非另有说明,“抗体”的单数术语可以包括在其结构或特异性上不一定为异源的抗体的集合。
如本文中所使用,术语“人源化”抗体指代具有抗原接合位点的分子,所述抗原接合位点大致衍生自来自非人类物种的免疫球蛋白,并且分子的剩余免疫球蛋白结构基于人免疫球蛋白的结构和/或序列。抗原接合位点可以包含融合于恒定域的全长可变域,或者只包含接枝至可变域中合适的框架区的互补性决定区(CDR)。抗原接合位点可以是野生型或被一个以上氨基酸取代修饰,如修饰为更加接近地类似人免疫球蛋白。人源化抗体的一些形式保留了全部CDR序列(如含有源自鼠抗体的全部6个CDR的人源化鼠抗体)。人源化抗体的其他形式具有相对原始抗体改造的一个以上CDR(一个、两个、三个、四个、五个、六个)。
在抗体结合的语境下术语“特异性结合”指代抗体向特异性表位的高亲和力(avidity)和/或高亲和性(affinity)结合。因此,特异性结合于一个表位(“第一表位”)并且不结合于另一个表位(“第二表位”)的抗体是“特异性抗体”。如果两个表位共有同源性或其他相似性,特异于第一表位的抗体可以与第二表位交叉反应并结合至第二表位。在多核苷酸的语境下术语“特异性结合”指代严格条件下的杂交。增加DNA/DNA杂交反应和DNA/RNA杂交反应两者的严格性的条件广泛已知,并且在本领域中发表(Curr.Prot.Molec.Biol.,John Wiley&Sons(2001))。
如本文中所使用,术语“抗原”指代能够被抗体结合的分子。抗原是此外还能够被免疫系统识别和/或能够诱导体液免疫应答和/或导致B-和/或T-淋巴细胞的激活的细胞免疫应答。然而,至少在某些情况下,这需要抗原包含或连接至Th细胞表位并且辅助给药。抗原可以具有一个以上表位(B-和/或T-细胞表位)。以上指代的特异性反应意欲表明抗原通常以高度选择的方式,优选地与其对应抗体或TCR反应,并且不与可以被其他抗原诱发的多数其他抗体或TCR反应。如本文中所使用的抗原也可以是几个单一抗原的混合物。
如本文中所使用,术语“表位”指代基本元素或由单一抗体或T-细胞受体识别的最小单元,并且因此是所述抗体或T-细胞受体结合的特定域、区或分子结构。抗原可以由大量表位组成,而半抗原通常可以具有较少表位。
如本文中所使用,术语“核酸分子”、“核苷酸”、“寡核苷酸”、“多核苷酸”和“核酸”在本文中可交换地使用,以指代任意长度的核苷酸的聚合形式。其能够包括双链和单链序列,并且包括但不限于,来自病毒、原核和真核来源的cDNA;mRNA;来自病毒(如DNA病毒和逆转录病毒)或原核来源的基因组DNA序列;RNAi;cRNA;反义分子;核糖酶;以及合成DNA序列。该术语也包括(capture)包括DNA和RNA的任意已知碱基类似物的序列。
如本文中所使用,“互补的”核苷酸序列酸分子是包含其碱基对互补体的核苷酸序列酸分子。具有碱基腺嘌呤的脱氧核醣核苷酸与具有碱基胸腺嘧啶的脱氧核糖核苷酸是互补的,并且具有碱基胸腺嘧啶的脱氧核糖核苷酸与具有碱基腺嘌呤的脱氧核醣核苷酸是互补的。具有碱基胞嘧啶的脱氧核醣核苷酸与具有碱基鸟嘌呤的脱氧核糖核苷酸是互补的,并且具有碱基鸟嘌呤的脱氧核糖核苷酸与具有碱基胞嘧啶的脱氧核醣核苷酸是互补的。具有碱基腺嘌呤的核醣核苷酸与具有碱基尿嘧啶的核糖核苷酸是互补的,并且具有碱基尿嘧啶的脱氧核糖核苷酸与具有碱基腺嘌呤的脱氧核醣核苷酸是互补的。具有碱基胞嘧啶的核醣核苷酸与具有碱基鸟嘌呤的核糖核苷酸是互补的,并且具有碱基鸟嘌呤的脱氧核糖核苷酸与具有碱基胞嘧啶的脱氧核醣核苷酸是互补的。
如本文中所使用,术语“启动子”指代能够结合哺乳动物细胞内的RNA聚合酶并且起始可操作地连接于其上的下游(3'方向)编码序列的转录的DNA调控区。为了本发明的目的,启动子序列包括在高于背景的可检测水平上起始感兴趣的基因的转录所需的最小数量的碱基或元素。在启动子序列中,可以存在转录起始位点,以及负责RNA聚合酶结合的蛋白结合域(共有序列)。真核启动子通常但不总是包含“TATA”框和“CAT”框。启动子包括天然邻接核酸分子的启动子和不天然邻接核酸分子的启动子。此外,术语“启动子”包括诱导型启动子、诸如cre-lox启动子的条件激活启动子、组成型启动子和组织特异性启动子。
如本文中所使用,术语“转染的”意指保持导入的DNA或RNA,使用或不使用任何诸如脂质体的补充促进剂。本领域中已知的转染方法包括磷酸钙转染、DEAE葡聚糖转染、原生质体融合、电穿孔和脂质转染。
如本文中所使用,术语“核酸分子的表达”指代将核酸分子中包含的信息转化至基因产物中。基因产物可以是基因的直接转录产物(如mRNA、tRNA、rRNA、反义RNA、核糖酶或任何其他类型的RNA),或者通过mRNA翻译产生的肽或多肽。基因产物也包括通过诸如封闭、聚腺苷酸化、甲基化和编辑的处理修饰的RNA;以及通过例如甲基化、乙酰化、磷酸化、泛素化、ADP核糖基化、肉豆蔻酰化和糖基化修饰的蛋白质。
如本文中所使用,术语“宿主细胞”指代单个细胞或能够作为或已经作为任何重组体载体或分离的多核苷酸的受体的细胞培养物。宿主细胞包括单一宿主细胞的后代,并且由于自然、偶然或故意突变和/或改变,该后代不一定是与原始亲代细胞完全相同的(形态上或总DNA互补链)。宿主细胞包括在体内或体外用本发明的重组体载体或多核苷酸转染或感染的细胞。包含本发明的重组体载体的宿主细胞可以称为“重组体宿主细胞”。
如本文中所使用,术语“分离的”或“大致分离的”分子(诸如多肽或多核苷酸)是被处理以在高于自然的浓度中存在或从其原生环境中移除的分子。例如,在至少10%、或20%、或40%、或50%、或70%、或90%的与经处理抗体天然相关的未经处理抗体材料被移除时,经处理抗体是分离的、纯化的、大致分离的或大致纯化的。例如,在活体动物中天然存在的多核苷酸或多肽不是“分离的”,但是从其天然状态的共存材料中分离的相同的多核苷酸或多肽是“分离的”。而且,出于本发明的目的,载体中包含的重组DNA分子被认为是分离的。分离的RNA分子包括DNA和RNA分子的体内或体外RNA复制产物。分离的核酸分子还包括合成产物分子。此外,重组体宿主细胞中包含的载体分子也是分离的。因此,并非所有“分离的”分子需要是“纯化的”。
如本文中所使用,在用于指代分子时,术语“纯化的”意指被纯化的分子的浓度相对于在其自然环境中、或在其产生、发现或合成其的环境中的与其相关联的分子增加。自然相关联的分子包括蛋白质、核酸、脂质和糖,但是一般不包括水、缓冲液和添加以保持被纯化的分子的完整性或促进其纯化的试剂。根据此定义,相对于物质的污染物考虑时,物质可以是5%以上、10%以上、20%以上、30%以上、40%以上、50%以上、60%以上、70%以上、80%以上、90%以上、95%以上、98%以上、99%以上或100%纯度。
如本文中所使用,术语“生物活性的”实体或具有“生物活性”的实体是具有自然发生的分子的结构、调控或生物功能或与新陈代谢或生理过程有关或相关联的任何功能的实体。生物活性多核苷酸片段是呈现与本发明的多核苷酸的活性相似但不必相同的活性的多核苷酸片段。生物活性可以包括提高的期望活性,或者降低的不期望活性。例如,在实体参加与其他分子的分子相互作用诸如杂交时、当其在减轻疾病状况中具有治疗价值时、当其在诱导免疫应答中具有预防价值时、当其在判定诸如多核苷酸的生物活性片段的分子存在中具有诊断和/或预后价值时,实体展现生物活性,其中所述多核苷酸的生物活性片段例如能够被作为多核苷酸分子的唯一物而检测,或者能够在聚合酶链式反应中用作引物。生物活性多肽或其片段包括能够参与生物反应的多肽或其片段。
如本文中所使用,术语“受试者”、“个体”和“患者”在本文中互换使用,用于指代活体动物,包括人类和非人类动物。受试者可以是例如拥有能够响应抗原刺激以及通过细胞表面受体结合传导刺激和抑制信号的免疫细胞的生物体。受试者可以是哺乳动物,诸如人类或非人类哺乳动物,例如狗、猫、猪、牛、绵羊、山羊、马、大鼠和小鼠。术语“受试者”不排除对于疾病完全正常、或者在所有方面正常的个体。
如本文中所使用,“患者样品”是源自患者的任意生物样本。该术语包括但不限于生物液体,诸如血液、血清、血浆、尿液、脑脊液、泪液、唾液、淋巴液、透析液、灌洗液、精液和其他液体样品,以及生物来源的细胞和组织。该术语也包括细胞或源自细胞的细胞和细胞的后代,包括培养基中的细胞、细胞上清液和细胞裂解物。其还包括源自器官或组织培养的液体、组织活检样品、肿瘤活检样品、粪便样品和从生理组织提取的液体,以及从固体组织、组织切片和细胞裂解物分离的细胞。此定义涵盖在样品获得后以任何方式处理的样品,诸如通过试剂处理、溶解、或富集诸如多核苷酸或多肽的某些组分。该术语还包括患者样品的衍生物和小部分(fraction)。患者样品可以用于诊断、预后或其他监控分析。
如本文中所使用,术语“调节”指代在与合适的对照相比时,测量的活性的增加或减少、刺激、抑制、干扰或阻断的直接或间接的产生。多肽或多核苷酸的“调节剂”或“试剂”在本文中是互换使用的术语,用于指代在与合适的对照相比时,影响多肽或多核苷酸的测量活性的物质,所述影响例如增加、减少、刺激、抑制、干扰或阻断。
如本文中所使用,术语“疾病”或“失调”指代病理状况,例如能够通过症状或偏离健康或正常状态的其他识别因素识别的病理状况。术语“疾病”包括失调、综合征、病症和损伤。疾病包括但不限于增殖性、炎症性、免疫性、代谢性、传染性和缺血性疾病。
如本文中所使用,术语“炎症性病症”指代一组病症,包括类风湿性关节炎、骨关节炎、幼年特发性关节炎、牛皮癣、过敏性呼吸道疾病(如哮喘、鼻炎)、炎症性肠病(如克罗恩氏病、结肠炎)、内毒素驱动的疾病状态(如搭桥手术后并发症或促使如慢性心力衰竭的慢性内毒素状态)和涉及诸如关节软骨的软骨的相关疾病。该术语特别指代类风湿性关节炎、骨关节炎、过敏性呼吸道疾病(如哮喘)和炎症性肠病。
如本文中所使用,术语“自体免疫疾病”指代一组疾病,包括阻塞性呼吸道疾病,包括诸如COPD(慢性阻塞性肺病)的病症、哮喘(如内源性哮喘、外源性哮喘、尘埃性哮喘、小儿哮喘),特别是慢性或宿疾哮喘(例如晚发哮喘和呼吸道高反应性)、支气管炎,包括支气管哮喘、系统性红斑狼疮(SLE)、多发性硬化症、I型糖尿病和与其相关联的并发症、异位性湿疹(异位性皮炎)、接触性皮炎以及进一步的皮炎湿疹、炎症性肠病(如克罗恩氏病和溃疡性结肠炎)、动脉粥样硬化和肌萎缩性侧索硬化症。该术语特别指代COPD、哮喘、系统性红斑狼疮、I型糖尿病和炎症性肠病。
如本文中所使用,术语“增殖性疾病”指代诸如癌症(如子宫平滑肌肉瘤或前列腺癌)、骨髓增生异常(如真性红细胞增多症、原发性血小板增多症和骨髓纤维化)、白血病(如急性髓性白血病或急性淋巴细胞白血病)、多发性骨髓瘤、牛皮癣、再狭窄、硬化性皮炎或纤维变性的病症。该术语特别指代癌症、白血病、多发性骨髓瘤和牛皮癣。
如本文中所使用,术语“癌症”和“患癌症的”指代或描述典型特征为无序细胞生长的哺乳动物的生理状况。癌症的实例包括但不限于癌、淋巴瘤、肉瘤、胚细胞瘤和白血病。这样的癌症更具体的实例包括鳞状细胞癌、肺癌、胰腺癌、宫颈癌、膀胱癌、肝癌、乳腺癌、结肠癌以及头颈癌。
如本文中所使用,术语“肿瘤”指代任何恶性或赘生性细胞。
如本文中所使用,术语“治疗”包括预防性和/或治疗性的治疗两者,包括对包括人类的哺乳动物给药或施用对于疾病的救治(remedy),并且包括对疾病的抑制。其包括控制疾病发展和缓解疾病,诸如通过致使丧失的、缺失的或缺陷的功能回归或恢复或修复丧失的、缺失的或缺陷的功能,或者刺激效率低的过程。如本文中所使用,术语“预防”包括对于疾病在受试者中的发生或复发提供预防疗法,所述受试者可能倾向于患病但是尚未诊断出患有疾病。治疗和预防疗法能够体内、体外或离体向包括人类的生物体或细胞给药,并且随后将细胞给药至受试者。
如本文中所使用,术语“有效量”指代实现期望的生物效果所需或足以实现期望的生物效果的量。组合物的有效量将是达到选择的结果的量,并且该量可以由本领域技术人员按常规判定。例如,用于治疗免疫系统缺陷的有效量可以是使免疫系统激活、使得在暴露于抗原时导致抗原特异性免疫应答发展所需的量。该术语也与“足够的量”同义。任意特定应用的有效量可以取决于下列因素而变化,诸如治疗的疾病或病症、给药的特定组合物、受试者的体型大小、和/或疾病或病症的严重性。本领域普通技术人员能够以经验为主地判定本发明的特定组合物的有效量,而不需要过度的实验。
如本文中所使用,术语“载体(carrier)”指代任意常规类型的固态、半固态或液态填充剂、稀释剂、囊封材料、制剂辅助物或赋形剂。“药学上可接受的载体”指代无毒性的“载体”。药学上可接受的载体在采用的剂量和浓度下是对接受者无毒性的,并且与制剂的其他成分是相容的。例如,药学上可接受的载体可以是溶媒、佐剂或稀释剂。
如本文中所使用,术语“多肽”和“蛋白质”在本文中互换使用,用于指代氨基酸残基的聚合物,并且不受最小长度局限。因此,多肽、寡肽、二聚体和多聚体等包含在此定义中。全长蛋白和其片段两者都被此定义涵盖。该术语也包括多肽的表达后修饰,例如糖基化、乙酰化、磷酸化等。另外,“多肽”可以指代包括对原始序列的修饰的蛋白质,只要该蛋白质保持期望的活性,所述修饰诸如删除、添加和取代。这些修饰可以是故意的或无意的。
如本文中所使用,术语“受体”指代能够与其他分子相互作用的蛋白质或糖蛋白或其片段,所述其他分子称为配体。配体可以属于任意类型的生化或化学化合物。配体通常是细胞外分子,其在结合于受体后通常起始细胞应答,诸如起始信号转导通路。受体不必须是膜结合蛋白。
如本文中所使用,术语“重组体”对应于核酸分子,意指基因组、cDNA、病毒、半合成和/或合成来源的多核苷酸,该多核苷酸由于其来源或处理,不与其自然相关联的多核苷酸的全部或部分相关联。所使用的术语“重组体”对应于蛋白质或多肽,意指通过重组体多核苷酸的表达产生的多肽。相对于宿主细胞使用的术语“重组体”意指已经向其中引入重组体多核苷酸的宿主细胞。
如本文中所使用,短语“重组体病毒”意指通过人力基因改造的病毒。该短语包含本领域已知的任意病毒。
如本文中所使用,术语“载体(vector)”指代用于向宿主细胞或生物体传递遗传物质的介质(如质粒或病毒)。载体可以由DNA或RNA组成。
如本文中所使用,术语“干扰RNA”或“RNAi”或“干扰RNA序列”指代当干扰RNA与靶基因处于相同细胞内时,能够减少或抑制靶基因表达(即通过介导mRNA的降解,所述mRNA与干扰RNA的序列互补)的双链RNA(即二倍体RNA)。因此,干扰RNA指代由两条互补的链或由单一的自补链形成的双链RNA。干扰RNA可以与靶基因大致或完全相同,或者可以包含不匹配域(即错配的基序)。干扰RNA序列能够对应于全长靶基因或其子序列。干扰RNA包括“小干扰RNA”或“siRNA”,如长度约15-60、15-50或15-40(二倍体)个核苷酸的干扰RNA,更典型的是长度约15-30、15-25或19-25(二倍体)个核苷酸。
如本文中所使用,术语“样品”指代来自人类、动物的样品,或者研究样品,如细胞、组织、器官、液体、气体、气溶胶、浆液、胶体或凝固材料。“样品”可以在体内检验,如不从人类或动物移除,或者可以在体外检验。样品可以在处理后检验,如通过组织学方法。“样品”也指代例如包含液体或组织样品的细胞,或者从液体或组织样品分离的细胞。“样品”也可以指代从人类或动物新鲜取得的细胞、组织、器官或液体,或者处理或储存的细胞、组织、器官或液体。
具体实施方式
本发明部分基于VentX在调控DC的成熟和功能中起重要作用的发现。VentX通过IL6介导机制调控DC分化和成熟。在从IBD患者的发炎粘膜分离的DC中VentX表达升高,并且VentX的敲低减少了DC的成熟应答。本文中公开的数据展示了VentX能够作为炎症性疾病和免疫疗法的干预目标。
本文中的公开表明,单核细胞中VentX表达的去除显著损害了它们向DC的分化。相反地,VentX在单核细胞THP1中的过度表达加速了它们向DC的分化。增加的IL6表达部分地导致在VentX敲低的单核细胞中DC分化缺陷。临床重要的是在来自炎症性肠病患者的发炎粘膜的肠粘膜固有层DC(LPDC)中VentX表达增加的发现。敲低实验示出VentX对于LPDC的成熟至关重要。除此之外,类固醇治疗明显降低VentX在LPDC中的表达,并且VentX的强制表达抵消了类固醇的效果。总而言之,本文中公开的数据展示了VentX是DC分化和成熟的关键转录调控子,并且是免疫调控和疗法的潜在干预目标。
DC分化和成熟的机制由于其在免疫调控和疗法中的潜在应用,成为重大兴趣的焦点。因为已经了解了影响单核细胞分化成DC的细胞因子的丰富信息,DC分化潜在的关键转录调控事件仍未完全明确。(Gabriele等,2004 Blood 103(3):p.980-7;Hart等,2005Gastroenterology 129(1):p.50-65;Dillon等,2010 J Immunol 184(12):p.6612-21;Bell等,2001 J Immunol 166(8):p.4958-67.)在本研究中示出人同源盒蛋白VentX是DC分化和成熟的关键调控子。提供了示出在从IBD患者的发炎粘膜分离的DC中VentX表达升高的证据。发现了VentX是通常用于治疗自体免疫炎症性病症的皮质类固醇的下游靶标。
近来的研究表明,VentX是关键造血转录因子,其表达在所有谱系的造血细胞的个体发育期间受到限制并被高度调控。发现了VentX在早期造血及终末分化和成熟两者期间支配造血细胞的增殖和分化。机制上来说,VentX被发现拮抗经典Wnt信号转导以及激活p53/p21和pRb/p16衰老途径,从而在细胞增殖上发挥其效果。同时,VentX在造血细胞的谱系分化期间,激活各种细胞分化信号转导。VentX敲低在早期和终末期两者期间阻断造血细胞的分化,表明VentX对于造血细胞的分化是通用容许因子。VentX是否在谱系发育中起作用仍然需要进一步明确。结果显示VentX在U937细胞中的异位表达促进巨噬细胞发育,而VentX在THP1细胞中的异位表达促进DC发育,表明VentX在造血细胞分化上的效果是细胞类型特异性的。
对VentX调控DC分化的潜在机制的探索导向IL6,其是DC分化中涉及的关键信号。(Chomarat等,2000 Nat Immunol 1(6):p.510-4;Mitani等,2000 Br J Haematol 109(2):p.288-95;Park等,2004 J Immunol 173(6):p.3844-54.)先前的研究显示出通过(-IFN、Wnt5a、HLA-G和转录因子ESE-3对DC分化的抑制导致增加的IL6分泌或信号转导[9,35-38]。(Delneste等,2003Blood 101(1):p.143-50;Liang等,2008 Proc Natl Acad Sci U S A105(24):p.8357-62;Appel等,2006 Blood 107(8):p.3265-70;Valencia等,2011 JImmunol 187(8):p.4129-39;Bharadwaj等,2007 Cancer Res 67(11):p.5479-88.)结果显示IL6表达在VentX敲低的DC中增加(图3A和C),而VentX的过度表达抑制IL6表达(图3B和C)。重要的是,发现在VentX敲低时受损的DC分化能够部分地通过用IL6抗体减少IL6信号转导来挽救。机制上的探索表明,VentX通过其在THP1细胞中NF(B结合至IL6启动子域的效果,部分调控IL6表达。与VentX在分化上的细胞类型特异性效果一致,先前的研究显示VentX促进IL在巨噬细胞U937细胞中的表达。对VentX调控的IL6表达的细胞类型特异性效果的潜在机制有着重大兴趣,并且是本调查研究的目标。
近来在DC生物学上的发展表明,循环单核细胞的异常成熟和激活涉及肠粘膜炎症的发病机制。VentX表达在从IBD患者的发炎粘膜中分离的DC细胞中异常升高的发现表明确定VentX作为DC分化和成熟的新颖关键调控子的临床相关性。在调节DC活性和自体免疫/炎症性病症中靶向VentX的潜在应用已经由VentX是皮质类固醇的下游靶标以及VentX介导类固醇在DC成熟和激活上的抑制效果指明。需要进一步的研究,以明确VentX调控的DC分化和激活如何通过宿主和微生物因子调节。这样的信息对于理解IBD中肠粘膜的区域性损伤将非常关键。皮质类固醇和其他现有可用的免疫抑制剂的难治性(refractory)是管理自体免疫和炎症性疾病的中心问题。
在一个方面,本发明大体上涉及一种调控树突状细胞的方法。该方法包括向需要给药的对象给药治疗上有效量的组合物,该组合物包含发挥人同源盒基因VentX的调节作用的生物或化学药剂。
在某些实施方式中,调控树突状细胞包括影响树突状细胞的成熟应答。
在某些实施方式中,生物或化学药剂发挥人同源盒基因VentX的抑制效果。
在某些实施方式中,生物或化学药剂是多肽。
在某些实施方式中,生物或化学药剂包含VentX突变体,该VentX突变体缺失作为封闭多肽(blocking polypeptide)的同源区。
在某些实施方式中,生物或化学药剂是抗体片段。
在某些实施方式中,生物或化学药剂是寡核苷酸。在某些实施方式中,寡核苷酸是RNAi。
在另一个方面,本发明大体上涉及一种筛选用于树突状细胞的调控功能的化合物的方法。该方法包括:(a)提供包含多核苷酸的细胞,该多核苷酸包含VentX启动子;(b)使细胞与候选化合物接触;以及(c)测量VentX表达的活性。
在某些实施方式中,候选化合物是小分子药剂。
在某些实施方式中,候选化合物是多肽。
在某些实施方式中,候选化合物是寡核苷酸。
在又一个方面,本发明大体上涉及一种药物组合物,该药物组合物包含通过人同源盒基因VentX的调节作用在树突状细胞上发挥调控作用的生物或化学药剂。
在药物组合物的某些实施方式中,调节作用包括与炎症性病症的治疗相关的抑制效果。
在药物组合物的某些实施方式中,炎症性病症是自体免疫疾病。
在药物组合物的某些实施方式中,生物或化学药剂是多肽。
在药物组合物的某些实施方式中,生物或化学药剂是抗体。
在药物组合物的某些实施方式中,生物或化学药剂是寡核苷酸。
在又一个方面,本发明大体上涉及一种诊断对象的与树突状细胞的成熟相关联的炎症性病症的方法,包括人同源盒基因VentX的表达谱的检测。在某些实施方式中,炎症性病症是自体免疫疾病。
在又一个方面,本发明大体上涉及一种抑制肿瘤的方法。该方法包括向需要给药的对象给药治疗上有效量的组合物,该组合物包含在树突状细胞功能上发挥调节作用的生物或化学药剂。
在某些实施方式中,在树突状细胞功能上的调节作用包括通过人同源盒基因VentX的调节作用(如,通过诱导或直接转染至DC细胞来增加VentX,从而增强DC功能。在某些实施方式中,生物或化学药剂是多肽。在某些实施方式中,生物或化学药剂是抗体片段。在某些实施方式中,生物或化学药剂是寡核苷酸。在某些实施方式中,寡核苷酸是RNAi。
VentX调控原代单核细胞向树突状细胞分化
随着近期发现VentX在人原代单核细胞中表达,在通过GM-CSF和IL-4处理诱导的原代单核细胞向树突状细胞分化期间检查了VentX表达。(Grassl等,1999 J Am SocNephrol 10(7):p.1466-77.)发现了在诱导的DC分化期间,VentX表达在mRNA和蛋白质水平两者都经历了显著的增加(图S1)。为了探索VentX在DC分化中的潜在作用,检查了VentX敲低在体外DC分化上的效果。在转染5天后,VentX siRNA在单核细胞中产生了最低限度的VentX敲低效能(未公布数据),而DC从单核细胞中的体外分化需要与GM-CSF和IL4温育5~7天。(Iwamoto等,2007 J Immunol 179(3):p.1449-57;Gabriele等,2004 Blood 103(3):p.980-7.)为了在单核细胞中获得持久的VentX敲低效果,开发了吗啉代(MO)介导的VentX敲低策略。设计了两个不同的吗啉代反义寡核苷酸(VentX MO和VentX MO-2),并且测试了对于VentX的敲低效能。如图S2所示,在转染后5天时,两个吗啉代序列与对照序列相比都抑制VentX在DC中的表达。新鲜分离的CD14+CD1a-单核细胞在用GM-CSF和IL-4培养时,在体外分化为未成熟的CD14-CD1a+DC。(Bell等,2001 J Immunol 166(8):p.4958-67.)在分别用对照MO或VentX MO转染的单核细胞中,检查了CD1a抗原的表面表达。如图1所示,与对照MO相比,VentX MO MO的转染导致了CD14-CD1a+细胞百分比的实质性减少(图1A-B)。被认为是单核细胞衍生的DC的附加分化标志物的CD1b、CD1c和CD11c的表面表达在VentX MO转染的细胞中被类似地下调(图1C-D,图S3)。(Osugi等,2002 Blood 100(8):p.2858-66.)VentX MO的转染不在DC中造成过度的细胞死亡(数据未示出),其排除了分化下降是由于细胞活性受到危害的可能性。诸如CD16、CD36和CD64的其他表面抗原的表达,甘露糖受体(MR)和Toll样受体4(TLR4)在VentX MO转染的DC中删除剩余的未变化物(图S3),进一步支持了VentX MO转染在DC分化上发挥了特异性效果。为了确证VentX在调控DC分化中的作用,在原代单核细胞中异位表达VentX,并且在细胞因子处理3天后确定CD14-CD1a+细胞。VentX在单核细胞中的过度表达极大地加速了DC分化(图1E-F)。总的来说,数据展示出VentX是原代单核细胞向DC分化的关键调控子。
VentX促进树突状细胞分化
人单核细胞系THP1在适当的分化条件下能够被诱导而分化为具有表型和功能性质类似于原代DC的未成熟的和成熟的DC。(Ogasawara等,2009 Biochem Biophys ResCommun 389(3):p.543-9;Menetrier-Caux等,1998 Blood 92(12):p.4778-91.)为了进一步探索VentX在树突状细胞分化上的效果及其潜在机制,在强力霉素可诱导启动子控制下,生成了表达GFP或GFP.VentX的稳定THP1细胞系。已经报道了VentX起抗增殖和促分化转录因子的作用。(Wu等,2011 J Clin Invest 121(7):p.2599-613;Kamada等,2008 J ClinInvest 118(6):p.2269-80;Berges等,2005 Biochem Biophys Res Commun 333(3):p.896-907;Grassl等,1999 J Am Soc Nephrol 10(7):p.1466-77.)因此,首先检查了VentX的过度表达是否能够在THP1细胞的增殖上发挥抑制作用。与先前的研究一致,VentX在THP1细胞中的表达引起了明显的生长抑制(图2A)和G1细胞周期停滞(图2B),其与通过VentX的c-myc表达的下调和p21表达的上调相关联(图2C-D)。为了判定VentX表达在THP1细胞的DC分化上的效果,开发了次优的诱导条件,在该条件下可以观察到GFP转导的THP1细胞的温和分化(图2E,上栏)。在此条件下,VentX的过度表达显著加速了THP1细胞的DC分化。如图2E所示,VentX转导的THP1细胞显示了CD1a、CD11c、CD40、CD86和HLA-DR的表面表达的明显增加,表明在这些细胞中的增强的DC分化。在VentX转导的THP1细胞中,CD36、CD64和CD80的表达维持基本不变(图S4)。突出的是,VentX转导的THP1细胞变得与大量的树突形成粘附并且扁平化(图2F,右栏),类似于从原代单核细胞衍生的DC的形态。相对而言,在GFP转导的细胞中没有观察到这样的形态变化(图2F,左栏)。总之,这些结果表明VentX可以在DC分化中起普遍的作用。
IL-6介导DC分化的VentX调控
为了阐明VentX调控DC分化和成熟的潜在机制,通过反转录PCR和细胞间染色分析了已知对于DC发育重要的细胞因子的表达水平。而IL1(、TNF(和IL10表达未检测出明显变化(数据未示出),IL6表达在用VentX MO转染的细胞中一致地增加。已经示出IL6抑制从CD14+单核细胞和CD34+祖细胞两者中的DC分化和成熟。(Gabriele等,2004 Blood 103(3):p.980-7;Dendorfer等,1994 Mol Cell Biol 14(7):p.4443-54;Isshiki等,1990 MolCell Biol 10(6):p.2757-64;Libermann等,1990 Mol Cell Biol 10(5):p.2327-34.)为了确证敲低实验的结果,检查了VentX的异位表达在IL6表达水平上的效果。与功能缺失型途径一致,VentX的过度表达在原代单核细胞中减少了IL6mRNA水平(图3C)并且抑制了IL6的生成(图3B)。为了判定VentX是否在转录水平抑制IL6表达,进行了具有IL6启动子的荧光素酶报告基因分析。评价了VentX对592bp IL6启动子荧光素酶报告基因和几个缺失突变体结构体活性的影响。(de Jong等,1999 J Leukoc Biol 66(2):p.201-4.)如图3D所示,VentX在原代单核细胞中显著抑制了-592bp IL6启动子活性。先前的研究已经描述了几个人IL-6启动子中的功能性顺式调控元件,包括AP1(-283至-277bp)、C/EBP(-158至-145bp以及-87至-76bp)和NF(B(-75至-63bp)33-36的结合位点。有趣的是,仅包含NF(B结合位点33的-80bp IL6启动子的活性被VentX抑制在与-592bp IL6启动子的活性类似的程度(图3E),说明VentX可以靶向NF(B结合位点从而调控IL6表达。为了检验这一假设,进行了IL6启动子的突变分析,并且发现-80bp IL6启动子上的NF(B结合位点的突变废除了VentX抑制启动子报告基因活性的能力(图3F)。为了判定NF(B是否涉及VentX调控的IL6表达,检查了VentX对NF(B表达上的效果及其与IL6启动子的相互作用的影响。发现了VentX不影响NF(B的表达(数据未示出)。然而,使用染色质免疫沉淀分析发现,VentX的异位表达导致NF(B向IL6启动子的结合减少(图3G),表明VentX通过抑制NF(B与IL6启动子的相互作用,在转录水平上抑制IL6表达。进一步调查在VentX MO转染的单核细胞中受损的DC分化是否能够归因于这些细胞中的IL6分泌增加。为了解决这个问题,将抗IL6的中和抗体添加至VentX MO转染的单核细胞的培养基中,并且通过CD1a表面表达的流式细胞术分析评价了DC的分化。如图4所示,用特异性抗体中和IL6活性显著地改善了VentX MO转染的单核细胞中的DC分化缺陷,指明VentX至少部分地通过调节IL6自分泌生产而调控DC分化。
VentX调控树突状细胞成熟
在用诸如LPS的细菌组分刺激时,DC经历成熟过程。为了探索VentX是否在DC成熟中起作用,使用PCR分析方法在LPS诱导的成熟过程中检查了VentX对DC基因表达谱的变化上的影响。PCR分析包括对于DC成熟重要的抗原摄取和呈递中涉及的基因、细胞因子/趋化因子和它们的受体。炎症中涉及的细胞表面受体和信号转导因子也包括在此分析中。如图5A-E所示,VentX的过度表达诱导很多种基因的上调,诸如CD1抗原、协同刺激因子CD80和CD86、HLA分子、促炎细胞因子IL12和TNF(、趋化因子CCL2(MCP-1)、CCL3(MIP-1()和CCL5(RANTES)、趋化因子受体CCR5和CXCR4以及Toll样受体(TLR)。除此之外,CSF1R(M-SCF受体)和CDKN1A(p21)的表达也如先前所展示的增加。(Wu等,2011 J Biol Chem 286(14):p.12693-701;Wu等,2011 J Clin Invest 121(7):p.2599-613.)通过独立PRC反应,使用不同的引物集进一步证实了PCR分析的结果。如图5F所示,独立的定量PCR实验产生了与PCR分析一致的结果;因此表明了VentX在DC成熟过程期间的潜在作用。
VentX在肠粘膜炎症期间调控DC成熟
在炎症性肠病的发病机制中,在粘膜炎症中已经涉及了DC的异常激活。为了解决这个问题,从患有溃疡性结肠炎和克罗恩氏病的患者发炎粘膜以及正常粘膜中分离了粘膜固有层DC(LPDC)。通过RT-PCR方法判定了这些DC中的VentX表达水平。如图6A所示,与来自非炎症区域的LPDC相比,VentX表达水平在来自炎症区域的LPDC中显著升高(图6A)。为了判定VentX的升高表达是否与DC的异常激活相关,检验了VentX敲低对LPDC的成熟和激活上的影响。如图6B-C所示,在检查的细胞表面标志物中间,在LPDC中的VentX敲低显著地下调了协同刺激因子CD40、CD80和CD86、趋化因子受体CCR7以及模式识别受体TLR2和TLR4的表达,先前已经示出其在来自IBD患者的发炎粘膜的DC中被上调。CD83的表达不被VentX敲低影响。细胞间染色展示出VentX敲低的LPDC产生较少的炎症性细胞因子IL12、TNF(和趋化因子CCL3、CCL5。一致地,与对照LPDC细胞相比,反转录PCR揭示了VentX敲低的LPDC细胞中的CCL2、CCL3、CCL5、CXCL12、IL12、TNF(和TLR的mRNA水平的显著下降(图6D)。除表型特征之外,功能性分析示出LPDC细胞中的VentX敲低显著地损害了DC刺激原代T细胞增殖的能力(图6E)。诸如皮质类固醇的免疫抑制剂在中度到重度IBD患者的粘膜炎症的管理中仍然作为主要治疗模式,并且已经报道了其调节DC成熟和功能。(de Jong等,1999 J Leukoc Biol66(2):p.201-4 Piemonti等,1999 J Immunol 162(11):p.6473-81.)为了判定VentX是否可能是IBD中类固醇治疗的下游靶标,检查了用强的松体外处理的LPDC中的VentX表达水平。如图6F所示,类固醇治疗显著地降低了LPDC中的VentX表达以及诸如IL2、TNF、CCL5的促炎性细胞因子的表达(图6G)。当VentX在DC中异位表达时,皮质类固醇在促炎性细胞因子的表达上的抑制效果显著地减少,表明类固醇通过在DC中下调VentX表达部分地发挥其抗炎性。
实验部分
材料和方法
人原代细胞的分离和处理
通过Ficoll密度梯度离心法分离来自达纳法博癌症研究所(Dana-Farber CancerInstitute)的健康成年供体的外周血单核细胞(PBMC)。使用抗CD14抗体包覆的磁性微珠(Miltenyi Biotec,Auburn,CA)从PBMC中纯化CD14+单核细胞。在12孔板中,用包含10%胎牛血清(FBS)、GM-CSF(100ng/ml)和IL4(20ng/ml)的RPMI 1640培养基(PeproTech,RockyHill,NJ),以1×106细胞/mL培养单核细胞。总共5天,每2或3天向培养基添加细胞因子,以诱导树突状细胞分化。从R&D Systems(Minneapolis,MN)购入抗Il6的中和抗体,并且以2.5(g/ml的每日剂量使用。从来自诊断患有炎症性肠病的患者的手术切除样本获得肠粘膜,所述炎症性肠病包括克罗恩氏病和溃疡性结肠炎。样本取自发炎的和非发炎的粘膜,并且宏观地及微观地确定。使用先前描述的技术分离粘膜固有层单核细胞。(Dillon等,2010 JImmunol 184(12):p.6612-21;Kamada等,2008 J Clin Invest 118(6):p.2269-80.)LPDC用磁性微珠(Miltenyi Biotec)纯化作为CD19-CD1c+细胞的组分以磁性微珠(MiltenyiBiotec)纯化。为了促进DC的成熟,向培养基中添加100ng/ml的大肠杆菌LPS(Sigma-Aldrich),并且进一步培养24小时。依照布莱根妇女医院(Brigham and Women’sHospital)的机构审查委员会批准的指导方针,用人类材料进行实验。
VentX敲低
使用人单核细胞核转染试剂盒(Human Monocyte Nucleofector Kit)(Lonza,Walkersville,MD),根据生产厂商说明,用吗啉代(MO)反义寡核苷酸转染人原代单核细胞。简要地说,将10×106个单核细胞在具有2.5nmol的VentX MO寡核苷酸(VentX MO:5'-TACTCAACCCTGACATAGAGGGTAA-3'或VentX MO-2:5'-GAGCCCGGTTTGCATACACGGCTAA-3')或标准对照MO寡核苷酸中的任一种的100(l核转染溶液中再悬浮,并且用核转染II装置(Nucleofector II Device)(Lonza)电穿孔。然后立刻从装置中移出细胞并且用1ml预先温热的含有2mM谷氨酰胺和10%FBS的人单核细胞核转染培养基温育过夜。然后在完全RPMI培养基中再悬浮细胞,并且用恰当的细胞因子处理以诱导向DC的分化。所有的MO寡核苷酸订购自Gene Tools(Philomath,OR)。如先前的研究中所描述,用靶向VentX的siRNA转染LPDC。(Wu等,2011 J Clin Invest 121(7):p.2599-613.)
条件表达VentX的THP1细胞系的产生
从美国模式培养物保藏所(American Type Culture Collection)(ATCC;Manassas,VA)获得人单核细胞白血病细胞系THP1。已经在先前的研究中描述了表达GFP.VentX或GFP的强力霉素可诱导的逆转录病毒。(Wu等,2011 J Clin Invest 121(7):p.2599-613.)条件表达GFP.VentX的THP1细胞系通过pRetroX-GFP.VentX和pRetroX-Tet-On advanced逆转录病毒共转导而产生,并且在用1.0(g/ml的强力霉素温育24小时后(达纳法博癌症研究所流式细胞术核心设施)通过FACSAria高速分选仪(BD Bioscience,SanJose,CA)分选GFP.VentX阳性细胞。然后在强力霉素缺失下,在RPMI 1640培养基中保持分选的细胞。类似地产生条件表达GFP的THP1细胞系作为对照。为了诱导THP1细胞向DC的分化,如先前所述及一些修改,用细胞因子鸡尾酒处理细胞。(Berges等,2005 BiochemBiophys Res Commun 333(3):p.896-907.)简要地说,细胞在供应有10%FBS、100ng/mlGM-CSF、50ng/ml IL4和TNF-(、100ng/ml Ionomycin的RPMI1640培养基中,在12孔板中生长2天。在这样的次优条件下,在表达GFP的THP1细胞中仅观察到温和的DC分化,其使我们确定在此模式细胞系中VentX表达对DC分化上的影响。
FACS分析
在用荧光染料偶联的抗体(eBioscience,San Diego,CA)免疫染色细胞后,用流式细胞术进行DC和THP1细胞的表型分析。使用了下列的FITC或PE偶联的抗体:抗-CD1a、CD1b、CD1c、CD11c、CD14、CD16、CD36、CD40、CD64、CD80、CD83、CD86、CD116、CCR7、HLA-DR、TLR2和TLR4。按照生产厂商提供的流程,用PE偶联的抗体进行CCL3、CCL5、IL6和IL12p70以及TNF(的细胞间染色。对于全部实验,并列进行同位素对照染色。通过碘化丙啶(PI)染色进行细胞周期分析。使用FlowJo软件,以FACScan流式细胞分析仪(BD Bioscience)分析染色的细胞。结果表达为阳性细胞百分比或者扣除从同位素对照抗体获得的MFI之后的平均荧光强度(MFI)值。
荧光素酶报告基因分析
用正向引物:5'-GTAACGCGTTTCTACAACAGCCGCTCACAG-3'和反向引物:5'-GATAGAGCTTCTCTTTCGTTC-3'放大人IL6启动子域的-592bp片段。用相同的反向引物和下列正向引物:5'-GTAACGCGTCAATGACGACCTAAGCTGCAC-3'和5'-GTAACGCGTGTGGGATTTTCCCATGAGTC-3',分别放大-225bp和-80bp启动子。用限制酶Mlu I和Xho I酶切放大的产物,并且随后将酶切片段克隆至pGL3荧光素酶报告基因。通过电穿孔,用pcDNA-VentX质粒或对照pcDNA-GFP质粒进行报告基因质粒向原代单核细胞中的转染。也将报告基因质粒转染至稳定表达四环素可诱导的VentX[18]的U2OS细胞,并且在四环素的缺失或存在下评估报告基因活性。在每个转染中包括10ng Renilla荧光素酶质粒,以标准化报告基因活性。在转染或四环素添加后48小时收集细胞,并且用双荧光素酶报告基因分析系统(Dual-Luciferase Reporter Assay System)(Promega,Madison,WI)分析细胞。通过Stratagene(Santa Clara,CA)的定点突变试剂盒(Site-DirectedMutagenesis Kit),实现荧光素酶报告基因的NF(B结合位点的突变。如先前所报道,野生型NF(B结合序列5'-GGGATTTTCC-3'突变为5'-GGGATTTTAG-3'。(Grassl等,1999 J Am SocNephrol 10(7):p.1466-77.)
染色质免疫沉淀(ChIP)分析
采用条件表达GFP或GFP.VentX的THP1细胞系,以检测VentX表达是否损害NF(B向IL6启动子域的结合。用1.0(g/ml强力霉素处理细胞2天,并且收集用于染色质免疫沉淀(ChIP)分析。按照生产厂商的说明,用Cell Signaling(Danvers,MA)的酶染色质免疫沉淀试剂盒(Enzymatic Chromatin IP Kit)进行ChIP程序。使用NF(B/p65抗体(Cell Signaling)用于免疫沉淀。用正向引物:5'-GGACGTCACATTGCACAATC-3'和反向引物:5'-GCCTCAGACATCTCCAGTCC-3',通过定量PCR放大包含NF(B结合位点的人IL6启动子域。
PCR分析、实时PCR、蛋白印迹和混合淋巴细胞反应
从SABiosciences(Valencia,CA)购买了树突状和抗原呈递细胞PCR分析仪(Dendritic&Antigen Presenting Cell PCR Array)。此分析的全部基因的GeneBank访问号在表S1中列出。在(480实时PCR系统;Roche,Indianapolis,IN)上进行实时PCR。用于VentX、IL6、IL12、p21、c-myc、CCL2、CCL3、CCL5、CCL19、CXCL1、CXCL12、CD1a、TNF(、TLR2和TLR4的mRNA水平检测的引物在表S2中示出。如先前研究中所描述,进行蛋白印迹分析。(Wu等,2011 J Clin Invest 121(7):p.2599-613.)抗p21和c-myc的原代抗体来自Cell Signaling,并且抗VentX抗体购买自Abcam(Boston,MA)。除向培养基添加BrdU以判定T细胞增殖之外,如先前所述进行混合淋巴细胞反应。(Wu等,2011 J Clin Invest 121(7):p.2599-613.)用PE偶联的抗BrdU抗体检测并入的BrdU,并且用流式细胞术分析。
统计分析
使用学生t检验计算统计显著性,并且p<0.05被认为是统计上显著的。
在本说明书和随附的权利要求书中,单数形式“一(a)”、“一(an)”和“所述(the)”包括复数的指代,除非上下文另有明确指示。
除非另有定义,本文中使用的所有技术和科学术语都与本领域普通技术人员普遍理解的意义相同。尽管也能够在所公开的实施方式的实践和测试中使用与本文中描述的方法和材料类似或等同的方法和材料,现在描述了优选的方法和材料。除所公开的特定顺序之外,本文中叙述的方法可以以逻辑上可行的任意顺序进行。
通过引用并入
在本公开中,已经对诸如专利、专利申请、专利公开、期刊、书籍、论文、网页内容的其他文件进行了参考和引用。所有这些文件通过引用以其整体并入本文。通过引用并入本文的任何材料或其部分,在其与本文中明确阐明的现有定义、陈述或其他公开材料相冲突时,仅以并入的材料和本公开的材料之间不出现冲突的程度并入。在冲突的情形中,以有利于本文中公开的实施方式作为优选公开来解决。
等同性
代表性实例意在帮助说明各方面,并且无意于也不应被理解为限制(发明的)范围。从本文件的全部内容,以及本文中包括的实例及对科学和专利文献的参考,除本文中描述并示出的实施例之外,其各种修改以及许多进一步的实施例对本领域技术人员确实将是显而易见的。这些实例以其各种实施方式及其等同物,包含了能够适应于本文中公开的实施例的实践的重要附加信息、范例和指导。
Claims (1)
1.一种用于将单核细胞分化成树突状细胞的体外方法,包括向单核细胞中引入用于表达人VentX多肽的表达构建体,其中VentX多肽在单核细胞中过表达,由此单核细胞分化成树突状细胞。
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WO2012170979A1 (en) * | 2011-06-10 | 2012-12-13 | Zhenglun Zhu | Human homeobox gene ventx and macrophage terminal differentiation and activation, compositions and methods thereof |
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WO2012071513A2 (en) * | 2010-11-24 | 2012-05-31 | Hong Gao | Expanding hematopoietic stem cells |
WO2012170979A1 (en) * | 2011-06-10 | 2012-12-13 | Zhenglun Zhu | Human homeobox gene ventx and macrophage terminal differentiation and activation, compositions and methods thereof |
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Title |
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IL-6 Regulates In Vivo Dendritic Cell Differentiation through STAT3 Activation;Sung-Joo Park等;《The Journal of Immunology》;20040915;第173卷(第6期);摘要 |
VentX, a Novel Lymphoid-Enhancing Factor/T-Cell Factor–Associated Transcription Repressor, Is a Putative Tumor Suppressor;Hong Gao等;《Cancer Research》;20091222;第70卷(第1期);摘要 |
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