CN117625536B - 一种人视网膜色素上皮细胞的纯化、培养方法 - Google Patents
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Abstract
本发明属于生物医药领域,具体涉及一种视网膜色素上皮细胞的纯化、培养方法。更具体地,本发明提供了ITGAV的特异性结合试剂在纯化和/或制备视网膜色素上皮细胞中的应用,通过ITGAV的特异性结合试剂纯化得到的细胞具有提升视网膜功能的功能。
Description
技术领域
本发明属于生物医药领域,具体涉及一种人视网膜色素上皮细胞的纯化、培养方法。
背景技术
视网膜退行性疾病是可导致患者视力不可逆性损伤的一类疾病,该疾病以视网膜色素上皮(retinal pigment epithelium,RPE)细胞及光感细胞进行性破坏及缺失为主要特点,主要包括年龄相关性黄斑变性(age-related macular degeneration,AMD)、视网膜色素变性(retinitis pigmentosa,RP)及Stargardt黄斑营养不良(SMD),其中AMD是老年人中主要的致盲疾病,而SMD好发于青少年。视网膜退行性疾病的发病机制决定了细胞替代疗法可能是一种可以从病因上控制和治疗该类疾病的方法。
视网膜色素上皮细胞移植是治疗年龄相关性黄斑变性及视网膜色素变性非常有前景的方案,目前在实验室模型动物以及临床前期实验中取得较好的效果。目前用于移植的视网膜色素上皮来源主要依赖于胚眼、人胚胎干细胞及诱导多能干细胞分化来源。胚眼来源的细胞数量有限,而且由于伦理限制,限制了产业化的应用。
多能干细胞体外分化来源的这些组织或者分化的细胞群体中视网膜色素上皮的产量较低,需要进行分离纯化。目前分离、纯化视网膜色素上皮的方案主要通过物理观察,从多能干细胞二维或者三维诱导分化的细胞群体中挑取带有黑色素的细胞团体。然而这种分离方式过度依赖于人工挑取,而且批次间差异较大;较为严重的问题是细胞纯度低,还不能充分去除未分化的细胞,存在潜在的成瘤风险,这些都是限制其产业化应用的重要瓶颈问题。
发明内容
为解决视网膜色素上皮细胞(RPE)难以被产业化应用的技术问题,本发明通过对8万个多能干细胞体外3D诱导分化来源的类视网膜组织的细胞悬液进行单细胞转录组测序,并通过生物信息学挖掘,鉴定出人视网膜色素上皮细胞特异的表面标记物ITGAV,并针对该分子使用单克隆抗体,偶联磁珠后,多能干细胞三维诱导分化的类视网膜组织及多能干细胞二维自分化细胞群体进行磁力筛选(MACS),有效去除混杂细胞,获取高纯度的视网膜色素上皮细胞。并且,针对不同发育阶段的人视网膜色素上皮细胞给与系列培养基支持,进行视网膜变性模型RCS大鼠视网膜下腔移植后,可显著改善动物的电生理功能。使用我们的细胞分化及培养体系,可获取高纯度、高质量的种子细胞,并可在液氮内保存,可根据需要随时复苏,促进人视网膜色素上皮的产业化应用。
具体技术方案如下:
第一方面,本发明提供了ITGAV的特异性结合试剂在纯化和/或制备视网膜色素上皮(retinal pigment epithelium,RPE)细胞中的应用。
优选地,所述视网膜色素上皮(retinal pigment epithelium,RPE)细胞是人的视网膜色素上皮细胞。
另一方面,本发明提供了ITGAV的特异性结合试剂在制备视网膜色素上皮细胞移植产品中的应用。
优选地,所述特异性结合试剂包括抗体、适配体、探针等。
最优选地,所述特异性结合试剂是抗体。
优选地,所述抗体包括Fab片段、Fab’片段、F(ab’)片段、双抗体或scFv。
本发明所述术语“抗体”包括涉及任何同型或子类的糖基化和非糖基化免疫球蛋白或涉及其与完整抗体竞争特异性结合的抗原结合区,包括人类、人源化、嵌合、多特异性、单克隆、多克隆抗体和其寡聚物或抗原结合片段。还包括具有抗原结合片段或区域的蛋白,例如Fab、Fab’、F(ab’)2、Fv、双体抗体、Fd、dAb、大型抗体、单链抗体分子、互补决定区(CDR)片段、scFv、二体抗体、三体抗体、四体抗体和多肽,其含有至少一部分足以赋予与标靶多肽的特异性抗原结合的免疫球蛋白。所述抗体的生产方法包括但不限于通过重组手段制备、表达、产生或分离的抗体,诸如从被转染以表达抗体的宿主细胞分离的抗体。
本发明所述ITGAV也可称Integrin Subunit Alpha V或整联蛋白αV,其EnsemblID为ENSG00000138448。
另一方面,本发明提供了一种制备视网膜色素上皮细胞的方法,所述方法包括通过ITGAV的特异性结合试剂分选视网膜色素上皮细胞。
优选地,所述分选所针对的细胞是胚胎干细胞(embryonic stem cells,ESC)或者诱导多能干细胞(induced pluorescent stem cells,iPSCS)诱导分化所得到的类视网膜组织的色素团细胞。
更优选地,所述方法还包括诱导胚胎干细胞分化出类视网膜组织的色素团细胞的步骤。
更具体地,所述方法是将ITGAV的特异性结合试剂与磁珠偶联,通过磁力柱筛选出ITGAV表达的细胞。
另一方面,本发明提供了前述方法所制备得到的细胞或高表达ITGAV的RPE细胞在制备治疗眼科疾病的药物、RPE移植产品中的应用。
另一方面,本发明提供了一种治疗眼科疾病的方法,所述方法包括向患者施用上述方法所制备得到的细胞。
具体地,所述施用方式包括视网膜下腔注射。
优选地,所述眼科疾病包括视网膜退行性疾病,或,RPE细胞异常而导致的眼科疾病。
更具体地,所述眼科疾病包括年龄相关性黄斑变性(age-related maculardegeneration,AMD)、视网膜色素变性(retinitis pigmentosa,RP)、Stargardt黄斑营养不良(Stargardt macular degeneration,SMD)等。
更具体地,所述AMD疾病可分为非渗出性AMD,也称干性AMD(dry-AMD),和渗出性AMD,也称湿性AMD(wet-AMD)。
另一方面,本发明提供了包含前述方法制备得到的细胞的组合物。
优选地,所述组合物中还可以包括药学上可接受的载体,所述可作为药学上可接受的载体的具体例子包括糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如羧甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸;乳化剂,如润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。本发明的组合物可根据需要制成各种剂型,并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
本发明所述“治疗”包括通过所述方法制备到的细胞来阻止或延缓疾病(如肿瘤)的症状和并发症的出现。治疗也可以是预防性。对肿瘤的治疗也指在个体控制肿瘤的进展,延长肿瘤患者的生存期,改善生活质量,减轻症状,使肿瘤缩小甚至消除,使肿瘤转移受到遏制。将所述方法制备到的细胞进行视网膜下腔注射后可以有效的提升视网膜功能。
本发明所述治疗所针对的”受试者”包括任何动物(例如,哺乳动物),包括但不限于人、非人灵长类动物、啮齿类动物等,落在本发明的范围内的合适的动物包括灵长目动物、啮齿动物(例如,小鼠、大鼠、豚鼠)、兔形目动物(例如,家兔、野兔)、牛科动物(例如,牛)、绵羊类动物(例如,绵羊)、山羊类动物(例如,山羊)、猪类动物(例如,猪)、马科动物(例如,马)、犬科动物(例如,狗)、猫科动物(例如,猫)、鸟类动物(例如,鸡;鸭;鹅;陪伴鸟类,诸如金丝雀、虎皮鹦鹉等)、海洋哺乳动物(例如,海豚、鲸鱼)、爬行动物(例如,蛇、青蛙、蜥蜴等)和鱼。其将成为特定治疗的接受者。通常,术语“受试者”和“患者”在涉及人受试者时在本文中可互换地使用。
优选地,所述受试者是人。
另一方面,本发明提供过了检测ITGAV表达量的试剂在判断目标细胞群体中视网膜色素上皮(RPE)细胞含量中的应用。
优选地,所述目标细胞群体是眼细胞,更优选地,是本发明具体实施例所诱导分化出的类视网膜组织的色素团细胞。
优选地,所述表达量包括蛋白表达量和mRNA表达量。
优选地,所述mRNA表达量的检测试剂是本领域公知的,包括但不限于特异性结合目标序列的核酸探针、扩增目标序列的引物、非特异性荧光染料(例如,SYBR Green I)或其组合。
优选地,所述蛋白表达量的检测试剂包括免疫学检测所需的试剂;所述免疫学检测包括ELISA检测、Elispot检测、Western印迹或表面等离子共振法。免疫学检测所需要的试剂是本领域熟知的,包括但不限于能够与ITGAV特异性结合的抗体、靶向多肽。
具体地,所述ITGAV表达量高则代表RPE细胞的含量高。
附图说明
图1是hESC相差显微镜图像。
图2是hESCs标志物免疫荧光染色的图像
图3是hESC体外二维分化为视网膜色素细胞的相差显微镜图像。
图4是 hESC体外三维诱导分化为第18-126天类视网膜组织的黑色素团细胞相差显微镜图像
图5是特异性细胞膜表达的蛋白ITGAV在RPE细胞中的表达情况。
图6是类视网膜组织的RPE细胞特异性表达ITGAV蛋白的免疫荧光染色结果图。
图7是MACS分选后RPE细胞的相差显微镜图像。
图8是MACS分选纯化的RPE细胞的标记物免疫荧光染色鉴定结果图。
图9是MACS分选纯化的RPE细胞移植入RCS大鼠视网膜下腔后8W的电生理检测的结果图。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1、RPE细胞表面标记物ITGAV的筛选
步骤1、hESC体外分化
(1)hESC进行体外二维培养,hESC传代接种至Vitronectin(VTN,A14700,ThermoFisher,US)预包被的6孔板内,待细胞完全长满后,更换为分化培养基,18天开始,即可出现黑色素颗粒的群落,StemPro™Accutase™细胞解离试剂消化细胞进行后续实验操作。hESC二维自发分化为色素细胞时在 DIC显微镜下观察可见部分色素颗粒细胞聚集(图3)。
(2)hESC进行体外三维培养,hESC消化解离,使用DMEM+IMDM+10%KSR分化培养液重悬细胞并以10000个/孔的细胞 接种至U型孔低吸附96孔板内,6天后加入BMP4,每3天半量换液,18天时转移到低吸附的直径为10cm的Petri dish中,培养基更换为DMEM/F12+10%KSR+N2+RA+Taurine培养基,3天换液一次,18天时即可观察到类视网膜的黑色素团细胞聚集(图4)。图4显示hESC 经体外三维诱导分化为类视网膜组织的图像。
步骤2、hESC体外分化的细胞进行单细胞测序及生物信息学分析
选取hESC分化不同时间点36d-186d的不同时间点的类视网膜组织进行细胞构成的解析及各类细胞的分子生物学鉴定,通过R package筛选出RPE细胞的特异性细胞膜表达的蛋白ITGAV,其他细胞群体未见该分子转录表达(图5)。
步骤3、验证RPE细胞表面标记物ITGAV
通过分化后30d的类视网膜组织进行免疫免疫荧光染色,发现ITGAV蛋白特异性在RPE细胞胞浆及胞膜表达(图6)。
实施例2、通过ITGAV磁力分选纯化RPE细胞
收集hESC分化后30d的类视网膜组织的色素团细胞,维纳斯剪刀在显微镜下将类视网膜组织剪碎,加入胶原酶I/Ⅳ(10mg/ml,Sigma)和木瓜蛋白酶(10mg/ml,Sigma),巴氏吸管吹打后在37℃水浴锅内进行消化,每隔5分钟充分吹打30次,30分钟后加入培养基终止消化,并在1200rpm离心,去上清,加入PBS溶液洗涤细胞后再次离心,去上清,并使用1mlPBS进行重选后,加入正常FBS室温封闭15min后,加入藕连磁珠的Mouse-anti-ITGAV单抗(1:200稀释)在4℃冷室内进行孵育15min,PBS洗涤细胞,过磁力柱分选ITGAV+细胞,并进行体培养,显微镜下观察细胞呈现典型的铺路石样结构,并且具备黑色素颗粒的形态(图7)。
收集hESC分化后30d的类视网膜组织以及分选后的RPEITGAV+细胞,4%多聚甲醛固定,免疫荧光染色发现RPEITGAV+细胞表达RPE细胞经典标记物ZO1、RPE65(图8)。
实施例3、RPE细胞视网膜下腔移植,检测RCS大鼠视功能改善
RCS大鼠是经典的视网膜色素变性模型动物,该动物由于视网膜色素上皮细胞功能异常,导致视网膜感光细胞逐渐变性、凋亡。RPEITGAV+细胞(实施例2所制备得到的细胞)消化成单细胞悬液后,按照5微升/只(1*108/ml)的细胞量进行视网膜下腔注射,注射后8W进行电生理检测,与PBS对照组比较,发现注射RPE细胞组的视网膜功能有显著提升(图9)。
Claims (6)
1.ITGAV的特异性结合试剂在纯化和/或制备视网膜色素上皮细胞、制备视网膜色素上皮细胞移植产品中的应用。
2.如权利要求1所述应用,所述特异性结合试剂包括抗体、适配体或探针。
3.如权利要求1所述应用,所述特异性结合试剂是抗体。
4.如权利要求3所述应用,所述抗体包括Fab片段、Fab’片段、F(ab’)片段、双抗体或scFv。
5.一种制备视网膜色素上皮细胞的方法,所述方法包括通过ITGAV的特异性结合试剂分选视网膜色素上皮细胞。
6.如权利要求5所述方法,所述特异性结合试剂包括抗体、适配体、探针。
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