CN113301915A - 用于促进眼中的血管生成的组合物和方法 - Google Patents
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Abstract
提供用IL‑6家族蛋白促进眼中血管生成的组合物和方法,包括白血病抑制因子(LIF)或心肌营养素‑1(CT‑1)。
Description
相关申请交叉引用
本申请要求2019年1月4日提交的美国临时申请号62/788,174的优先权权益,所述申请通过引用并入本文。
技术领域
本发明涉及促进血管生成以减轻眼睛病症。
背景技术
血管生成是胚胎发育、成人血管稳态和组织修复所需的生理过程(1)。然而,血管生成也会导致各种病理状况,例如肿瘤和几种眼内病症,包括湿性年龄相关的黄斑变性(AMD)(1)。在肿瘤进展期间,新血管为肿瘤组织提供营养和氧气,并且因此发挥重要作用;在眼内病症中,异常渗漏血管的生长可能会破坏视网膜并导致失明(1,2)。深入剖析血管生成的分子基础并确定肿瘤和其他疾病的治疗靶点的广泛努力导致发现参与血管发育和分化的关键信号通路(1,3)。具体地,大量研究已经确定VEGF通路在生理性血管生成中的关键作用,并且针对该通路的疗法在癌症和眼部病症(如湿性AMD)的治疗中取得成功(4,5)。反过来,刺激血管生成有望通过改善灌输改善各种缺血性病症的患者的预后(6)。该假设导致过去几十年的一系列临床试验,测试血管生成因子如VEGF或bFGF,这些因子通过基因治疗或作为重组蛋白在冠状动脉或肢体缺血患者中递送。不幸的是,尽管有希望的临床前研究,这些研究都没有成功(7)。因此,需要识别新的策略来改善血管生成治疗。
胶质母细胞瘤细胞分泌多种血管生成因子,这些因子导致此类肿瘤的高度血管表型(8)。尽管VEGF表达非常低,但源自LN-229胶质母细胞瘤细胞系的异种移植肿瘤已充分血管化(9,10)。因此,LN-229分泌组对表征假定的内皮有丝分裂原很感兴趣。
IL-6细胞因子超家族包括白血病抑制因子(LIF)。由于它能够保持胚胎干细胞的多能性,它被广泛用于实验干细胞生物学。还观察到LIF在不同类型的细胞和组织中的多种作用,包括胚胎植入、造血细胞发育、炎症反应、肿瘤进展等。(67)。
LIF在血管生成中的作用仍然存在争议。它最初被表征为牛主动脉内皮细胞的抗血管生成因子,并且对牛肾上腺皮质毛细血管内皮细胞没有影响(35),表明LIF对不同类型的内皮细胞有不同作用。随后的研究显示相当大的复杂性。过度表达LIF的转基因小鼠显示眼中的血管系统减少和视网膜血管发育抑制(14);而携带纯合LIF敲除等位基因的小鼠视网膜血管密度增加(16)。在出生后早期向幼鼠注射重组LIF也导致发育中视网膜的无血管区域略有增加(22)。
发明内容
本发明提供IL-6超家族的成员及其功能片段,其可用于增加需要治疗性治疗病症例如但不限于年龄相关的黄斑变性和视网膜病变(ROP)的受试者眼中的血管生成早产儿。在实施方案中,受试者是人。
在实施方案中,本发明提供一种治疗与受试者眼中的血管化不足相关的病症的方法,包括向有需要的受试者施用有效量的IL-6家族蛋白或其功能片段,以促进血管生成。在实施方案中,本发明规定IL-6家族蛋白是白血病抑制因子(LIF)或心肌营养素-1(CT-1)。
在实施方案中,本发明规定施用增加视网膜微血管密度。在实施方案中,本发明规定施用增加脉络膜内皮细胞的增殖。
在实施方案中,本发明规定病症是年龄相关的黄斑变性。在实施方案中,本发明提供病症是早产儿视网膜病(ROP)。
在实施方案中,本发明规定施用是经由玻璃体内注射。在实施方案中,本发明规定有效量不会诱导血管渗漏。在实施方案中,本发明规定有效量不会诱导水肿。
在实施方案中,本发明提供一种诱导受试者眼中血管形成的方法,包括向有需要的受试者施用有效量的IL-6家族蛋白或其功能片段。
在实施方案中,本发明规定施用增加视网膜血管生成。在实施方案中,本发明规定施用增加脉络膜内皮细胞的增殖。
在实施方案中,本发明规定受试者患有年龄相关的黄斑变性。在实施方案中,本发明规定受试者患有早产儿视网膜病(ROP)。
在实施方案中,本发明规定施用是经由玻璃体内注射。在实施方案中,本发明规定有效量不会诱导血管渗漏。在实施方案中,本发明规定有效量不会诱导水肿。
在实施方案中,本发明规定IL-6家族蛋白是白血病抑制因子(LIF)。在实施方案中,本发明规定IL-6家族蛋白是心肌营养素-1(CT-1)。
附图说明
图1A-1F显示LIF是来自LN-229条件培养基的内皮细胞有丝分裂原。LN-229条件培养基刺激牛脉络膜内皮细胞的生长,n=3(图1A)。VEGF中和抗体无法抑制LN-229CM诱导的BCE细胞生长,n=3(图1B)。LN-229CM的反相色谱级分诱导BCE细胞生长。如图所示,BCE细胞与级分(2μl/孔)一起孵育,n=3(图1C)。抗LIF中和抗体消除由反相级分诱导的BCE细胞生长,n=3(图1D)。重组人LIF蛋白以剂量依赖性方式刺激BCE细胞的生长。BCE细胞在载体、VEGF(10ng/ml)和指定浓度的重组人LIF(rhLIF)存在下培养,n=3(图1E)。LIF和VEGF协同刺激BCE细胞生长。在6天后使用阿拉玛蓝分析细胞增殖,n=3。条和误差条代表平均值±SD。*,p<0.05;**,p<0.01;#,P≤0.0001;ns,无统计学意义(图1F)。
图2A-2E显示LIF经由JAK-STAT3途径促进BCE细胞生长。JAK抑制剂巴瑞替尼(Ba)可以阻断LIF对STAT3的激活。BCE细胞与DMSO、巴瑞替尼(2μM)、考比替尼(Co)(150nM)或BEZ235(BE)(5nM)预孵育1h,并且然后用载体或LIF(10ng/ml)处理15分钟。Ctrl,无抑制剂预孵育(图2A)。巴瑞替尼抑制LIF诱导的BCE细胞生长。BCE细胞与DMSO、巴瑞替尼、考比替尼或BEZ235预孵育1h,然后用载体、LIF(10ng/ml)或VEGF(10ng/ml)处理。在6天后分析细胞增殖,n=3(图2B)。图2C和2D显示BCE细胞中的STAT3敲下。用靶向STAT3的siNegative和siRNA转染BCE细胞。进行qRT-PCR以检查STAT3mRNA水平。siNegative中的STAT3水平设置为1。来自三个独立实验的数据被平均并显示在图2C中。在图2D中,用siRNA转染的细胞用LIF(10ng/ml)或载体处理15分钟。用指定的抗体对全细胞裂解物进行蛋白质印迹。LIF诱导的BCE细胞生长被STAT3敲下消除。将STAT3敲下的BCE细胞与LIF(10ng/ml)或载体一起培养。在3天后分析细胞增殖。每个载体组在590nm处的荧光读数设置为1,n=3。siNegative阴性对照siRNA,不靶向任何已知基因。**,p<0.01;***p<0.001;#,P≤0.0001;ns,无统计学意义(图2E)。
图3A-3J显示LIF在离体和体内模型中促进血管生成。图3A和3B显示LIF对小鼠脉络膜发芽的诱导。图3A的代表性图片显示接种后6天的初级脉络膜外植体的血管增殖。如所示将补充剂加到每个样品。使用Axiovision软件对血管芽的生长进行量化,n=5。图3C和3D显示玻璃体内注射LIF增加小鼠眼中的血管密度。成年小鼠玻璃体内注射指定量的VEGF和LIF。在注射后7天,PFA固定的脉络膜巩膜复合物和视网膜接受CD31 IF。CD31阳性血管的代表性图像在图3C中显示。用ImageJ软件确定的血管密度在图3D中显示,n=5-8。图3E和3F显示LIF处理的小鼠视网膜的OCTA成像。成年小鼠玻璃体内注射1μl LIF(50ng)或载体溶液(PBS)。在注射后7天获得视网膜OCTA图像,其代表在图3E中显示。血管密度使用ImageJ软件用血管覆盖面积/总面积百分比确定并且在图3F中显示,n=7-8。图3G和3H显示LIF治疗增加小鼠视网膜中的血管密度。成年小鼠玻璃体内注射LIF(10ng)或载体溶液。在注射后7天,对小鼠眼睛的冷冻切片进行H&E染色和CD31 IF染色。代表性图像在图3G中显示。使用ImageJ软件对CD31阳性进行量化在图3H中显示,n=4。在图3I和3J中,五天大的新生小鼠玻璃体内注射LIF(50ng)或载体溶液(PBS)。在处理3天后,用Dyight-488标记的凝集素对小鼠视网膜进行IF染色。图3I显示类似眼位点的代表性图像。使用ImageJ软件对凝集素标记区域进行量化在图3J中显示,n=4。*,p<0.05;**,p<0.01。
图4A-4F显示LIF通过JAK-STAT3途径抑制BAE细胞生长。重组人LIF以剂量依赖性方式抑制BAE细胞的生长。BAE细胞在载体和指定浓度的重组人LIF(rhLIF)存在下培养。在6天后分析细胞增殖,n=3(图4A)。JAK抑制剂巴瑞替尼可以阻断LIF对STAT3的激活。用DMSO和抑制剂预孵育1h的BAE细胞用载体和LIF(10ng/ml)处理15分钟。用指定的抗体对全细胞裂解物进行蛋白质印迹。Ctrl,无抑制剂预培养;Ba,巴瑞替尼(2μM);Co,考比替尼(150nM);BE,BEZ235(5nM)(图4B)。JAK抑制剂巴瑞替尼可逆转LIF诱导的BAE生长抑制。将与抑制剂预孵育1h的BAE细胞用载体、LIF(10ng/ml)和VEGF(10ng/ml)处理。在6天后使用阿拉玛蓝分析细胞增殖,n=3(图4C)。图4D和4E显示BAE细胞中STAT3的敲下。用靶向STAT3的siRNA转染BAE细胞。进行qRT-PCR以检查STAT3 mRNA水平。siNegative中的STAT3水平设置为1。对来自三个独立实验的数据进行平均并显示在图4D中。在图4E中,用siRNA转染的细胞用LIF(10ng/ml)和载体处理15分钟。用指定的抗体对全细胞裂解物进行蛋白质印迹。图4F显示STAT3的敲下消除LIF诱导的BAE细胞生长抑制。将STAT3敲下的BAE细胞与LIF(10ng/ml)和载体一起培养。在3天后分析细胞增殖。每个载体组的荧光读数设置为1,n=3。条和误差条代表平均值±SD。siNegative阴性对照siRNA,不靶向任何已知基因。**,p<0.01;***p<0.001;#,p≤0.0001;ns,无统计学意义。
图5A-5B显示LIF不会诱导豚鼠皮肤和小鼠视网膜的血管通透性。在图5A中,无毛雄性豚鼠(Crl:HA-Hrhr/IAF,450–500g,Charles River Laboratories)通过腹膜内(ip)施用甲苯噻嗪(5mg/kg)和氯胺酮(75mg/kg)进行麻醉。然后动物接受1ml 1%伊文思蓝染料的静脉内注射(阴茎静脉)。在15min后,将不同剂量(每个注射部位1、5、25、100、200ng)的PBS中的rhLIF皮内注射(0.05ml/每个部位)注射到肩部后方的躯干区域。注射0.05ml PBS和0.05ml PBS中的25ng VEGF作为阴性和阳性对照。在皮内注射后30min,通过腹腔注射戊巴比妥(200mg/kg)对动物实施安乐死。从结缔组织解剖皮肤组织并拍照,n=2。在图5B中,小鼠视网膜中显示血管渗漏。将LIF(10ng)或VEGF(100ng)注射到玻璃体腔(0.1%BSA/PBS作为对照)中。TRITC-葡聚糖用于指示血管渗漏。视网膜血管系统由FITC-凝集素标记,n=5。
图6A-6F显示LIF经由组织蛋白酶L的上调诱导细胞死亡。图6A和6B显示LIF处理诱导BAE细胞中的细胞死亡。在用LIF(10ng/ml)或载体处理24小时后,BAE细胞被膜联蛋白V-Cy5染色。代表性图像在图6A中显示。计算膜联蛋白V阳性面积与总细胞覆盖面积的百分比,并显示在图6B中,n=3。图6C和6D显示BAE细胞中LIF诱导的组织蛋白酶L表达。在用LIF(10ng/ml)或载体处理24小时后,进行qRT-PCR以检查BAE细胞中的组织蛋白酶L(CTSL)mRNA水平。载体组CTSL水平设置为1。将每个样品中的CTSL mRNA水平与载体组进行比较,并在图6C中显示为倍数变化,n=3。来自LIF处理的BAE细胞的总蛋白用于牛组织蛋白酶L ELISA。载体处理组的组织蛋白酶L蛋白水平设置为1。计算组织蛋白酶L蛋白的诱导倍数变化(LIF处理的样品与载体组),并且来自三个独立实验的倍数变化在图6D中显示。图6E和6F显示组织蛋白酶L抑制剂CA074me和CAA0225减轻LIF诱导的BAE细胞生长抑制。用指定浓度的CA074me和CAA0225预孵育1h的BAE细胞用载体、LIF(10ng/ml)和VEGF(10ng/ml)处理。在6天后分析细胞生长,n=3。条和误差条代表平均值±SD。*,p<0.05;**,p<0.01;***p<0.001;#,p≤0.0001;ns,无统计学意义。
图7A-7C显示LIF在BAE细胞中诱导细胞周期停滞。图7A和7B显示LIF处理减少BAE细胞中的BrdU掺入。在用LIF(10ng/ml)和载体处理48小时后,BAE细胞与10μM BrdU一起孵育4小时。图7A显示用Alexa Fluor-488偶联的BrdU抗体检测到的BrdU掺入的代表性图像。BrdU阳性细胞核与DAPI染色的总细胞核的百分比计算并显示在图7B中,n=3。图7C显示BAE中LIF对细胞周期蛋白A和B表达的抑制。BAE和BCE细胞用LIF(10ng/ml)和载体处理24小时。进行qRT-PCR以检查CTSL1、CCNA2、CCNB1和MYC mRNA水平。对于每个基因探针,载体处理组水平设置为1。LIF处理样品中的mRNA水平标准化为载体组,n=3。条和误差条代表平均值±SD。*,p<0.05;***p<0.001;#,p≤0.0001;ns,无统计学意义。
图8A-8D显示其他IL-6家族蛋白对小鼠眼模型的影响。将重组LIF(50ng)和1μl中不同剂量的CT-1和PBS载体对照玻璃体内注射到小鼠眼中(图8A)。视网膜血管系统用活小鼠OCT-A成像和CD31免疫荧光染色表示,n=5(图8A)。使用共聚焦显微镜对视网膜平面染色进行成像(图8B)。使用Image J对血管进行量化。图8C和8D显示碘酸钠用于诱导小鼠脉络膜毛细血管损伤。在注射碘酸钠后,在眼内注射指定量的LIF、CT-1或OSM。脉络膜毛细血管在OCT-A系统下成像,n=5。脉络膜中的无血管区域使用Image J测定和量化。
具体实施方式
本说明书中提到的所有出版物、专利和专利申请均通过援引并入本文,其程度如同每个单独的出版物、专利或专利申请被具体地且单独地指出通过援引并入一样。
除非另有定义,否则本文中所用的所有技术和科学术语以及任何首字母缩略词均具有与本发明领域的普通技术人员通常所理解的相同的含义。尽管与本文所述的那些类似或等同的任何方法和材料都可以用于本发明的实践中,但是本文描述了示例性方法、装置和材料。
除非另外指明,否则本发明的实践将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术,这些都在本领域的技术范围内。这样的技术在以下文献中充分解释:分子克隆(Molecular Cloning):实验室手册(A LaboratoryManual),第2版(Sambrook等人,1989);寡核苷酸合成(Oligonucleotide Synthesis)(M.J.Gait编辑,1984);动物细胞培养(Animal Cell Culture)(R.I.Freshney编著,1987);酶学方法(Methods in Enzymology)(学术出版社有限公司);分子生物学实验指南(Current Protocols in Molecular Biology)(F.M.Ausubel等人编著,1987,并且定期更新);PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)(Mullis等人编著,1994);Remington,药学科学与实践(The Science and Practice of Pharmacy),第20版,(利平科特,威廉姆斯&威尔金斯2003)以及Remington,药学科学与实践,第22版,(费城理工大学医药出版社和药学院(Philadelphia College of Pharmacy at University of theSciences)2012)。
本发明提供IL6超家族的成员及其功能片段,可用于增加需要治疗性治疗病症例如但不限于年龄相关的黄斑变性和早产儿视网膜病的受试者眼中的血管生成(ROP)。在实施方案中,受试者是人。
在实施方案中,本发明提供一种治疗与受试者眼中的血管化不足相关的病症的方法,包括向有需要的受试者施用有效量的IL-6家族蛋白或其功能片段,以促进血管生成。在实施方案中,本发明规定IL-6家族蛋白是白血病抑制因子(LIF)或心肌营养素-1(CT-1)。
在实施方案中,本发明规定施用增加视网膜微血管密度。在实施方案中,本发明规定施用增加脉络膜内皮细胞的增殖。在实施方案中,本发明提供施用刺激血管生成。
在实施方案中,本发明规定病症是年龄相关的黄斑变性。在实施方案中,本发明规定病症是早产儿视网膜病(ROP)。
在实施方案中,本发明规定施用是经由玻璃体内注射。在实施方案中,本发明规定有效量不会诱导血管渗漏。在实施方案中,本发明规定有效量不会诱导水肿。
在实施方案中,本发明提供一种诱导受试者眼中血管形成的方法,包括向有需要的受试者施用有效量的IL-6家族蛋白或其功能片段。
在实施方案中,本发明规定施用增加视网膜血管生成。在实施方案中,本发明规定施用增加脉络膜内皮细胞的增殖。
在实施方案中,本发明规定受试者患有年龄相关的黄斑变性。在实施方案中,本发明规定受试者患有早产儿视网膜病(ROP)。
在实施方案中,本发明规定施用是经由玻璃体内注射。在实施方案中,本发明规定有效量不会诱导血管渗漏。在实施方案中,本发明规定有效量不会诱导水肿。
在实施方案中,本发明规定IL-6家族蛋白是白血病抑制因子(LIF)。在实施方案中,本发明规定IL-6家族蛋白是心肌营养素-1(CT-1)。
定义
为了促进对本发明的理解,如本文使用的许多术语和缩写如下定义:
当介绍本发明或其(一种或多种)优选的实施方式的要素时,冠词“一个或一种(a)”、“一个或一种(an)”、“所述或该(the)”以及“所述(said)”旨在意指表示存在一个(种)或多个(种)要素。术语“包括(comprising)”、“包括(including)”和“具有”旨在是包括性的并且意指可以存在除所列要素之外的额外的要素。
当在两个或更多个条目的列表中使用时,术语“和/或”意指所列条目中的任何一个可以单独使用或与所列条目中的任何一个或多个组合使用。例如,表述“A和/或B”旨在意指A和B中的任何一个或两者,即仅A、仅B或A和B组合。表述“A、B和/或C”旨在意指仅A、仅B、仅C、A和B组合、A和C组合、B和C组合或A、B和C组合。
应当理解,本文所述的本发明的方面和实施方式包括“由方面和实施方式组成”和/或“基本上由方面和实施方式组成”。
应当理解,以范围格式的描述仅是为了方便和简洁,而不应当被解释为对本发明的范围的不灵活的限制。因此,应当认为对范围的描述已经具体公开所有可能的子范围以及所述范围内的各个数值。例如,对范围诸如从1至6的描述应当被认为已经具体公开子范围,诸如从1至3、从1至4、从1至5、从2至4、从2至6、从3至6等,以及所述范围内的各个数字,例如1、2、3、4、5和6。无论范围的广度如何,这都适用。值或范围在本文中也可以表达为“约”,从“约”一个特定值和/或至“约”另一个特定值。当表达这样的值或范围时,公开的其他实施方式包括所叙述的具体值,从一个特定值和/或至其他特定值。类似地,当通过使用先行词“约”将值表达为近似值时,应当理解,特定值形成另一个实施方式。还将进一步理解,存在其中公开的多个值,并且每个值在本文中还被公开为除所述特定值本身以外的“约”所述特定值。在实施方案中,“约”可以用于意指例如在所叙述值的10%内、在所叙述值的5%内或在所叙述值的2%内。
如本文所用,“患者”或“受试者”意指待治疗的人类或动物受试者。
本文所用的术语“药物组合物”是指药物可接受的组合物,其中该组合物包括药学上活性剂,并且在一些实施方式中进一步包括药学上可接受的载体。在一些实施方式中,药物组合物可以是药学上活性剂和载体的组合。
术语“组合”是指以一种剂量单位形式的固定组合或用于联合施用的部分的试剂盒,其中一种或多种活性化合物和组合伴侣(例如,下文所解释的另一种药物,也称为“治疗剂”或“共剂”)可以同时独立施用或在时间间隔内分别施用。在一些情况下,组合伴侣示出合作,例如协同效应。本文所用的术语“共同施用”或“联合施用”等意在涵盖将所选择的组合伴侣施用到有此需要的单个受试者(例如,患者),并且旨在包括其中不一定通过相同的施用途径或在同一时间施用的治疗方案。如本文所用,术语“药物组合”意指由于混合或组合多于一种活性成分得到的产品,并且包括活性成分的固定组合和非固定组合两者。术语“固定组合”意指活性成分(例如,化合物和组合伴侣)均以单一实体或剂量的形式同时施用到患者。术语“非固定组合”意指活性成分(例如,化合物和组合伴侣)均作为单独的实体同时地、并发地或相继地以无特定时间限制的形式施用到患者,其中这样的施用在患者体内提供治疗有效水平的两种化合物。后者也适用于鸡尾酒疗法,例如,施用三种或更多种活性成分。
如本文所用,“有效”或“治疗有效”是指足以治疗或改善或以某种方式减轻与疾病和医学病症相关的症状的一种或多种药学上活性化合物的量。当参考一种方法使用时,方法足以有效地治疗或改善或以某种方式减轻与疾病或病症相关联的症状。例如,关于年龄相关的眼睛疾病的有效量是足以阻断或预防发作;或者如果疾病病理已开始,则是足以缓和、改善、稳定、逆转或减慢疾病进展,或者以其他方式减轻疾病的病理后果的量。在任何情况下,有效量可以以单剂量或分次剂量给予。
本文所用的术语“治疗(treat)”、“治疗(treatment)”或“治疗(treating)”至少涵盖与患者的疾病相关联的症状的改善,其中改善在广义上用于指至少参数(例如,与所治疗的疾病或病症相关联的症状)量级的降低。因此,“治疗(treatment)”还包括疾病、疾患或病理状况、或者至少与之相关联的症状被完全抑制(例如,阻止发生)或停止(例如,终止),使得患者不再患有所述病症,或至少表征所述病症的症状。
如本文所用的并且除非另外指明,否则术语“预防(prevent)”、预防(preventing)是指预防疾病或病症或者其一种或多种症状的发作、复发或传播。在某些实施方式中,术语是指在症状发作先前,特别是针对具有本文提供的疾病或疾患的风险的受试者,在具有或不具有一种或多种其他额外的活性剂的情况下,用本文提供的化合物或剂型治疗或者施用本文提供的化合物或剂型。此术语涵盖抑制或减轻特定疾病的症状。在某些实施方式中,具有家族病史的受试者是预防方案的潜在候选者。在某些实施方式中,具有复发症状史的受试者也是预防方案的潜在候选者。在这方面,术语“预防(prevention)”可以与术语“预防性治疗(prophylactic treatment)”互换使用。
如本文所用并且除非另外指明,否则化合物的“预防有效量”是足以预防疾病或疾患或者防止其复发的量。化合物的预防有效量意指单独的或者与一种或多种其他剂组合使用的治疗剂的量,其在疾病的预防中提供预防益处。术语“预防有效量”可以涵盖改善总体预防或增强其他预防剂的预防功效的量。
如本文所用,术语“药学活性”是指物质对生物体,并且特别是对人体细胞和组织的有益生物活性。“药学活性剂”或“药物”是具有药学活性的物质,而“药学活性成分”(API)是药物中的药学活性物质。
如本文所用,术语“药学上可接受的”是指除在动物,并且更具体地在人和/或非人类哺乳动物中安全使用的其他制剂外,由联邦或州政府的监管机构批准的或在美国药典、其他普遍认可的药典中列出的。本发明考虑配制用于眼科递送(包括玻璃体内注射)的用于治疗眼睛的组合物。
本文所用的术语“药学上可接受的载体”是指与一种或多种脱甲基化合物一起施用的载体、稀释剂、防腐剂、增溶剂、乳化剂、佐剂和/或载体。这样的载体可以为无菌液体,诸如水和油,包括石油、动物、植物或合成来源的那些,诸如花生油、大豆油、矿物油、芝麻油等;聚乙二醇、丙三醇、丙二醇或其他合成溶剂。抗菌剂,诸如苯甲醇或对羟苯甲酸甲酯;抗氧化剂,诸如抗坏血酸或亚硫酸氢钠;螯合剂,诸如乙二胺四乙酸;以及用于调整张力的试剂,诸如氯化钠或右旋糖也可以是载体。用于生产组合物与载体的组合的方法是本领域的技术人员已知的。在一些实施方式中,词语“药学上可接受的载体”旨在包括与药物施用相容的任何和所有溶剂、分散介质、包衣、等渗剂和吸收延迟剂等。使用这样的介质和剂用于药学活性物质在本领域中是众所周知的。参见例如Remington,药学科学与实践(TheScience and Practice of Pharmacy),第20版,(利平科特,威廉姆斯&威尔金斯2003)。除非到任何常规介质或剂与活性化合物不相容的程度,否则设想在组合物中使用这样的物质。
如本文所用,术语“药学上可接受的盐”是指本公开中化合物的酸加成盐或碱加成盐,例如多药物偶联物。药学上可接受的盐是任何盐,其保留母体试剂或化合物的活性并且不会对其施用的受试者和在施用的上下文中产生任何有害或不期望的影响。药学上可接受的盐可以源自氨基酸,包括但不限于半胱氨酸。用于产生作为盐的化合物的方法是本领域技术人员已知的(参见例如Stahl等人,Handbook of Pharmaceutical Salts:Properties,Selection,and Use,Wiley-VCH;Verlag Helvetica Chimica Acta,Zurich,2002;Berge等人,J Pharm.Sci.66:1,1977)。在一些实施方案中,“药学上可接受的盐”旨在表示本文表示的试剂或化合物的游离酸或碱的盐,其无毒、生物学上可耐受或在生物学上适合施用于受试者。一般参见Berge等人,J.Pharm.Sci.,1977,66,1-19。优选的药学上可接受的盐是药理学上有效的并且适合与受试者的组织接触而没有过度毒性、刺激或过敏反应的盐。本文所述的试剂或化合物可具有足够酸性的基团、足够碱性的基团、两种类型的官能团或每种类型中的多于一种,并且因此与多种无机或有机碱以及无机和有机酸反应,形成药学上可接受的盐。
药学上可接受的盐的实例包括硫酸盐、焦亚硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物、乙酸盐、丙酸盐、癸酸盐、辛酸盐、丙烯酸盐、甲酸盐、异丁酸盐、己酸盐、庚酸盐、丙炔酸盐、草酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸盐、富马酸盐、马来酸盐、丁炔-l,4-二酸盐、己炔-l,6-二酸盐、苯甲酸盐、氯苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、羟基苯甲酸盐、甲氧基苯甲酸盐、邻苯二甲酸盐、磺酸盐、甲基磺酸盐、丙磺酸盐、苯磺酸盐、二甲苯磺酸盐、萘-1-磺酸盐、萘-2-磺酸盐、苯乙酸盐、苯丙酸盐、苯丁酸盐、柠檬酸盐、乳酸盐、γ-羟基丁酸盐、乙醇酸盐、酒石酸盐和扁桃酸盐。
术语“氨基酸”是指天然存在的和合成的氨基酸,以及以类似于天然存在的氨基酸的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的氨基酸,以及那些后来被修饰的氨基酸,例如羟脯氨酸、α-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构的化合物,即与氢、羧基、氨基和R基团结合的α碳,例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如正亮氨酸)或修饰的肽骨架,但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有不同于氨基酸的一般化学结构的结构但以类似于天然存在的氨基酸的方式起作用的化合物。
用于本发明的IL-6蛋白家族包括白血病抑制因子(LIF)或心肌营养素-1(CT-1)。用于本发明的IL-6蛋白家族还可以包括其他IL-6细胞因子以促进血管生成,例如白细胞介素11(IL-11)、睫状神经营养因子(CNTF)、心肌营养素样细胞因子(CLC)、白细胞介素27(IL-27),即一种也可归入IL-12家族的异质二聚体细胞因子。然而,制瘤素M(OSM)具有相反的作用。本领域技术人员可以根据在此描述的本发明的知识,针对用于本发明的血管生成促进活性常规地筛选另外的IL-6家族成员。IL-6家族蛋白质可以是分离的或部分纯化的天然存在的蛋白质或重组产生的蛋白质。
这种天然存在的IL-6家族成员的氨基酸序列是本领域众所周知的。对于氨基酸序列,本领域技术人员将认识到对改变、添加或删除编码序列中的单个氨基酸或小百分比的氨基酸的核酸、肽、多肽或蛋白质序列的个别替换、缺失或添加是“保守修饰的变体”,其中改变导致氨基酸被化学上类似的氨基酸取代。提供功能类似的氨基酸的保守取代表是本领域公知的。这种保守修饰的变体是本发明的多态性变体、种间同源物和等位基因的补充并且不排除本发明的多态性变体、种间同源物和等位基因。
在实施方案中,本发明涉及促进血管生成以预防或治疗以血管化不足或不够为特征的疾病或病症。此类疾病或病症包括但不限于早产儿视网膜病变(ROP)、年龄相关的黄斑变性、糖尿病视网膜病变、青光眼、糖尿病足溃疡、肺动脉高压、缺血、慢性溃疡、秃顶或头发变白、皮肤再生皮瓣、伤口和烧伤愈合、人造皮肤植入、胚胎发育和移植血管的准备。
本发明将LIF识别为原代脉络膜内皮细胞的有丝分裂原。在本发明前,LIF长期以来一直被表征为内皮细胞生长/血管生成的负调节剂,尽管其确切机制在很大程度上仍然未知。1992年,首次报道LIF是BAE细胞生长的抑制剂(35)。随后的研究将LIF描述为bFGF和VEGF诱导的内皮细胞增殖的抑制剂(15,41)。唯一的例外是研究显示LIF在通过SV40大T抗原生成的永生化内皮细胞系中具有一些促有丝分裂作用(42)。
本发明首次证明,LIF可以在体外刺激初级内皮细胞生长。此外,本发明公开LIF-JAK-STAT3信号轴负责内皮细胞的促有丝分裂作用。玻璃体内注射重组LIF显著增加成年小鼠视网膜的血管密度,证实LIF的促血管生成作用。有趣的是,CT-1还诱导视网膜血管生成,并且在NaIO3模型中也具有保护性。
在基因工程小鼠模型(GEMM)中,LIF表达水平与视网膜血管发育呈负相关(14,16)。然而,先前有报道称LIF会影响多种细胞类型(16,43)并且甚至完全破坏GEMM的视网膜发育(44)。具体地,LIF对视网膜星形胶质细胞的成熟产生负面影响,进而促进未成熟星形胶质细胞的VEGF表达,这可能有助于增加血管密度(16,31,32,45)。因此,GEMM中视网膜血管系统的改变可能不是LIF对内皮细胞的直接影响。在另一项研究中,腹腔内和玻璃体内注射LIF均导致新生大鼠眼血管密度中度降低(22);LIF的这种抑制作用也可以通过其对视网膜发育的影响来解释。此外,该研究没有明确指出玻璃体内注射LIF的剂量(22)。考虑到本发明公开的紧密的钟形剂量反应,很难将本发明与任何先前的研究进行比较。事实上,至少文献中的一些差异可能是由于不同研究中使用的LIF剂量差异很大,从几纳克到几百纳克(16,22)。
早产儿视网膜病变(ROP)是早产儿常见的致盲疾病,其特征是血管系统发育延迟和现有血管退化,随后是缺氧引起的视网膜新生血管形成(46)。眼中VEGF表达的急剧下调与ROP的发生和进展有关(47)和施用外源性VEGF可减轻小鼠ROP的严重程度(47)。然而,使用VEGF作为治疗剂的担忧仍然存在,因为VEGF有助于病理性新血管形成与血管通透性增加(48)。在本发明中,与VEGF不同,LIF不会诱导豚鼠皮肤的血管通透性(图5A)。此外,TRITC标记的葡聚糖用于测定小鼠视网膜微血管渗漏。在TRITC-葡聚糖注射前15min玻璃体内注射LIF(10ng)或VEGF(100ng)。结果表明,与VEGF不同,LIF不会诱导视网膜微血管渗漏(图5B)。因此,可以在ROP的某些阶段使用LIF来防止血管回归。
与先前的报道一致(35),本发明表明LIF导致BAE细胞生长抑制。本发明表明,这至少部分归因于细胞死亡,如通过LIF处理后的膜联蛋白V染色的增加所证明。有趣的是,溶酶体半胱氨酸蛋白酶组织蛋白酶L的两种抑制剂(即CA-074me和CAA0225),而不是半胱天冬酶抑制剂,逆转LIF诱导的细胞死亡,表明参与非半胱氨酸蛋白酶非依赖性细胞死亡。此外,组织蛋白酶B特异性抑制剂CA074未能挽救BAE细胞死亡,并且组织蛋白酶L(而非组织蛋白酶B)在LIF处理的BAE细胞中上调,表明组织蛋白酶L是LIF诱导的溶酶体细胞死亡的执行者。
组织蛋白酶B和L的诱导与自噬和细胞死亡有关(49,50)。本发明首次将LIF-组织蛋白酶L途径与内皮细胞死亡的诱导联系起来。这提出这样一个信号通路是否参与特定的生理或病理过程的问题。有趣的是,LIF和组织蛋白酶L都与血管疾病例如腹主动脉瘤和动脉粥样硬化的发生和发展有关(51-53)。这些数据共同表明LIF-组织蛋白酶L通路在调节病理环境中的血管系统中的作用。
在本发明中,LIF还导致BAE细胞中BrdU掺入减少,伴随细胞周期蛋白A/B表达降低,表明LIF诱导的细胞周期停滞在BAE生长抑制中起作用。先前有报道称细胞周期蛋白A1和细胞周期蛋白B1是STAT3的直接靶点(54)。此外,根据特定设置,STAT3与细胞周期蛋白A/B的上调和下调有关(55-58),并且STAT3对细胞周期蛋白A表达的抑制是由其直接靶标PIM1介导的(58)。这解释为什么LIF抑制BAE细胞中细胞周期蛋白A/B的表达,而不抑制BCE细胞中的表达,因为LIF仅在BAE细胞中诱导PIM1。
本发明公开在两种类型的内皮细胞中由相同信号通路引起的相反反应(增殖与生长抑制)。激活的STAT3在这两种细胞类型中反式激活不同的基因组。事实上,在BCE和BAE细胞中,在LIF处理后一些基因的表达存在差异,包括S期和G2/M细胞周期蛋白基因CCNA2和CCNB1的下调,以及BAE细胞中溶酶体半胱氨酸蛋白酶CTSL的上调但增殖基因MYC的上调仅在BCE细胞中。不同类型的内皮细胞具有其独特的基因表达模式/表观遗传谱,这决定它们对相同刺激的不同反应(59-61)。本发明公开的LIF在不同内皮细胞中的相反作用例示这种多样性的新方面:相同的信号通路介导不同作用,这取决于内皮细胞类型特异性转录程序。本发明首次报道由LIF诱导的溶酶体蛋白酶组织蛋白酶L导致内皮细胞的细胞死亡。
本发明在实施方案中公开LIF在脉络膜和视网膜内皮细胞中出人意料的促有丝分裂作用,并表明LIF和CT-1两者在体内都增加视网膜微血管密度。事实上,保护具有湿性或干性AMD的患者的眼血管(如脉络膜毛细血管层)是有益的,因为它可以预防萎缩(62)。LIF和CT-1在NaIO3模型中都具有保护作用,表明这些试剂在保护视网膜色素上皮和脉络膜毛细血管方面具有治疗价值,从而预防AMD萎缩。在这方面,LIF和CT-1缺乏直接通透效应将特别有用。值得注意的是,OSM具有相反作用,表明LIF和CT-1的作用具有特异性。
实施例
材料和方法
试剂
抗体:人PDGF-AA抗体(R&D Systems,CAT#AF-221-NA),人CCL2/MCP-1(R&DSystems,CAT#AF-279-NA),人LIF抗体(Sigma,CAT#L9277),正常山羊IgG同种型对照(R&DSystem,CAT#AB-108-C)和Alexa Fluor-488偶联的BrdU抗体3D4(Biolegend,CAT#364106)
小分子抑制剂:巴瑞替尼(Apexbio Technology,CAT#A414150),考比替尼(MedChemExpress,CAT#HY-13064),BEZ235(Selleckchem,CAT#S1409),Z-VAD-FMK(R&DSystems,CAT#FMK001),Z-DEVD-FMK(R&D Systems,CAT#FMK004),Q-VD(OMe)-OPh(ApexbioTechnology,CAT#A8165),5-AIQ盐酸盐(Sigma,CAT#A7479),CA-074me(Calbiochem,CAT#205531)、CA-074(Tocris,CAT#4863)和CAA0225(Calbiochem,CAT#219502)
重组蛋白:人LIF(Sigma,CAT#SRP9001),人LIF(Biolegend,CAT#593902),人PDGF-AA(Peprotech,CAT#100-13A),人过氧化物酶1(Abcam,CAT#ab74172),人IL-8(Biolegend,CAT#574202)和人VEGF 165(R&D Systems,CAT#293-VE)
细胞培养
LN-229人胶质母细胞瘤细胞保存在补充有5%FBS的高糖DMEM中。牛脉络膜内皮(BCE)(P5-P9)和牛视网膜内皮(BRE)(P5-P9)细胞保存在纤连蛋白涂覆的培养板上的补充有10%小牛血清(BCS)、2mM谷氨酰胺、5ng/ml bFGF和10ng/ml VEGF的DMEM低葡萄糖中。牛主动脉内皮(BAE)细胞(P5-P10)保存在补充有10%BCS的DMEM低葡萄糖中。人视网膜微血管内皮(HRME)细胞(P4-P9)保存在明胶涂层培养板上的具有抗生素的EGM2培养基中。所有细胞均在37℃下保存在具有5%CO2的湿润气氛中。
内皮细胞增殖测定
基本上如先前所述进行牛内皮细胞增殖测定(63,64)。将BCE(1×103细胞/孔)或BRE(5×102细胞/孔)细胞接种在96孔板的培养基(补充有10%BCS、2mM谷氨酰胺和抗生素的DMEM-低葡萄糖)和测试材料中,每孔总体积为200μl。将BAE细胞以2×103个细胞的密度接种在96孔板的培养基(补充有1%BCS和抗生素的DMEM-低葡萄糖)和测试材料中,每孔总体积为200μl。将HRME细胞以1×103细胞/孔的密度接种在明胶涂覆的96孔板中的测定培养基(补充有20%FBS和抗生素的DMEM-低葡萄糖)和测试材料中,使每孔总体积为200μl。对于涉及抗体或小分子抑制剂的测定,首先加入抑制剂或载体对照,然后在一小时后加入测试材料。在6天后(除非另有说明),将细胞与阿拉玛蓝一起孵育4小时。在530nm激发波长和590nm发射波长下测量荧光。每个实验一式两份/一式三份进行并重复至少三次。
LN-229细胞条件培养基
将5×106个LN-229细胞接种在具有35ml培养基(具有0.5%FBS和1%抗生素的DMEM-高糖)15cm培养皿中,并且在37℃下培养72h。通过离心收集LN-229CM,用0.22μm过滤器过滤并且在-80℃下保存备用。
在LN-229CM中内皮有丝分裂原的色谱富集
约400ml的LN-229CM通过色谱纯化的顺序进行内皮有丝分裂原的富集。使用GEAKTA Explorer系统(GE Healthcare)将CM缓冲液交换为20mM Tris,pH 8.0,过滤(0.2μm)并加载到5ml HiTrap QTM HP柱(GE Healthcare,Pittsburgh,PA)。在用Tris缓冲液中的0.2M、0.5M、1M和2M NaCl逐步洗脱后,在如上所述的BCE细胞生长测定中测试洗脱级分的等分试样。然后合并促有丝分裂级分,在0.1%三氟乙酸/H2O(TFA,ThermoFisher)中稀释并施加到SynChropak RP C4反相柱(4.6x 100mm,Eichrom Technologies,Darien,IL)。级分用乙腈/0.1%TFA的线性梯度洗脱。使用MiVac DUO浓缩器(Genevac,Ipswich,UK)蒸发洗脱的级分,洗涤,重悬于PBS中,并如上测试。对有丝分裂级分和相邻的阴性级分进行质谱分析。
ELISA
LN-229CM样品中的VEGF和LIF水平分别根据制造商的说明通过人VEGF ELISA试剂盒(R&D Systems,CAT#DVE00)和人LIF ELISA试剂盒(Biolegend,CAT#443507)测定。根据制造商的说明,使用牛组织蛋白酶L ELISA试剂盒(MyBioSource,Inc,CAT#MBS2887609)测量BAE细胞中的组织蛋白酶L水平。
STAT3被siRNA敲下
BCE和BAE细胞以1.5×105个细胞/孔的密度接种到6孔培养板上。BCE细胞在2ml补充有10%BCS、2mM、5ng/ml bFGF、10ng/ml VEGF和抗生素的DMEM低葡萄糖中培养过夜,而BAE细胞在2ml补充有10%BCS和抗生素的DMEM低葡萄糖中培养过夜。使用2ml不含抗生素的培养基代替旧培养基。siRNA,包括siNegative(Ambion,CAT#AM4611)、siSTAT3-915(Invitrogen,CAT#361146C04)、siSTAT3-1492(Invitrogen,CAT#361146C05)和siSTAT3-454(CAT#5A4618),根据制造商的说明与在Opti-MEMTM I减少血清培养基(Gibco,CAT#31985062)中的Lipofectamine RNAiMAX试剂(ThermoFisher Scientific,CAT#13778150)混合。简而言之,将含有25pmol siRNA、7.5ul RNAiMAX试剂和125ul Opti-MEM培养基的混合物用于转染每孔中的细胞,使最终siRNA浓度为12.5nM。RNAiMAX和Opti-MEM的混合物用作无siRNA对照。将细胞与siRNA孵育8小时并且然后使用新鲜的正常培养基替换含有siRNA的培养基。在用siRNA转染24小时后,将细胞用于内皮增殖测定和RNA/蛋白质提取。
蛋白质印迹
BCE和BAE细胞在生长培养基中培养过夜。去除生长培养基并且然后用PBS洗涤细胞两次。将重组人LIF加入细胞15分钟,然后在以下培养基中孵育3小时:补充有10%BCS、2mM谷氨酰胺和用于BCE细胞的抗生素的DMEM-低葡萄糖,和补充有1%BCS和用于BAE细胞的抗生素的DMEM-低葡萄糖。如果适用,在LIF处理前1h将小分子抑制剂(即巴瑞替尼、考比替尼、BEZ235和载体对照DMSO)加到细胞中。然后用RIPA裂解缓冲液(Life Technologies,CAT#89901)加上蛋白酶和磷酸酶抑制剂混合物(ThermoFisher Scientific CAT#78440)裂解细胞。细胞裂解物中的蛋白质浓度用BCA测定法(ThermoFisher Scientific CAT#23227)测量。等量的蛋白质在NuPAGE 4-12%Bis-Tris凝胶(ThermoFisher Scientific,CAT#NW04125BOX)中进行电泳,然后转移到PVDF膜上。膜在室温下用5%脱脂牛奶的TBST封闭1h,与以下所示的主要抗体在含有0.5%脱脂牛奶的TBST中在4℃下孵育过夜,然后与HRP偶联的二级抗体(1:2000,GE Healthcare)在室温下孵育1h。用SuperSignalTM West Pico PLUS化学发光底物(ThermoFisher Scientific)显示信号。使用的主要抗体:抗磷酸化STAT3(细胞信号传导,CAT#9131,1:3000),抗STAT3(细胞信号传导,CAT#4904,1:3000),抗磷酸化ERK(细胞信号传导,CAT#4376,1:5000),抗ERK(细胞信号传导,CAT#4695,1:5000),抗磷酸化AKT Ser473(细胞信号传导,CAT#4060,1:2000),抗AKT(细胞信号传导,CAT#)4691,1:2000)和HRP偶联的抗β-肌动蛋白(Sigma,CAT#AC-15,1;10000)。
RNA提取和qRT-PCR
BCE和BAE细胞,在指定的处理后,用Trizol试剂(Invitrogen,CAT#15596026)裂解,并按照制造商的说明进行RNA提取。用Nanodrop 2000(ThermoFisher Scientific)测定RNA浓度并使用高容量cDNA逆转录试剂盒(Applied Biosystems,CAT#4368814)将1μg总RNA逆转录为cDNA。使用TaqMan Fast Advanced Master Mix(Applied Biosystems,CAT#4444557)和ViiA7实时PCR系统对等量(通常10ng/反应)的cDNA进行qRT-PCR分析。将检查基因的相对mRNA水平标准化为内部对照RPLP0(核糖体蛋白侧茎亚基P0),通过与对照样品组比较来确定,并报告为倍数变化。使用TaqMan基因表达测定探针:牛RPLP0(Bt03218086_m1),牛STAT3(Bt03259865_m1),牛CTSL1(Bt03257307_m1和Bt03257309_m1),牛CTSB(Bt03259161_m1),牛MYC(Bt03260377_m1),牛JunB(Bt03246919_s1),牛CCNA2(Bt03240503_g1),牛CCNB1(Bt03237853_g1)和牛PIM1(Bt03212957_m1)。实验一式三份进行并重复三次。
细胞死亡的膜联蛋白V染色
将BAE细胞以2×104个细胞/孔的密度与1ml培养基(DMEM-低葡萄糖加10%BCS)一起接种在12孔板中,并且然后在37℃下孵育过夜。在去除培养基后,将细胞在0.5ml DMEM-低葡萄糖加1%BCS中培养。将LIF(10ng/ml)和载体对照(PBS中的0.1%BSA)加到细胞中。在LIF处理24小时后,根据制造商的说明,使用膜联蛋白V-Cy5细胞凋亡染色检测试剂盒(Abcam,CAT#ab14150)检查细胞的细胞死亡标记物膜联蛋白V。简而言之,去除细胞培养基并将0.5ml膜联蛋白V结合溶液铺在细胞上。在加入5μl膜联蛋白V-Cy5后,将细胞在室温下孵育5min。然后,弃去染色溶液并用0.5ml膜联蛋白V结合溶液代替。使用KeyenceMicroscope BZ-X710(Keyence Corporation,Osaka,Japan)对膜联蛋白V染色进行成像。选择四个随机视野并使用ImageJ软件确定作为细胞死亡指标的膜联蛋白V染色面积占总细胞覆盖面积的百分比。使用Keyence Microscope BZ-X710(Keyence Corporation,Osaka,Japan)对膜联蛋白V染色进行成像。实验一式三份进行并重复三次。
BrdU掺入测定
将BAE细胞以2×104细胞/孔的密度与1ml培养基(DMEM-低葡萄糖加10%BCS)一起铺在每孔中具有18-mm聚D-赖氨酸处理的盖玻片的12孔板中,在37℃下孵育过夜。在去除培养基后,将细胞在0.5ml DMEM-低葡萄糖加1%BCS中培养。将LIF(10ng/ml)和载体对照(PBS中的0.1%BSA)加到细胞中。在LIF处理48小时后,通过将2.5μl 2mM BrdU的DMSO溶液加到每个孔中至终浓度10μM并孵育4小时,使细胞进行BrdU掺入。然后,使用与Fluor alexa-488(Biolegend,CAT#364106,1:400)偶联的BrdU的抗体使细胞进行BrdU免疫荧光染色。简而言之,从培养板中去除BrdU标记培养基,并用3.7%甲醛的PBS溶液在室温下固定细胞15分钟。在用PBS(PBST)中的0.1%Triton X-100进行细胞透化后,细胞DNA在冰上用1N HCl变性10分钟,并在室温下用2N HCl变性10分钟。将细胞盖玻片与荧光alexa-488偶联的BrdU抗体在5%山羊血清-PBST中在4℃下孵育过夜。然后,用带有DAPI的Fluoroshield封固培养基(Abcam,CAT#ab104139)将盖玻片封固到载玻片上。使用Keyence Microscope BZ-X710(Keyence Corporation,Osaka,Japan)对BrdU染色进行成像。针对每个样品随机选择四个视野,并且手动计数BrdU阳性细胞核和总细胞核(DAPI-阳性);通过将BrdU阳性细胞核数除以总细胞核数确定BrdU阳性细胞的百分比。实验一式两份/一式三份进行,并且重复三次。
小鼠脉络膜外植体测定
在48孔板中,将60μL生长因子减少的基底膜提取物(GFR-BME)(Corning,CAT#354230)加入到每个孔中,并使其在37℃下固化20分钟。从雄性C57BL/6J小鼠(年龄P20)解剖的外周脉络膜-巩膜复合体的小块(约1mm×1mm)被加到每个孔的中心,如前所述(23)。将60μL GFR-BME的顶层加到每个孔,然后在37℃下孵育30分钟。在加入500μL补充有2%FBS和抗生素的内皮细胞生长基础培养基EBM-2(Lonza,CAT#CC3156)后,脉络膜外植体的内源性VEGF活性被5μg/ml抗VEGF Mab B20-4.1.1减弱。与抗体孵育90分钟后,在测试孔中加入10ng/ml LIF或PBS对照。组织在具有5%CO2的标准细胞培养条件下培养,并且每48小时更换一次新鲜培养基。使用Keyence显微镜在第5天拍摄每个外植体的相差Z-stack图像。使用ImageJ软件量化血管发芽面积。实验重复3次,并且每次分析每个条件5次重复来获得数据。
小鼠眼中玻璃体内注射重组蛋白
用氯胺酮/甲苯噻嗪混合液麻醉雄性C57BL/6J小鼠(6-8周和P5)。用33号Hamilton注射器玻璃体内注射1μl PBS中的指定量的重组LIF(Sigma,CAT#SRP9001)和PBS载体对照。在注射后7(对于成年小鼠)或3(对于新生小鼠)天,将动物安乐死,然后摘除眼睛并在4%多聚甲醛(PFA)中固定15min。分离脉络膜巩膜复合物和视网膜,并进行抗CD31免疫荧光(IF)或凝集素标记以通过视网膜和脉络膜组织的整片染色或视网膜平片来证明血管系统。对于CD31 IF,将大鼠抗小鼠抗体(BD Biosciences,CAT#550274)稀释1:100,并且在4℃下孵育过夜。在与Alexa Fluor-488偶联的抗大鼠抗体(Life Technologies,CAT#A11006)孵育4小时后,使用Keyence Microscope BZ-X710(Keyence Corporation,Osaka,Japan)或A1RConfocal STORM超分辨率系统(尼康)经由488nm通道对整片进行成像。对于凝集素染色,将Dylight-488标记的凝集素(Vector Laboratories,CAT#DL-1174)以1:200稀释,并使用A1RConfocal STORM超分辨率系统(尼康)获得图像。脉络膜和视网膜中血管密度的量化通过Image J进行。Student's t检验用于统计分析。每个实验重复3次,每次结果类似,并且每个处理组由4或5个个体样品组成。所有动物实验程序均通过加州大学圣地亚哥分校机构动物护理和使用委员会(IACUC)批准,并按照动物护理计划(ACP)的指导方针进行。
碘酸钠模型
八周大的C57BL/6J小鼠用氯胺酮/甲苯噻嗪混合液麻醉。无菌NaIO3作为单次静脉注射施用(20mg/kg体重)(28)(29)。对照小鼠用PBS注射。PBS、LIF(50ng)、CT-1(不同剂量)或OSM(10ng)在五只小鼠组中进行玻璃体内注射。在注射后第5、7和9天,通过OCT-A系统监测脉络膜毛细血管。在注射后9天,处死小鼠,并且摘除眼睛进行H&E和免疫荧光染色。脉络膜毛细血管中的无血管区域使用ImageJ进行分析。
测量视网膜血管渗漏
将重组人VEGF(100ng)或LIF(10ng)注射到玻璃体内(0.1%BSA PBS溶液作为载体对照)。然后将TRITC-葡聚糖(50mg/ml,100ul)注射到尾静脉中。在十分钟后,处死动物并摘除眼睛。视网膜平片在显微镜下成像(65)。
光学相干断层扫描血管造影(OCTA)成像
在LIF注射后7天,使用与先前描述的方法论一致的由华盛顿大学西雅图分校的R.K.Wang博士小组开发的1300nm光学相干断层扫描(OCT)系统对成年小鼠的视网膜进行光学相干断层扫描血管造影(OCTA)成像(66)。简而言之,在单经度模式下以1300nm和200kHzA线速率为中心的90nm带宽为中心操作的扫频激光用于扫描小鼠视网膜并在1.5×1.5mm2的视野中生成血管系统图像。在500个横截面捕获2500个B帧,每个横截面具有五个重复的B帧。为了量化视网膜血管密度,3D结构OCT扫描中的视网膜和脉络膜层被超反射视网膜色素上皮(RPE)分开。然后生成正面最大强度投影。然后通过使用ImageJ软件计算血管覆盖面积占总视野面积的百分比来确定血管密度。
统计分析
除了质谱分析外,实验至少重复三次,结果类似。条形图代表平均值±标准偏差(sd)。为了比较研究中仅有的两组,进行双尾Student’s t检验。对于具有多于两组数据的研究中的组间比较,进行多重比较的单向方差分析。为了在具有两个或更多个变量的研究中进行组间比较,进行多重比较的双向方差分析。p<0.05被认为具有统计学意义。所有统计分析均使用Graphpad Prism软件包进行。
结果
识别LIF作为脉络膜内皮细胞的有丝分裂原
LN-229细胞条件培养基(LN-229CM)能够刺激牛脉络膜内皮(BCE)细胞的生长(图1A)。然而,与先前的研究一致(9,10),LN-229细胞在培养基中分泌非常少的VEGF。抗VEGF抗体B20-4.1(11)不抑制LN-229CM的促有丝分裂作用(图1B),表明VEGF非依赖性途径的参与。使用特异性抗体阵列检查LN-229CM的血管生成因子谱。该分析表明,除在CM中含量丰富的PDGF-AA、CCL2(也称为MCP-1)和白细胞介素8(IL-8)外,大多数已知的血管生成因子都无法检测到。然而,中和PDGF-AA或CCL2的抗体未能抑制LN-229CM诱导的BCE细胞生长。此外,重组PDGF-AA和IL-8未能刺激BCE细胞生长(表1)。
表1重组人PDGF-AA、IL-8和PRDX1在体外不刺激BCE细胞生长。BCE细胞用指定浓度的PDGF-AA/IL-8/PRDX1处理。在第6天测定细胞生长。每个处理组中的细胞生长均被标准化为载体对照组,n=3。ns,无统计学意义。
为了识别LN-229CM中的促有丝分裂因子,采用蛋白质组学方法。BCE促有丝分裂活性通过两个连续的色谱步骤,即阴离子交换和反相色谱富集。在各个步骤,只有一个由4至5个连续的部分组成的吸光度峰显示有丝分裂活性。在反相柱步骤后,对促有丝分裂峰级分(R26和R27)、最小促有丝分裂(R25和R28)和相邻的阴性(R24和R29)级分(图1C)进行质谱分析。通过筛选出细胞内蛋白质生成5种候选蛋白质的简短列表(表2)。
等级 | 蛋白质身份 |
1 | PRDX1_HUMAN过氧化物酶-1 |
2 | PRDX2_HUMAN过氧化物酶-2 |
3 | PRDX6_HUMAN过氧化物酶-6 |
4 | LIF_HUMAN白血病抑制因子 |
5 | A2MG_HUMANα-2-m巨球蛋白 |
表2从LN-229CM反相级分的质谱分析中产生的候选蛋白质。通过排除细胞内蛋白质和与促有丝分裂级分相比在非活性级分中显示更高丰度的蛋白质来识别候选物。如方法中所述,根据相对丰度对蛋白质进行排序。
列出的5种蛋白质中有四种是血清组分并且起氧化还原酶的作用,包括过氧化还原酶(PRDX)-1、-2和-6以及α-2-巨球蛋白,而LIF作为细胞因子表现突出。LIF是白细胞介素6(IL-6)家族蛋白的成员,在多种细胞类型和组织中广泛表达并发挥作用,并参与各种关键生理过程,包括胚胎干细胞自我更新、囊胚植入、星形胶质细胞分化(12,13)。本文中的LIF的存在是出乎意料的,因为这种细胞因子先前已被表征为内皮细胞生长抑制剂和抗血管生成剂(14-16)。然而,针对LIF的抗体完全抑制由反相级分诱导的BCE细胞的生长(图1D)。每个级分的LIF水平与促有丝分裂活性密切相关:最具生物活性的级分R26和R27显示最高的LIF浓度,R25和R28具有痕量的LIF,而无活性级分R24和R29没有LIF(表3)。
级分编号 | R24 | R25 | R26 | R27 | R28 | R29 |
LIF浓度(ng/ml) | 0.2 | 5.9 | 72.9 | 44.8 | 4.9 | 0 |
表3用人LIF ELISA试剂盒测量来自反相色谱的促有丝分裂级分(R26和R27)和相邻的阴性级分(R24、R25、R28和R29)中的LIF浓度(ng/ml)。
这些观察结果表明LIF可能是有丝分裂作用的原因。事实上,重组LIF刺激BCE细胞的生长(图1E),而另一个候选物PRDX1没有效果(表1),进一步证实LIF作为有丝分裂因子。在牛视网膜内皮(BRE)细胞上进行测试时,LIF也发挥促有丝分裂活性。有趣的是,VEGF和LIF一起导致BCE(图1F)和BRE细胞中的有丝分裂效应大于累加效应,表明LIF和VEGF之间存在协同关系。事实上,虽然LIF没有在人视网膜微血管内皮细胞中引起强烈的促有丝分裂反应,但它的加入显著增强VEGF刺激的生长。
LIF对内皮细胞生长的作用由JAK-STAT3通路介导
尽管IL-6家族的所有成员共享受体组分gp130,但LIF信号通过gp130:LIFR受体二聚体转导,而IL-6通过IL6Rα:gp130:gp130:IL6Rα四聚体激活其下游信号(12)。在与gp130相关的四种Janus激酶(JAK1、JAK2、JAK3和TYK2)中,LIF信号通过转磷酸化选择性激活JAK1(12,17,18)。在LIF被激活后,JAK引发三种不同的信号级联:JAK-STAT、PI3K-AKT-mTOR和RAS-MAPK,它们以细胞类型特定的方式发挥不同的功能(12,19)。对于JAK-STAT通路,LIF信号优先激活STAT3,尽管STAT1和STAT5也可以被JAK1磷酸化(19,20)。为了检查BCE细胞中哪些通路负责LIF诱导的生长刺激,采用一组小分子抑制剂巴瑞替尼、考比替尼和BEZ235,分别专门针对JAK1/2、MEK1/2(MAPK通路)和PI3K/mTOR。在BCE细胞中,LIF处理15分钟引发STAT3和ERK的磷酸化,但对AKT磷酸化几乎没有影响(图2A)。与JAK1/2抑制剂巴瑞替尼预孵育几乎完全抑制LIF诱导的STAT3和ERK MAPK磷酸化(图2A),而考比替尼预处理阻断ERK磷酸化,但对STAT3和AKT磷酸化没有影响(图2A)。无论LIF处理如何,BEZ235对AKT磷酸化仅具有中等影响(图2A)。此外,巴瑞替尼完全阻断LIF诱导的细胞生长,而考比替尼显示影响很小,而PI3K/mTOR抑制剂BEZ235对LIF刺激的细胞生长没有影响(图2B)。这些观察结果表明MAPK和PI3K通路可能不是BCE细胞中LIF刺激的主要贡献物,并且因此涉及JAK-STAT。由于STAT3是LIF诱导的JAK-STAT信号级联中的优先介质(19,20)并且涉及多种细胞类型的增殖和存活(21),因此STAT3通过siRNA敲下在BCE中的作用进一步被检查。siRNA成功抑制BCE细胞中RNA和蛋白质水平的STAT3水平(图2C和2D)。STAT3的下调在体外阻止LIF诱导的BCE细胞生长(图2E)。这些观察结果表明JAK-STAT3信号轴介导BCE细胞中LIF的促有丝分裂作用。
LIF促进内皮细胞离体和体内生长
LIF可诱导脉络膜和视网膜内皮细胞体外增殖。然而,先前的报告表明LIF可能会对发育中的眼睛的血管功能产生负面影响(14,16,22)。为了解决这些明显的差异,研究LIF在离体和体内内皮细胞中的功能是否不同,尤其是在眼睛中。LIF对脉络膜内皮细胞的影响在由先前报告修改的离体脉络膜外植体模型中检查(23)。作为对LIF的反应,与对照相比,从外植体到基质胶的微血管生长显著增强(图3A和3B)。接下来,通过玻璃体内注射在6-8周龄小鼠中检查体内LIF效应。如通过用内皮细胞表面标记物CD31的抗体的免疫组织化学(IHC)评估,每只眼施用10ng剂量的LIF显著增加视网膜微血管密度,而100ng的剂量则不太有效(图3C和3D),与对许多细胞因子观察到的钟形反应一致(24)。光学相干断层扫描血管造影(OCTA)还记录LIF注射后视网膜血管密度的显著增加(图3E和3F)。小鼠眼睛横截面中CD31的免疫荧光染色也表明,LIF注射增加成年小鼠视网膜中的血管密度(图3G和3H)。
为了验证这种促血管生成作用确实是由LIF诱导的,而不是由痕量污染物如内毒素或与注射相关的非特异性事件诱导的,重组LIF通过暴露到95℃2小时热灭活,这不影响内毒素稳定(30)。这种治疗消除LIF促进体外有丝分裂和体内血管生成的能力。然而,先前使用LIF基因敲除小鼠的研究表明,LIF表达与视网膜血管密度呈负相关(16)。这种观察结果与本发明数据之间的差异增加LIF在不同发育阶段调节视网膜血管生成中发挥不同作用的可能性。重要的是,LIF在视网膜星形胶质细胞成熟中也起着关键作用,这可能会继发影响视网膜血管系统的发育(31,32)。为了检查LIF对发育中的视网膜血管系统的影响并最大程度减少其对星形胶质细胞发育的影响,将LIF玻璃体内注射到出生后5天的(P5)小鼠中,其中视网膜血管系统正在发育,但星形胶质细胞网络已经建立并正在成熟(33,34)。如在注射后三天评估,这种新生小鼠中的LIF治疗也导致血管密度显著增加(图3I和3J),证实LIF在视网膜血管系统中的促血管生成作用。
由于LIF是白细胞介素-6(IL-6)家族的成员(25),对另外两个家族成员心肌营养素-1(CT-1)(26)和制瘤素M(OSM)的视网膜血管形成的影响(27)进行测试。与50ng LIF相比,20ng和100ng CT-1分别使视网膜密度增加约30%和50%。然而,OSM治疗的小鼠视网膜中的血管密度降低,而不是促进。OSM与LIF和CT-1对视网膜血管系统的不同作用表明OSM可能不会激活与LIF和CT-1相同的信号通路,因为OSM可以结合到gp130::LIFR和gp130::OSMR受体复合物,而LIF和CT-1仅利用gp130::LIFR复合物。
NaIO3小鼠模型已被广泛用作萎缩性AMD的临床前模型(28)。在该模型中,RPE层和脉络膜毛细血管均严重受损(29)。因此,测试LIF、CT-1和OSM在该模型中促进脉络膜毛细血管恢复的能力。在静脉注射NaIO3后,玻璃体内注射LIF、CT-1或OSM。与对视网膜血管系统的影响一致,与PBS组相比,LIF和CT-1减少无血管区域。相比之下,OSM处理的脉络膜中的无血管区域大于PBS组(图8C和8D)。LIF和CT-1对视网膜血管系统对NaIO3治疗的保护作用可能归因于它们在视网膜内皮细胞中的直接有丝分裂活性并且也可能归因于它们保护视网膜RPE细胞免受氧化应激诱导的损伤的能力,这继而经由促血管生成因子(例如VEGF)的分泌支持视网膜血管系统的维持。
LIF经由JAK-STAT3通路抑制生长
与先前的研究一致(35),LIF导致BAE细胞的生长抑制(图4A),表明LIF在调节内皮功能中的复杂作用。为了研究BAE细胞中的LIF诱导的信号级联,使用巴瑞替尼、考比替尼和BEZ235抑制LIF-gp130:LIFR下游组分JAK1/2、MEK1/2和PI3K/mTOR。在BAE细胞中,LIF处理15分钟导致STAT3、ERK(MAPK)和AKT磷酸化(图4B)。巴瑞替尼预处理显著抑制LIF诱导的STAT3、ERK和AKT磷酸化,而考比替尼和BEZ235预处理也分别有效地抑制ERK和AKT的磷酸化(图4B)。有趣的是,巴瑞替尼是唯一一种能够逆转BAE细胞中被LIF诱导的生长抑制的抑制剂(图4C),表明JAK-STAT通路介导LIF在BAE细胞中的作用。为了进一步检查LIF对BAE细胞的抑制是否归因于JAK-STAT3级联,在BAE细胞中用3种不同的siRNA将STAT3敲下大约80%(图4D和4E)。有趣的是,BAE细胞中STAT3的敲下改善LIF的生长抑制(图4F)。这些观察结果表明,LIF-JAK-STAT3信号通路在调节内皮细胞生长方面可以发挥相反的作用,这取决于内皮细胞的类型。
LIF经由组织蛋白酶L依赖性细胞死亡和细胞周期停滞抑制BAE细胞生长
接下来检查通过LIF在BAE细胞中诱导哪些生长抑制作用(例如细胞周期停滞、细胞衰老或程序性细胞死亡)。由于IL-6-STAT3信号传导与细胞衰老密切相关(36-38),首先假设LIF-STAT3轴也诱导BAE细胞衰老。然而,在衰老相关的β-半乳糖苷酶测定中,未观察到用LIF处理48小时的BAE细胞中衰老细胞数量增加,这表明衰老不是被LIF在BAE细胞中引起的主要影响。有趣的是,细胞死亡标记物膜联蛋白V的染色显示,在用LIF处理24小时的BAE细胞中膜联蛋白V阳性的细胞比例增加(图6A和6B),表明LIF处理诱导细胞死亡。令人惊讶地,与半胱天冬酶抑制剂(Q-VD-OPH、Z-VAD-fmk和Z-DEVD-fmk)或聚(腺苷5'-二磷酸核糖)聚合酶(PARP)抑制剂(5-AIQ)共孵育无法拯救通过LIF诱导的细胞死亡表型。这些数据表明,在BAE细胞中,LIF介导的细胞死亡可能涉及半胱天冬酶非依赖性途径。为了调查LIF在BAE和BCE细胞中分化作用的分子基础,通过在与LIF孵育6小时的BAE和BCE细胞中的RNA-seq分析被LIF诱导/抑制的基因。值得注意的是,LIF处理在这两种细胞类型中导致不同的基因表达模式。具体地,IGFBP3,一种先前显示至少在某些情况下是血管生成抑制剂的分泌蛋白(39)在BAE细胞中上调大约8倍,但在BCE细胞中没有,该发现随后被qRT-PCR证实。然而,重组IGFBP3对BAE细胞生长没有影响。此外,在存在LIF中和抗体的情况下,用LIF处理72小时的BAE细胞的条件培养基不抑制BAE细胞的生长,这与LIF诱导的BAE生长抑制是由分泌因子介导的假设相反。先前有报道称STAT3可以经由溶酶体蛋白酶组织蛋白酶B和L的上调诱导非半胱天冬酶依赖性细胞死亡(40)。因此,研究LIF是否可能在BAE细胞中引发这种信号级联。有趣的是,通过在BAE细胞中处理24小时,通过LIF在mRNA和蛋白质水平上显著上调CTSL而不是CTSB(图6C和6D)。与CA074me,即一种拮抗组织蛋白酶B和L的抑制剂共同孵育,以CA074me的剂量依赖性方式减轻BAE细胞中的LIF诱导的生长抑制(图6E)。此外,另一种组织蛋白酶L特异性抑制剂CAA0225也抑制BAE细胞中的LIF诱导的生长抑制,尽管程度较小(图6F)。相比之下,即使在测试的最高剂量,即50μM下,组织蛋白酶B选择性抑制剂CA074也无法抑制BAE细胞中的LIF诱导的作用。有趣的是,无论细胞何时与载体或LIF一起孵育,qRT-PCR都无法检测到BCE细胞中的组织蛋白酶LmRNA(CTSL)水平。这些数据共同表明,LIF诱导的BAE细胞中组织蛋白酶L的上调,并且进而导致不依赖于半胱天冬酶的细胞死亡。此外,与LIF孵育48小时后,BAE细胞显示与载体对照相比显著减少的BrdU掺入(图7A和7B),表明LIF引发细胞周期停滞。该观点被LIF处理的BAE中细胞周期蛋白A/B下调的支持,但在BCE细胞中则不然(图7C)。
在实施方案中,本发明提供IL-6细胞因子例如LIF和CT-1的新的和出乎意料的活性以诱导血管生长,即血管生成。
例如,本发明提供LIF,一种先前已被表征为内皮细胞生长的抑制剂的分子,通过体外、离体和体内研究评估,在眼中具有出乎意料的促血管生成性质。
本发明提出LIF能够直接刺激脉络膜内皮细胞增殖,同时它抑制主动脉内皮细胞的生长,强调其对内皮细胞作用的特异性和独特性。当注射到小鼠玻璃体中时,LIF还促进脉络膜外植体的内皮发芽和血管生成。
LIF是一种特征明确的细胞因子,是IL6家族的成员。它与LIF受体相互作用,LIF受体继而与GP130形成异源二聚体,从而导致Stat3激活等效应。
本发明提供LIF可以促进内皮细胞亚群的生长,为在各种条件下进行治疗干预提供机会,包括视网膜/脉络膜、冠状动脉和心肌疾病中的低灌输(Reboucas等人,2016年;Simon-Yarza等人,2012年;Wang等人,2013年)。LIF不会诱导血管通透性的观察结果表明,施用该因子将避免与VEGF相关的不良血管渗漏(Niu等人,2016)。
本发明表明,IL-6家族成员如LIF和CT-1可以保护RPE免受损伤,包括氧化应激引起的损伤。这应当代表一种用于治疗与RPE损伤或变性相关的视网膜疾病的新的治疗策略。
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Claims (20)
1.一种治疗与受试者眼中的血管化不足相关的病症的方法,所述方法包括向有需要的受试者施用有效量的IL-6家族蛋白或其功能片段以促进血管生成。
2.根据权利要求1所述的方法,其中所述施用增加视网膜微血管密度。
3.根据权利要求1所述的方法,其中所述施用增加脉络膜内皮细胞的增殖。
4.根据权利要求1所述的方法,其中所述病症是年龄相关的黄斑变性。
5.根据权利要求1所述的方法,其中所述病症是早产儿视网膜病(ROP)。
6.根据权利要求1所述的方法,其中所述施用是经由玻璃体内注射。
7.根据权利要求1所述的方法,其中所述有效量不会诱导血管渗漏。
8.根据权利要求1所述的方法,其中所述有效量不会诱导水肿。
9.根据权利要求1所述的方法,其中所述IL-6家族蛋白是白血病抑制因子(LIF)。
10.根据权利要求1所述的方法,其中所述IL-6家族蛋白是心肌营养素-1(CT-1)。
11.一种诱导受试者眼中血管形成的方法,所述方法包括向有需要的受试者施用有效量的IL-6家族蛋白或其功能片段。
12.根据权利要求11所述的方法,其中所述施用增加视网膜血管生成。
13.根据权利要求10所述的方法,其中所述施用增加脉络膜内皮细胞的增殖。
14.根据权利要求10所述的方法,其中所述受试者患有年龄相关的黄斑变性。
15.根据权利要求10所述的方法,其中所述受试者患有早产儿视网膜病(ROP)。
16.根据权利要求10所述的方法,其中所述施用是经由玻璃体内注射。
17.根据权利要求10所述的方法,其中所述有效量不会诱导血管渗漏。
18.根据权利要求10所述的方法,其中所述有效量不会诱导水肿。
19.根据权利要求10所述的方法,其中所述IL-6家族蛋白是白血病抑制因子(LIF)。
20.根据权利要求10所述的方法,其中所述IL-6家族蛋白是心肌营养素-1(CT-1)。
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