CN112375735A - Crx、c-Myc、Nr2e1和Mitf-a转录因子组合的应用 - Google Patents
Crx、c-Myc、Nr2e1和Mitf-a转录因子组合的应用 Download PDFInfo
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Abstract
本发明提供Crx、c‑Myc、Nr2e1和Mitf‑a转录因子组合的应用,本发明还提供一种制备iRPE细胞的方法。本发明将四种因子组合,具有抗TGFβ诱导的EMT的功能,并且能够诱导脱分化RPE细胞分化为功能性RPE细胞。
Description
技术领域
本发明涉及一种Crx、c-Myc、Nr2e1和Mitf-a转录因子组合的应用,属于生物医药领域。
背景技术
年龄相关性黄斑变性(Age-related macular degeneration,AMD)是以视网膜色素上皮细胞(Retinal pigment epithelial cells,RPE)和视网膜光感受器细胞功能丧失而引起视功能损害为共同表现。我国流行病学调查显示,50岁以上人群中,AMD的发病率高达11.4%-15.6%。AMD往往会导致严重的视力损害,最终会致盲,从而严重地影响患者的生活质量,并成为家庭和社会的负担。尽管临床上已开展针对AMD的治疗,但仍缺乏有效的治疗和预防措施。
考虑到RPE细胞是单层细胞,且不需要同感光细胞一样要建立突触连接,因而临床上最早采用人眼中分离的RPE细胞来移植治疗以RPE病变为主的AMD,但限于取材困难,一直不能获得广泛的应用。胚胎干细胞(Embryonic stem cells,ESCs)和诱导多能性干细胞(Induced pluripotent stem cells,iPSCs)可以高效分化为RPE细胞,多个临床试验将ESCs和iPSCs来源的RPE细胞用于治疗AMD。但是研究显示,移植治疗的效果并不明显,主要的原因可能同RPE易发生上皮间质转化(Epithelial-mesenchymal transition,EMT)相关。
RPE细胞具有典型的上皮细胞结构,体内形成单层柱状六边形结构,并维持顶端到基底端的极性。EMT过程包含上皮细胞之间的胞间连接解体,粘附力降低,细胞失去顶端-基底端极性,获得前沿-后延极性,细胞骨架重塑,形态发生变化,获得间质细胞标记,迁移能力增强。在干性AMD起始阶段,RPE细胞即可发生病变并发生EMT,特别是随着AMD的进展,在地图样萎缩区域周边的RPE细胞发生不规则形态的改变,RPE细胞从Bruch膜上脱离,形成多层结构或迁移进入神经视网膜或进入Bruch膜,RPE细胞表达间质细胞标记分子,细胞极性丢失,上皮细胞功能减弱,获得较强的迁徙能力。而在湿性AMD中,在视网膜下腔及RPE下方会形成脉络膜新生血管组织,导致视网膜下纤维化组织形成。手术剥离的脉络膜新生血管膜组织中包含RPE细胞及RPE细胞来源的肌成纤维细胞,证实RPE细胞发生了EMT。一旦发生EMT,RPE细胞丧失极性,对感光细胞的支持功能降低,诱发感光细胞凋亡,促进疾病的发展,因而RPE细胞的EMT过程一直是治疗AMD的靶点。转化生长因子-β(Transforming growthfactor-β,TGFβ)信号通路是经典的诱导EMT的信号通路,是促进AMD疾病发展的重要细胞因子。TGFβ可以激活SMAD2/3的磷酸化,后者入核调控EMT相关蛋白的表达,诱导RPE细胞发生EMT。
发明内容
本发明的目的在于提供Crx、c-Myc、Nr2e1和Mitf-a转录因子组合的应用,从而为年龄相关性黄斑变性提供新的治疗方案。
本发明采用了如下技术方案:
本发明提供一种Crx、c-Myc、Nr2e1和Mitf-a转录因子的组合在制备诱导脱分化RPE细胞分化为成熟RPE细胞的试剂中的应用。
本发明还提供一种Crx、c-Myc、Nr2e1、Mitf-a转录因子组合在制备抗上皮间质转化的试剂中的应用。
上述的应用,还具有这样的特征:上皮间质转化指视网膜退行性疾病中体内上皮细胞的病理过程,所述上皮细胞包括视网膜色素上皮细胞。
本发明还提供过表达Crx、c-Myc、Nr2e1、Mitf-a转录因子组合的试剂在制备诱导间质上皮转化获得具有抗EMT功能的iRPE细胞的试剂中的应用。
本发明还提供Crx、c-Myc、Nr2e1和Mitf-a在制备抑制TGFβ诱导的SMAD2/3磷酸化及入核的试剂中的应用。
本发明还提供Crx、c-Myc、Nr2e1、Mitf-a转录因子的组合在制备减少间质细胞标记分子的表达的试剂中的应用。
本发明还提供Crx、c-Myc、Nr2e1、Mitf-a转录因子组合在制备具有视网膜保护作用的药物中的应用。
上述的应用,还具有这样的特征,上调细胞中Crx、c-Myc、Nr2e1、Mitf-a的量,或采用Crx、c-Myc、Nr2e1、Mitf-a蛋白。
本发明还提供一种制备iRPE细胞的方法,其特征在于,包括:
步骤一、制备包含Crx、c-Myc、Nr2e1或Mitf-a的逆转录病毒:将293FT细胞培养在细胞培养皿中,达到预定融合度时,每皿换成第一预定量的DMEM,添加10%FBS;然后配制脂质体和质粒的混合液:将10质量份连接有转录因子Crx、c-Myc、Nr2e1或Mitf-a的载体质粒、7.5质量份包装质粒pMXs-VSVG、3质量份包装质粒pMXs-G/P加到第二预定量的DMEM中吹打混匀,得到质粒混合液;将第三预定量的脂质体滴加到DMEM中,混匀,得到脂质体混合液,将配制好的质粒混合液滴加到脂质体混合液中,混匀;将脂质体和质粒的混合液滴加到293FT细胞,37℃培养第一预定时间后,每皿细胞换DMEM添加5%FBS的新鲜培液,第二预定时间后收集病毒上清液,过滤后-80℃冻存备用,用于感染目的细胞;
步骤二:转染细胞,将脱分化RPE细胞接种到培养皿中,用含有10%FBS的DMEM/F12培养,当细胞密度达到30%-50%时撤掉细胞培养液,加入制备的Crx、c-Myc、Nr2e1和Mitf-a病毒上清液,并加入第四预定量的polybrene;
步骤三、挑选iRPE细胞克隆:第三预定时间后撤掉病毒液,换成含有10%FBS的DMEM/F12培养液,第四预定时间后挑选iRPE细胞克隆。
进一步,本发明的制备iRPE细胞的方法,还具有这样的特征:所述脱分化RPE细胞为来源于iPS-RPE细胞、ESC-RPE细胞或人原代RPE细胞。
RPE细胞移植是治疗因RPE细胞损伤所致的AMD疾病的最优种子细胞,但疾病微环境中存在的EMT诱导因子可以诱导移植的RPE细胞发生EMT,一旦发生EMT,RPE细胞改变其功能,导致移植细胞丧失治疗作用。采用Crx、c-Myc、Nr2e1、Mitf-a转录因子组合将脱分化的RPE细胞诱导分化为具有抗EMT功能的RPE细胞的方法,完全克服病变微环境对RPE细胞的EMT诱导作用。该方法适合于iPSC-RPE细胞、ESC-RPE细胞及人自体RPE细胞,我们将其转化为iRPE细胞为临床治疗AMD提供功能更强的供体细胞。
发明的有益效果:Crx被证明是感光细胞特异性转录因子,c-Myc在RPE细胞中表达,Nr2e1是一个孤儿核受体,调节pax2的表达,促进视觉发育,Mitf-a能够调节RPE细胞多个功能蛋白的表达,采用这四种转录因子组合诱导脱分化RPE细胞发生间质上皮转化未见报道,而采用这四种转录因子诱导获得的iRPE细胞具有抗TGFβ诱导的EMT的功能也未见报道。
本发明证实将四种因子组合,具有抗TGFβ诱导的EMT的功能。并且能够诱导脱分化RPE细胞分化为功能性RPE细胞。
附图说明
图1显示Crx、c-Myc、Nr2e1、Mitf-a转录因子组合诱导脱分化的iPSC-RPE细胞分化为iPSC-iRPE细胞。
其中,图1A:iPSC-iRPE细胞具有多边形结构,表达RPE特异性标记分子RPE65和Cralbp,不表达间质标记分子FN1及α-SMA,F-actin呈环形带状。
图1B:Q-PCR结果显示iPSC-iRPE细胞高表达RPE特异性标记分子pedf、cralbp、mertk、rpe65、bestrophin和tyrosinase,不表达间质标记分子fn1及α-sma。
图1C:iPSC-iRPE细胞具有吞噬感光细胞外节盘膜的作用。
图1D:iPSC-iRPE细胞吞噬感光细胞外节盘膜的定量数据。
图1E:iPSC-iRPE细胞长期培养后能够合成色素颗粒。
图2显示iPSC-iRPE细胞抗TGF-β1和TGF-β2诱导的EMT的功能。
其中,图2A:iRPE细胞可以抵抗TGF-β1和TGF-β2的EMT诱导作用,维持多边形结果,抑制p-SMAD2/3入核。
图2B:Westernblot结果显示iRPE细胞中SMAD2/3磷酸化受到抑制。图2C:iRPE细胞中SMAD2/3磷酸化的定量数据。
图3显示脱分化人RPE细胞来源的iRPE细胞抗TGF-β1和TGF-β2诱导的EMT的功能。
图4显示脱分化人ESC-RPE细胞来源的ESC-iRPE细胞抗TGF-β1和TGF-β2诱导的EMT的功能。
图5显示iPSC-iRPE细胞体内移植对视网膜的保护作用。
其中,图5A:移植iPSC-iRPE细胞的RCS大鼠具有更强的ERG电生理反应。
图5B:ERG的b波振幅定量数据。
图5C:移植iPSC-iRPE细胞的RCS大鼠能够维持更多的外核层厚度。
图5D:iPSC-iRPE细胞对RCS大鼠外核层厚度维持的定量数据。
图6.iPSC-iRPE细胞体内抗EMT的功能。移植的细胞仍然表达RPE细胞的特异性标记分子RPE65,不表达间质细胞标记分子α-SMA。
具体实施方式
以下结合附图来说明本发明的具体实施方式。
以下实施例中,293FT购自ATCC。iPSC-RPE细胞、ESC-RPE细胞由本实验室从iPSC细胞和ESC细胞分化获得,人原代RPE细胞由本实
验室从捐献的眼球分离获得。
实施例1.制备包含Crx、c-Myc、Nr2e1或Mitf-a的逆转录病毒,转染细胞,采用克隆环挑选iRPE细胞克隆。
病毒制备:
采用的是Invitrogen公司的lipofectamine 2000脂质体,主要步骤如下:293FT细胞培养在10cm的细胞培养皿中,70%融合时,提前3小时每皿换成10ml DMEM(含4.5g葡糖糖/L)添加10%FBS;接着配制脂质体和质粒的混合液:将10μg连接有转录因子Crx、c-Myc、Nr2e1或Mitf-a的载体质粒、7.5μg包装质粒pMXs-VSVG、3μg包装质粒pMXs-G/P加到500μl的DMEM(含4.5g葡糖糖/L)中吹打混匀;将41μl脂质体轻轻滴加到500μl DMEM(含4.5g葡糖糖/L)中,颠倒混匀,静置5分钟,将配制好的质粒混合液滴加到脂质体混合液中,颠倒混匀,静置15分钟;将混合液滴加到293FT细胞,轻轻摇匀,37℃培养4小时后,每皿细胞换15ml DMEM(含4.5g葡糖糖/L)添加5%FBS的新鲜培液,60h后收集病毒上清液,0.45μm针式过滤器过滤,-80℃冻存备用,用于感染目的细胞。
转染细胞:
将脱分化RPE细胞(iPSC-RPE细胞、ESC-RPE细胞及人原代RPE细胞)接种到10cm培养皿中,用含有10%FBS和100U/mL青霉素/100mg/mL链霉素的DMEM/F12培养,当细胞密度达到30%-50%时撤掉细胞培养液,加入制备的Crx、c-Myc、Nr2e1和Mitf-a病毒液上清,每个病毒液2ml,并加入8μg/mL polybrene;12小时以后撤掉病毒液,换成含有10%FBS和100U/mL青霉素/100mg/mL链霉素的DMEM/F12新鲜培养液,观察细胞形态的变化,7天后挑选iRPE细胞克隆,传代培养。
组织学染色:
采用4%PFA固定细胞10min,3%BSA室温封闭1小时,加3%BSA稀释的抗人ZO-1、RPE65、Cralbp、FN1、α-SMA等一抗(abcam公司),置于湿盒内防止干燥,4℃孵育过夜。PBS洗3次,每次5分钟;加3%BSA稀释的荧光素标记的的二抗,置于湿盒内,避光4℃孵育过夜,PBS洗3次,每次5分钟;用0.5μg/ml DAPI染细胞核5分钟,用荧光封片剂封片后,在荧光显微镜下观察。
定量PCR检测iRPE细胞基因的表达:
提取RNA采用的是Trizol裂解的方法,主要步骤如下:
1.将细胞用PBS缓冲液洗1-2次,按比例加入Trizol裂解液,例如六孔板的一个孔对应1mL裂解液。
2.细胞离壁后,转移到1.5mL的离心管,加入五分之一体积的氯仿,剧烈混匀后,以12000rpm的转速4℃离心15分钟。
3.离心后,将上清转移到新的离心管中,注意不要取到中间的蛋白层,加入等体积的异丙醇,冰浴20分钟。
4. 12000rpm的转速4℃离心15分钟,弃去上清,将沉淀用75%的乙醇洗1-2次。
5.将沉淀室温干燥后,用适量的DEPC水溶解。测浓度。
RNA反转录主要步骤如下:
反转体系为:5×RT Master Mix 8μl,RNA 1μg,ddH2O补至20μl。反转的PCR条件为:37℃,15min;85℃,5s,4℃保存。
引物见下表1。
表1用于检测基因的引物
定量PCR步骤如下:
将RNA反转录后得到的cDNA第一链作为模板,设计引物。利用天根公司的SYBRGreen实时荧光定量PCR检测试剂盒,检测目的基因的表达量。PCR扩增条件如下:94℃变性10分钟,进入循环(95℃5秒,60℃60秒),一共40个循环,并收集溶解曲线。
结果参见图1A和1B。图1A显示Crx、c-Myc、Nr2e1和Mitf-a可诱导脱分化的iPSC-RPE细胞分化为具有多边形结构的iPSC-iRPE细胞,表达RPE细胞特异性标记分子RPE65和Cralbp,不表达间质标记分子FN1及α-SMA,F-actin呈环形带状。图1B显示iPSC-iRPE细胞高表达RPE特异性标记分子pedf、cralbp、mertk、rpe65、bestrophin和tyrosinase,不表达间质标记分子fn1及α-sma。
iPSC-iRPE细胞对POS吞噬功能的检测步骤如下:
将iPSC-iRPE细胞接种到预置在培养皿中的玻片上,当细胞形成融合单层时,将用Biotin标记好的POS加入细胞培养基中,37℃孵育4小时,用PBS清洗3次。经4%多聚甲醛固定,加3%BSA稀释的抗人的ZO-1一抗,置于湿盒内防止干燥,4℃孵育过夜;加3%BSA稀释的FITC标记的二抗以及CY3标记的Avidin,置于湿盒内,4℃孵育过夜;用荧光封片剂封片后,在激光共聚焦显微镜下观察。ZO-1被染成绿色,POS被生物素标记成红色。
结果参见图1C和1D。图1C和1D显示iPSC-iRPE细胞吞噬更多的POS,显著高于脱分化的RPE细胞。
细胞色素产生的步骤如下:
iPSC-iRPE细胞培养在DMEM/F12添加N2B27的培养基中,隔天换液,培养2个月。
结果参见图1E。图1E显示iPSC-iRPE细胞能够产生黑色素,类似于成熟RPE细胞。
实施例2.iPSC-iRPE细胞抗TGFβ1和TGFβ2诱导的EMT的功能确定。
将iPSC-iRPE细胞接种到6孔板中,用含有10%FBS的DMEM/F12培养,每两天换一次液,当细胞达到60%融合时,换成无血清的DMEM/F12饥饿培养细胞12小时,在TGF-β1或TGF-β2刺激下,Smad2/3的磷酸化及入核验证:在细胞被处理1h后,多聚甲醛固定,进行免疫荧光染色检测及Westernblot检测。同时,分别用10ng/ml的TGF-β1或TGF-β2处理2-8天,观察细胞EMT的进展情况。
结果参见图2A和2B。图2A显示iPSC-RPE细胞在TGF-β1或TGF-β2处理下,磷酸化Smad2/3迅速入核,而iRPE细胞未发生Smad2/3的磷酸化及入核现象。iPSC-RPE细胞在TGF-β1或TGF-β2处理下发生EMT,而iPSC-iRPE细胞仍然维持多边形结构。说明iPSC-iRPE细胞可以抵抗TGFβ1和TGFβ2的EMT诱导作用,维持多边形结构。图2B显示TGF-β1和TGF-β2均能促进Smad2/3在iPSC-RPE细胞中的磷酸化,但在iPSC-iRPE细胞中,Smad2/3的磷酸化水平几乎没有变化,证实iPSC-iRPE细胞具有抗EMT的功能。
实施例3.人原代培养的RPE细胞来源的hiRPE细胞抗TGF-β1和TGF-β2诱导的EMT的功能确定。
实验处理方案同实施例2相同。
结果参见图3A。图3A显示人源RPE细胞在TGF-β1或TGF-β2处理下,磷酸化Smad2/3迅速入核,而hiRPE细胞未发生Smad2/3的磷酸化及入核现象。人源RPE细胞在TGF-β1或TGF-β2处理下发生EMT,而hiRPE细胞仍然维持多边形结构。证实hiRPE细胞也具有抗EMT功能。
实施例4.ESC细胞来源的ESC-iRPE细胞具有抗TGF-β1和TGF-β2诱导的EMT的功能确定。
实验处理方案同实施例2相同。
结果参见图4A.图4A显示ESC-RPE细胞在TGF-β1或TGF-β2处理下,磷酸化Smad2/3迅速入核,而iRPE细胞未发生Smad2/3的磷酸化及入核现象。ESC-RPE细胞在TGF-β1或TGF-β2处理下发生EMT,而ESC-iRPE细胞仍然维持多边形结构。
实施例5.iPSC-iRPE细胞体内移植对视网膜的保护作用
视觉电生理检查:
视网膜下腔移植3×105细胞,在不同时间点用视觉电生理(ERG)检测来评估细胞移植的治疗效果。方法如下:RCS大鼠暗适应过夜(12h-16h),腹腔注射20%的戊巴比妥钠麻醉,用速眠新进行肌肉松弛,0.5%托吡卡胺散瞳和0.4%盐酸奥布卡因麻醉眼表,等大鼠瞳孔扩散后,随后连接电极,角膜电极接触大鼠角膜,参考电极合并后连接在大鼠两耳之间的皮下,接地电极接于大鼠尾部皮下。依据视觉电生理仪器厂家(重庆康华)提供的仪器使用说明操作仪器,用6.325e-2cd×s/m2的光强来刺激大鼠,采集到的信号,统计b波振幅来评价视觉功能的好坏。整个记录过程都在完全黑暗的房间内完成,只能采用红光照明。
结果参见图5A和5B.图5A显示细胞移植2周后,iPSC-RPE细胞和iPSC-iRPE细胞均具有保护作用给,但iPSC-iRPE具有更强的保护作用。在移植6周后,iPSC-RPE细胞丧失保护作用,ERG的b波振幅降低到和PBS组没有差异,而移植iPSC-iRPE细胞组,ERG的b波振幅仍然同PBS组及ES-RPE组存在显著差别。
细胞移植后核层厚度的检测:
细胞移植后第六周,收集的眼球样品制备冰冻切片,用0.5μg/ml DAPI染细胞核,用荧光封片剂封片后采用荧光显微镜拍摄整个视网膜的结构,在鼻侧和颞侧各取10个不同位置,对视网膜的外核层(ONL)进行核层厚度的测量。
结果参见图5C和D.图5C和D显示iPSC-RPE细胞在第6周时已经丧失对视网膜外核层的保护,同PBS组相似仅剩1-2层细胞,而iPSC-iRPE细胞组中外核层仍然维持4-5层细胞,证实iPSC-iRPE细胞对视网膜的保护作用。
iPSC-iRPE细胞体内抗EMT功能:
细胞移植后第2周,收集的眼球样品制备冰冻切片,加3%BSA稀释的抗人RPE65、α-SMA等一抗,置4℃孵育过夜。加3%BSA稀释的荧光素标记的二抗,置4℃孵育12h;用0.5μg/mL DAPI染细胞核,用荧光封片剂封片后,在荧光显微镜下观察。
结果参见图6.图6显示移植的iPSC-RPE细胞移植后第2周时已经不表达RPE65细胞,但表达间质细胞标记分子α-SMA,而移植的iPSC-iRPE细胞仍然表达RPE65,且不表达α-SMA,证实iPSC-iRPE细胞体内具有抗EMT的功能。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 上海优祺生物医药科技有限公司
<120> Crx、c-Myc、Nr2e1和Mitf-a转录因子组合的应用
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Claims (10)
1.一种Crx、c-Myc、Nr2e1和Mitf-a转录因子的组合在制备诱导脱分化RPE细胞分化为成熟RPE细胞的试剂中的应用。
2.一种Crx、c-Myc、Nr2e1、Mitf-a转录因子组合在制备抗上皮间质转化的试剂中的应用。
3.根据权利要求1或权利要求2所述的应用,其特征在于,所述上皮间质转化指视网膜退行性疾病中体内上皮细胞的病理过程,所述上皮细胞包括视网膜色素上皮细胞。
4.过表达Crx、c-Myc、Nr2e1、Mitf-a转录因子组合的试剂在制备诱导间质上皮转化获得具有抗EMT功能的iRPE细胞的试剂中的应用。
5.Crx、c-Myc、Nr2e1和Mitf-a在制备抑制TGFβ诱导的SMAD2/3磷酸化及入核的试剂中的应用。
6.Crx、c-Myc、Nr2e1、Mitf-a转录因子的组合在制备减少间质细胞标记分子的表达的试剂中的应用。
7.Crx、c-Myc、Nr2e1、Mitf-a转录因子组合在制备具有视网膜保护作用的药物中的应用。
8.根据权利1-7中任意一项所述的应用,其特征在于,上调细胞中Crx、c-Myc、Nr2e1、Mitf-a的量,或采用Crx、c-Myc、Nr2e1、Mitf-a蛋白。
9.一种制备iRPE细胞的方法,其特征在于,包括:
步骤一、制备包含Crx、c-Myc、Nr2e1或Mitf-a的逆转录病毒:将293FT细胞培养在细胞培养皿中,达到预定融合度时,每皿换成第一预定量的DMEM,添加10%FBS;然后配制脂质体和质粒的混合液:将10质量份连接有转录因子Crx、c-Myc、Nr2e1或Mitf-a的载体质粒、7.5质量份包装质粒pMXs-VSVG、3质量份包装质粒pMXs-G/P加到第二预定量的DMEM中吹打混匀,得到质粒混合液;将第三预定量的脂质体滴加到DMEM中,混匀,得到脂质体混合液,将配制好的质粒混合液滴加到脂质体混合液中,混匀;将脂质体和质粒的混合液滴加到293FT细胞,37℃培养第一预定时间后,每皿细胞换DMEM添加5%FBS的新鲜培液,第二预定时间后收集病毒上清液,过滤后-80℃冻存备用,用于感染目的细胞;
步骤二:转染细胞,将脱分化RPE细胞接种到培养皿中,用含有10%FBS的DMEM/F12培养,当细胞密度达到30%-50%时撤掉细胞培养液,加入制备的Crx、c-Myc、Nr2e1和Mitf-a病毒上清液,并加入第四预定量的polybrene;
步骤三、挑选iRPE细胞克隆:第三预定时间后撤掉病毒液,换成含有10%FBS的DMEM/F12培养液,第四预定时间后挑选iRPE细胞克隆。
10.如权利要求9所述的制备iRPE细胞的方法,其特征在于:
所述脱分化RPE细胞为来源于iPSC-RPE细胞、ESC-RPE细胞或人原代RPE细胞。
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