CN116536260A - 一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法 - Google Patents
一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法 Download PDFInfo
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Abstract
本发明公开一种JAK/STAT信号通路抑制剂预处理hESC‑RPE细胞的方法,包括在移植前体外条件下,通过Ruxolitinib的预处理,阻断hESC‑RPE细胞的JAK1磷酸化过程,从而抑制细胞JAK1‑STAT通路的信号转导,以调控移植后hESC‑RPE细胞对依赖JAK‑STAT信号通路的细胞因子的应答,通过调控干细胞中关键的细胞因子信号通路,在不需要系统性使用免疫抑制剂的情况下,改善干细胞产品在宿主体内的存活和功能;这不仅有助于推进干细胞产品在视网膜变性疾病治疗领域的临床转化,也为解决其他异基因移植免疫排斥反应提供一类新方法,具有优秀的临床应用前景。
Description
技术领域
本发明涉及细胞生物学技术领域,具体为一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法。
背景技术
视网膜变性疾病主要指年龄相关性黄斑变性、视网膜色素变性和Stargardt’s综合征等一系列致盲性眼病,每年我国因此类疾病而失明的患者新增达20余万人,对国民视觉健康构成严重威胁,对的社会经济造成明显负担。病变累及包括感光细胞及色素上皮细胞在内的多种视网膜功能细胞,临床表现为进行性视力降低、视野缩窄、夜盲等。由于上述细胞自身缺乏再生修复能力,病理进程不可逆转,目前尚无明确有效的药物可以改善视力或延缓病情。近年来新兴的细胞替代疗法为视网膜变性疾病患者带来了曙光,这种疗法将外源性的种子细胞移植到患者玻璃体腔、视网膜下腔等位置,补充替代原本受损的视网膜功能细胞以挽救患者的视觉功能。由于干细胞具备强大的自我更新以及多向分化能力,成为了细胞替代疗法中种子细胞的最佳来源。当今再生医学领域大量涌现干细胞及其衍生物向临床转化的研究成果,干细胞替代治疗策略已经成为了治疗视网膜变性疾病最有希望的方向。
由于干细胞独特的表型:(1)低表达HLA-Ⅰ/Ⅱ这类重要的抗原信号,可逃避淋巴细胞的识别和攻击;(2)几乎不表达ICAMs、B7及CD40等共刺激分子,限制了其对淋巴细胞的趋化和激活作用;(3)通过分泌TGF-β、IL-10细胞因子,表达CTLA-4、PD-1等负向调节抗原而诱导免疫耐受;常规的体外培养情况下,干细胞的免疫原性较低。但在细胞替代治疗的临床应用中,被输送到受体内的干细胞及其衍生物仍会发生显著的免疫排斥反应,主要缘于以下两部分原因:(1)干细胞来源的功能细胞自身还保持着一定程度的干细胞特性,容易在外界因素的调控下改变表型,包括免疫原性;(2)当疾病发生后,组织微环境中原本的免疫平衡被打破,呈现出免疫细胞浸润和激活的“炎症状态”,组织中的细胞因子水平发生剧烈改变,穿刺移植、注射手术等治疗手段又可进一步刺激组织炎症的发生。输送到病变组织的干细胞在微环境的作用下诱导表达上述免疫活化抗原,并激活受体的免疫排斥反应。
为减少免疫排斥反应,临床上目前主要以全身使用传统免疫抑制剂来保护移植后的种子细胞。这些免疫抑制剂包括糖皮质激素、钙神经磷酸酶和抗代谢药物等,这些药物对机体免疫系统的作用缺乏特异性和选择性,既可抑制免疫病理过程,又会干扰机体正常的免疫应答,极大地增加了肿瘤和感染发生的概率,还可对肝、肾等重要脏器带来不可逆的损害。此外,研究发现多种免疫抑制剂均呈现出对干细胞的毒性作用,导致其增殖降低、凋亡增加、迁移及代谢能力受损等,严重影响干细胞进入体内后发挥功能。
因而,干细胞的临床转化亟待开发一种新型的免疫调节方式取代传统的免疫抑制剂。我们认为,从干细胞供体方面进行免疫表型的干预是较为合理的解决途径,即:在不影响种子细胞正常功能的前提下,通过调控其对微环境中关键细胞因子的应答来阻断干细胞免疫原性的上调,可有效减少移植后受体的对干细胞的免疫排斥反应。
Janus激酶(Janus kinases,JAKs)是一种非受体型酪氨酸蛋白激酶,大多数重要细胞因子包括干扰素(IFN-γ)、白介素、粒细胞/巨噬细胞集落刺激因子等,都可通过JAKs的磷酸化、激活信号转导子和转录激活子(signal transducers and activators oftranscription,STATs)并启动下游靶基因,进而调控细胞的免疫学表型和其他细胞功能。本发明拟通过药物抑制干细胞产品JAK-STAT信号通路的激活,减少免疫微环境中细胞因子的作用而维持较低的免疫原性,改善细胞存活、促进细胞整合,以期延长干细胞在治疗视网膜变性疾病中的有效性。
发明内容
本发明的目的在于提供一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,从而改善该细胞移植后的存活和功能。
本发明公开了JAK1信号通路抑制剂Ruxolitinib在制备hESC-RPE细胞产品中的应用。
本发明中Ruxolitinib是一种选择性的JAK1抑制剂,于2011年经美国食品与药物管理局批准上市,临床主要用于治疗骨髓纤维化,目前尚未见其在干细胞相关产品中的应用报道,Ruxolitinib的结构式如下:
本发明中的干细胞产品为人胚胎干细胞H1细胞系经诱导分化获得的人视网膜色素上皮细胞(hESC-RPE细胞),第3代次hESC-RPE细胞高表达视网膜色素上皮细胞特征性标记物RPE65,BEST1,CRALBP,具备相对成熟且稳定的视网膜上皮细胞功能。
hESC-RPE细胞产品可应用于治疗包括但不限于年龄相关性黄斑变性(age-related macular disease,简称AMD)、视网膜色素变性(retinitis pigmentosa,简称RP)及Stargardt’s综合征(简称SD)的视网膜变性疾病。
Ruxolitinib的预处理可有效降低hESC-RPE细胞对细胞因子IFN-γ的应答,减少IFN-γ的对hESC-RPE细胞的直接毒性作用,并抑制IFN-γ引起的细胞免疫原性的上调,以减少其移植后受到的免疫排斥反应,特别是淋巴细胞介导的特异性免疫应答。
根据本专利技术制备的hESC-RPE细胞因具有较低的免疫原性,可有效逃避受体免疫细胞对其识别和细胞毒性作用,不需系统使用免疫抑制剂亦可有效降低hESC-RPE细胞受到的免疫排斥反应,并促进其移植后与受体视网膜组织的整合及功能发挥,延长细胞替代疗法的有效性,从而改善视网膜变性疾病患者的长期视觉功能。
为实现上述目的,本发明的技术方案如下:在移植前体外条件下,通过Ruxolitinib的预处理,阻断hESC-RPE细胞的JAK1磷酸化过程,从而抑制细胞JAK1-STAT通路的信号转导,以调控移植后hESC-RPE细胞对依赖JAK-STAT信号通路的细胞因子的应答,其特征在于Ruxolitinib的预处理在抑制细胞因子作用、降低免疫原性的同时,本身不会对hESC-RPE细胞的存活和功能产生明显副作用。
技术方案用于评估hESC-RPE细胞成熟度的方法为:从第3代次(P3)、培养7天后的hESC-RPE细胞中选取部分,经流式细胞学检测PAX6、MITF、BEST1、RPE65、CRLBP等视网膜色素上皮特征性指标表达阳性率均≥96%,以确认该批次细胞可满足临床使用要求。
技术方案所述的Ruxolitinib浓度为4μM,预处理细胞的时间为48小时。
技术方案所述IFN-γ浓度为100ng/mL,刺激细胞的时间为24小时。
技术方案所述的JAK1-STAT1通路的分子靶点包括:JAK1,STAT1,p-JAK1,p-STAT1。
技术方案所述的免疫原性指的是:表达的抗原可致敏T淋巴细胞、自然杀伤细胞(简称NK细胞),表现为上述免疫细胞的活化、增殖,并发挥对hESC-RPE细胞的毒性作用。
进一步地,用于评估hESC-RPE细胞免疫原性的抗原包括:HLA-A,HLA-B,HLA-C,HLA-E,HLA-G,HLA-DRA,HLA-DRB1,HLA-DRB3,HLA-DRB4,HLA-DRB5,IFN-γR1中的一种或几种。
进一步地,用于评估T/NK细胞致敏及毒性作用的方法为:hESC-RPE细胞与T/NK细胞的离体共培养,靶细胞(hESC-RPE细胞):效应细胞(免疫细胞)=10:1。
进一步地,通过ELISA实验测定共培养后CD4+T淋巴细胞产生IFN-γ的水平评估hESC-RPE细胞对CD4+T淋巴细胞的致敏作用;通过LDH实验评估CD8+T淋巴细胞及NK细胞对hESC-RPE细胞的杀伤作用;通过细胞计数评估hESC-RPE细胞对效应细胞的趋化作用。
技术方案所述的细胞功能包括:细胞存活、吞噬功能、紧密连接、迁移功能。
进一步地,用于评估细胞存活的方法为:流式细胞学技术测定细胞的7-AAD/Annexin-Ⅴ水平;用于评估细胞吞噬功能的方法为:分离LE大鼠感光细胞外节(POS)与hESC-RPE细胞共培养,以流式细胞学技术测定被hESC-RPE细胞吞噬的POS数量;用于评估紧密连接的方法为:以细胞免疫荧光化学技术测定hESC-RPE细胞紧密连接标记物ZO-1的表达水平;用于评估细胞迁移功能的方法为:在100%密度的hESC-RPE细胞片层制造划痕,以显微镜观察及测定划痕的愈合速率。
技术方案所述的移植模型包括:人源化动物模型的皮下移植,视网膜变性人源化模型的视网膜下腔移植。
进一步地,人源化动物模型是基于重度免疫缺陷NPG小鼠构建的人源化免疫系统小鼠模型,皮下移植用于评估hESC-RPE细胞对小鼠体内人源免疫细胞的趋化作用。
进一步地,视网膜变性人源化模型是基于人源化免疫系统小鼠构建的视网膜色素上皮细胞损伤模型。具体实施方法为:。。。视网膜下腔移植用于评估hESC-RPE细胞在变性视网膜微环境中的整合和功能。
所述对JAK-STAT信号通路表达上调或者下调的定量,指的是干预后靶细胞中的JAK-STAT信号通的基因或蛋白靶点的至少一种相对于靶细胞指定对照组上调或者下调了1~1500倍中的任意一个倍数。
技术方案中在JAK-STAT信号通路上被抑制表达或上调表达的基因靶点选自:JAK1,STAT1,HLA-A,HLA-B,HLA-C,HLA-E,HLA-G,HLA-DRA,HLA-DRB1,HLA-DRB3,HLA-DRB4,HLA-DRB5,IFN-γR1中的一种或几种。
所述方法在JAK-STAT信号通路上被抑制表达或上调表达的蛋白靶点选自:JAK1,STAT1,HLA-ABC,HLA-DR,HLA-E,HLA-G,ZO-1,Rhodopsin,7-AAD,Annexin-Ⅴ中的一种或几种。
所述的用于鉴定细胞发育和增殖的基因或蛋白质靶点选自:AKT1,PMP22,DVL2,TPM1,PAK1,NME2,SGMA4A,UGT8,NDRG3,ACTN1,中的一种或几种。
所述的用于鉴定细胞迁移的基因或蛋白质靶点选自:DDR1,CAL1,SH2B1,MFN2,CTBP1,MICAL1,FSCN2,SEMA4A,NCKAP5,DDHD1,YAP1,ABL2,ACTN1,MYLK中的一种或几种。
所述的用于鉴定细胞发育和增殖的基因或蛋白质靶点选自:SLC1A5,NAV1,LBR,EPB41L2,PFLIM2,GNAI1,PLS3,MYLK,ACTN1,MICAL1,FSCN2,SEMA4A,NCKAP5,DDHD1,ADD1,TTC12,AKT1,ARAP1,PPHLN1,CYFIP2,COL5A1,INPP5F,STST5A,PTK7中的一种或几种。
根据现有报道,从未有关于干预细胞JAK-STAT信号通路以调控干细胞免疫原性的研究被公开。我们的研究发现:通过Ruxolitinib预处理的hESC-RPE细胞,可以有效抵抗IFN-γ的直接毒性作用及其引起的免疫原性上调,从而减少T/NK细胞的识别和攻击,显著延长hESC-RPE细胞在视网膜变性疾病动物模型体内的存活和功能,这为克服该细胞在临床应用中的免疫排斥反应提供了新思路。
与现有技术相比,本发明的有益效果为:通过调控干细胞中关键的细胞因子信号通路,在不需要系统性使用免疫抑制剂的情况下,改善干细胞产品在宿主体内的存活和功能;这不仅有助于推进干细胞产品在视网膜变性疾病治疗领域的临床转化,也为解决其他异基因移植免疫排斥反应提供一类新方法,具有优秀的临床应用前景。
附图说明
图1是H1人胚胎干细胞系(hESC)向RPE细胞诱导分化的时间轴和示意图。
图2是P3代次第7天hESC-RPE细胞上视网膜色素上皮特异性抗原的表达情况。
图2中A是细胞免疫荧光检测Bestrophin、CRALBP、MITF、RPE65的表达;
图2中B是流式细胞术检测Bestrophin、CRALBP、MITF、RPE65的表达。
图3是流式细胞学技术检测HLA-ABC,HLA-DR,HLA-E和HLA-G抗原在hESC细胞和hESC-RPE细胞表面的表达情况;(A-B)图中蓝色曲线表示同型对照组,红色表示特异性抗体标记组,右上方数字分别表示阳性细胞比例(Percentage,%)和平均荧光强度(MFI);(C-D)上述数值在hESC细胞和hESC-RPE细胞两组间进行非配对t检验的统计学结果(ns=无统计学差异,*p<0.05,**p<0.01,***p<0.001)
图4是IFN-γ(100ng/mL)刺激hESC-RPE细胞24小时后以流式细胞学技术检测HLA-ABC,HLA-DR,HLA-E和HLA-G抗原的表达情况。
图5是IFN-γ(100ng/mL)刺激hESC-RPE细胞24小时后进行转录组测序的结果,其中对照组为IFN-γ溶剂去离子水。
图5中A是基于GO和KEGG数据库构建的蛋白互相作用关系网络图展示被IFN-γ上调的通路;
图5中B是热图展示展示被IFN-γ上调的差异表达基因富集情况,其中V代表对照组,I代表IFN-γ刺激组。
图6是.Ruxolitinib(4μM)预处理hESC-RPE细胞48小时后再以IFN-γ(100ng/mL)刺激24小时再进行转录组测序的结果,其中对照组为Ruxolitinib溶剂DMSO。
图6中A是蛋白互相作用关系网络图展示Ruxolitinib预处理组(Ruxolitinib+IFN-γ)比对照组(IFN-γ)下调的通路;
图6中B是GO富集分析Ruxolitinib预处理组(Ruxolitinib+IFN-γ)后相比对照组(IFN-γ)下调的通路,其中BP表示生物学过程,CC表示细胞结构,MF表示分子学功能;
图7是经上述方法Ruxolitinib及IFN-γ处理后用蛋白质印迹法(Western Blot)对hESC-RPE细胞中JAK1、STAT1表达及其磷酸化情况的检测结果,以管家蛋白β-actin作为上样内参;直方图表示两组之间p-JAK1和p-STAT1磷酸化水平的非配对t检验结果(**p<0.01,***p<0.001)。
图8是经上述方法Ruxolitinib及IFN-γ处理后检测hESC-RPE细胞上HLA-ABC,HLA-DR,HLA-E和HLA-G抗原的表达情况,对照组(Vehicle)为IFN-γ溶剂去离子水。
图8中A-B是.RT-PCR检测HLA-ABC,HLA-DR,HLA-E和HLA-G抗原在转录水平表达情况,用单因素方差分析及Tukey法检验比较组间差异(**p<0.01,***p<0.001,****p<0.001);
图8中C是细胞免疫荧光技术显示HLA-ABC,HLA-DR,HLA-E和HLA-G抗原在细胞的表达及定位,阳性表达的抗原以红色标示;
图8中D-E是流式细胞学技术检测HLA-ABC,HLA-DR,HLA-E和HLA-G的细胞比例,并以单因素方差分析及Tukey法检验比较组间差异(**p<0.01,***p<0.001)。
图9是Ruxolitinib(4μM)预处理hESC-RPE细胞48小时后分别与人CD4+T淋巴细胞、CD8+T淋巴细胞和CD56+NK细胞进行共培养的结果;对照组为Ruxolitinib溶剂DMSO。
图9中A-B是细胞免疫荧光显示共培养后细胞形态,hESC-RPE细胞表达绿色GFP蛋白,免疫细胞以红色荧光标记,以免疫细胞/靶细胞(hESC-RPE细胞)比例表示趋化作用,以非配对t检验比较组间差异(***p<0.001,****p<0.001);
图9中C式流式细胞学技术检测hESC-RPE细胞与CD4+T淋巴细胞共培养后被激活的IFN-γ+CD4细胞比例;
图9中D是ELISA技术定量检测hESC-RPE细胞与CD4+T淋巴细胞共培养后上清中IFN-γ的含量,以非配对t检验比较组间差异(****p<0.001);
图9中E是G.流式细胞学技术检测与(E)CD8淋巴细胞及(G)NK细胞共培养后hESC-RPE细胞凋亡情况;
图9中F是H.LDH实验检测与(F)CD8淋巴细胞及(H)NK细胞共培养后被杀伤的hESC-RPE细胞比例,以非配对t检验比较组间差异**p<0.01,***p<0.001)。
图10是IFN-γ(100ng/mL)刺激hESC-RPE细胞24小时后进行转录组测序的结果,其中对照组为IFN-γ溶剂去离子水。
图10中A是基于GO和KEGG数据库构建的蛋白互相作用关系网络图展示被IFN-γ下调的的通路;
图10中B是热图显示IFN-γ刺激后hESC-RPE细胞中生物学过程(GO数据库)相关基因的表达情况,包括细胞发育(Cell Development)、细胞吞噬(Phagocytosis)和伤口愈合(Wound Healing)其中V代表对照组,I代表IFN-γ刺激组
图11是Ruxolitinib(4μM)预处理hESC-RPE细胞48小时后再以IFN-γ(100ng/mL)刺激24小时,用感光细胞外节(POS)孵育hESC-RPE细胞48小时以评估细胞吞噬功能,(A)通过细胞免疫荧光及流式细胞学技术检测被hESC-RPE细胞吞噬的POS,其中Vehicle指的是Ruxolitinib溶剂0.01M PBS平衡盐溶液;(B)统计各组吞噬POS(Rhodopsin阳性)细胞的比例,运用单因素方差分析及Tukey法检验比较组间差异(*p<0.05,***p<0.001)
图12是上述方法Ruxolitinib及IFN-γ处理后以划痕实验测定hESC-RPE细胞的迁移能力,并按时间点(0小时、12小时、24小时、36小时)统计各组划痕愈合的比例,在各时间点运用双因素方差分析及Tukey法检验比较组间差异(**p<0.01,***p<0.001,****p<0.001)。
图13是Ruxolitinib(4μM)预处理hESC-RPE细胞48小时后移植于人源化小鼠皮下的检测结果,对照组(Vehicle)为DMSO预处理的细胞。
图13中A是携带Luciferase报告基因的hESC-RPE细胞团显像,分别在移植后第2、4、8、12天通过活体成像系统采集图像;
图13中B是移植后第4天,移植区皮下组织免疫荧光,其中hESC-RPE表达绿色GFP蛋白,人源免疫细胞hCD45、hCD3及hCD56以红色荧光标记;
图13中C-D是移植后第4天,移植区皮下组织细胞行流式细胞学检测,并统计组织中源免疫细胞hCD45、hCD3及hCD56的比例,组间以非配对t检验计算差异是否具有统计学意义(*p<0.05,**p<0.01)。
图14是Ruxolitinib(4μM)预处理hESC-RPE细胞48小时后移植于RPE损伤(NaIO3)的人源化小鼠视网膜下腔的检测结果。其中hESC-RPE+Ruxo为经Ruxolitinib(4μM)预处理48小时的hESC-RPE细胞,hESC-RPE为未经处理的hESC-RPE细胞,CTRL为DMSO预处理的细胞。
图14中A是移植后1-12周小鼠视觉电生理fERG波形示意图;
图14中B是移植后1-12周小鼠视觉电生理fERG的b波和a波峰值统计,在各时间点运用双因素方差分析及Tukey法检验比较组间差异(*p<0.05,**p<0.01,***p<0.001)
图14中C是移植后2周小鼠视网膜冰冻切片,hESC-RPE细胞表达绿色GFP蛋白;
图14中D-E是移植后2周小鼠视网膜组织细胞的流式细胞学检测,(D)hCD45标记人源白细胞浸润情况,GFP门内标记视网膜中存留的hESC-RPE细胞及其凋亡情况;(E)用非配对t检验比较两组视网膜组织细胞中的hCD45细胞比例、GFP阳性的hESC-RPE细胞及其晚期凋亡比例(*p<0.05,***p<0.001,****p<0.0001)。
具体实施方式
下面结合附图对本发明涉及的具体实施方式作详细说明。除特殊说明外,实例中涉及的试剂、材料等均可从商业途径得到。
实施例1,诱导人胚胎干细胞系(hESC)向RPE细胞分化。
(1)Q-CTS-hESC-2人胚胎干细胞系由中国科学院干细胞与生殖生物学国家重点实验室提供,培养于无血清的Essential 8TM培养基(Gibco公司),换液间隔时间为24小时;
(2)在hESC细胞生长至超融合时转入RPE细胞诱导阶段,所用到的诱导培养基组成为:KnockOut DMEM CTS(Invitrogen公司)基础培养+20%血清替代物(Invitrogen公司)+0.1mM NEAA(Gibco公司)+1mM GlutaMAX-1(Invitrogen公司)+0.1mM 2-Mercaptoethano(Gibco公司),RPE细胞诱导期间换液间隔为48小时,约21天后可观察到细胞中出现棕褐色色素灶,并继续培养约14天使色素细胞扩增;
(3)于显微镜下以玻璃电极挑出色素灶,并接种于Vitronectin基质胶(Gibco公司)包被的6孔板中,此时胚胎干细胞诱导而来的视网膜色素上皮(hESC-RPE)细胞为P0代次,其后进入hESC-RPE细胞扩增纯化阶段;
(4)在hESC-RPE细胞生长至80%密度时进行细胞传代,用胰酶替代物CTS TrypleSelect(Invitrogen公司)于37℃消化细胞15分钟,将细胞轻轻吹离孔板并转移至离心管中,1500rpm室温离心15分钟,收集细胞沉淀、诱导培养基重悬并以1:3比例传代接种于Vitronectin基质胶包被的6孔板中,此时hESC-RPE细胞为P1代次。依此类推,本实例中应用的hESC-RPE细胞为P3代次;
(5)本发明体外细胞培养均处于条件为37℃、5% CO2的培养箱(Thermofisher公司)中进行,涉及细胞换液、消化、预处理、共培养等操作步骤均为超净工作台中完成。
实施例2,hESC-RPE细胞的视网膜色素上皮特异性抗原鉴定。
(1)细胞免疫荧光鉴定。(a)取P3代次第7天的hESC-RPE细胞爬片,依次以4%PFA(Boster公司)室温固定15分钟、0.3%Triton X-100(Sigma-Aldrich公司)+3%BSA(Solarbio公司)室温破膜封闭60分钟;(b)用该破膜封闭混合液分别稀释RPE65(Abcam公司,1:200)、Bestrophin(Abcam公司,1:500)、CRALBP(Abcam公司,1:400)、MITF(美国Millipore公司,1:500),孵育hESC-RPE细胞爬片4℃过夜;(c)次日以0.01M PBS 1:500稀释AF488标记的山羊抗兔二抗(Invitrogen公司)和AF568标记的山羊抗小鼠二抗(Invitrogen公司),室温孵育细胞爬片2小时,DAPI(Beyotime公司)室温孵育5分钟;(d)抗荧光淬灭封片剂(Beyotime公司)封片后以激光共聚焦显微镜(ZEISS公司)采集图像。
(2)流式细胞学鉴定。(a)Tryple法消化收获P3代次第7天的hESC-RPE细胞;将细胞分为2组:第1组分为3份用于检测细胞膜抗原Bestrophin、CRALBP和RPE65,第2组用于检测细胞核抗MITF;(b)其中第1组各管用流式染色试剂Staining buffer(BD公司)按100μL/106细胞量重悬细胞;第2组用Fix/Perm buffer室温固定破膜20min后,再以Perm/Wash buffer(BD公司)按100μL/106细胞量重悬细胞;(c)第1组样本中分别加入Staining buffer稀释的Bestrophin(1:100)、CRALBP(1:100)和RPE65(1:300)抗体(均为Abcam公司),向第2组样本中加入Perm/Wash buffer稀释的MITF抗体(1:100),4℃孵育30分钟;(d)根据一抗来源以AF488标记山羊抗小鼠二抗(1∶500)或AF488标记的山羊抗兔二抗(1∶500)4℃避光孵育30min,0.01M PBS漂洗并重悬调整细胞浓度至1×106/mL后用流式细胞仪(BD公司)检测。
(3)除特殊说明外,其他实例中涉及其他处理方法的细胞,在进行细胞荧光免疫及流式细胞学检测时均参照本实例操作。
实施例3,细胞凋亡的流式细胞学检测。
(1)Tryple法收集hESC-RPE细胞并进行细胞计数后,样本以用凋亡染色试剂Binding buffer(BD公司)按300μL/106细胞量重悬细胞;
(2)加入5μL的Annexin Ⅴ(BD公司)室温避光孵育15min,上机前5min加入5μL的7AAD或PI试剂(BD公司)染色,400目细胞滤网过滤后上机检测,上机前补加200的Binidngbuffer(BD公司);
(3)其他实例中细胞凋亡的流式细胞学检测参照本实例操作。
实施例4,流式细胞学检测HLA抗原的表达。
(1)EDTA消化液(Gibco公司)37℃孵育hESC细胞并收集单细胞悬液,Tryple法收集P3代hESC-RPE单细胞悬液;
(2)流式染色试剂Staining buffer重悬细胞至100μL/106浓度,并分别以2μl/106细胞量加入PE标记的HLA-ABC流式抗体、APC标记的HLA-DR流式抗体、APC标记的HLA-G流式抗体和PE标记的HLA-E流式抗体(均为Biolegend公司),4℃避光孵育30分钟,Stainingbuffer漂洗并重悬调整细胞浓度至1×106/mL后用流式细胞仪(BD公司)检测。
实施例5,Ruxolitinib预处理及IFN-γ刺激hESC-RPE细胞的方法。
(1)用诱导培养基将Ruxolitinib(Invivogen公司)稀释至浓度4μM,在常规细胞培养箱中孵育处理hESC-RPE细胞(P3代第7天)48小时,其后以正常的诱导培养基洗涤贴壁的hESC-RPE细胞;对照组为Ruxolitinib溶剂DMSO(Sigma-Aldrich公司);
(2)用诱导培养基将人重组hrIFN-γ(R&D systems)稀释至使用浓度100ng/mL,在常规细胞培养箱中孵育处理hESC-RPE细胞(P3代第7天)24小时,其后以正常的诱导培养基洗涤贴壁的hESC-RPE细胞;对照组为Ruxolitinib溶剂去离子水;
(3)若需要验证上述两种药物的共同影响,则先以4μM Ruxolitinib预处理48小时,再以100ng/mL IFN-γ刺激细胞;
(4)除特殊说明外,其他实例中有关上述两种药物处理的情况均参照本实例操作。
实施例6,hESC-RPE细胞转录组测序和生物信息学分析。
(1)参照实例5药物处理hESC-RPE细胞后Tryple法收集单细胞悬液,1500rpm室温离心15分钟并收集细胞沉淀,Trizol(Takara公司)法抽提细胞总RNA,-80℃超低温冰箱保存样本;
(2)采用Illumina Hiseq 4000平台对转录组样本进行测序建库;
(3)应用HTSeq v0.6.1软件计算每个基因的read数量,计算每百万reads中来自于某基因每千碱基长度的reads数(RPKM),并以RPKM值来代表基因表达水平;
(4)应用EDGseq软件分析差异表达基因(DEG)时采取有参考基因序列的方式进行,并将阈值设置为矫正后P值Padj<0.05,相对变化倍率大于1.5且差异概率大于0.8的基因被认定为DEG;
(5)基于KEGG数据库、GO数据库对差异基因进行功能和通路富集,借助STRING网站(string-db.org)和Cytoscape软件(3.7.1版本)分析并构建蛋白相互作用关系,利用R语言(4.2.1版本)及相关语言包对生物信息学结果进行可视化呈现。
实施例7,hESC-RPE细胞基因表达的荧光实时定量PCR检测。
(1)参照实例5中Trizol法抽提药物处理后的hESC-RPE细胞总RNA,分光光度计测定RNA纯度,OD260/280为合格样品;
(2)使用逆转录试剂盒(Takara)去除样品中基因组DNA并反转录为cDNA;
(3)对样本进行荧光实时定量PCR检测,用到的特异性引物序列如下:
实施例8,hESC-RPE细胞基因表达的蛋白质印迹WB检测。
(1)参照实例5药物处理hESC-RPE细胞后,Tryple法消化细胞并收集细胞沉淀;
(2)用含蛋白酶和磷酸化酶抑制剂的RIPA裂解液(Beyotime公司)按200μL/106细胞量重悬细胞,冰浴中吹打裂解细胞20分钟;
(3)BCA法测定各组蛋白样品浓度,按照蛋白样品体积:蛋白上样缓冲液(5×,Beyotime公司)体积=4:1比例稀释样品,并置于沸水中加热变性10分钟;
(4)按照SDS-PAGE试剂盒(Beyotime公司)说明书配制分离胶和上层浓缩胶,每孔按照20μg总蛋白等质量上样,恒压电泳使各样本中不同分子量蛋白分离;
(5)冰水浴中电泳使凝胶中蛋白转移至PVDF膜,以5%BSA室温封闭PVDF膜2小时;
(6)一抗稀释液(Beyotime公司)分别稀释JAK1、p-JAK1、STAT1和p-STAT1以及内参β-actin,先后孵育于电转后的PVDF膜,置于4℃恒温摇床过夜;
(7)次日以HRP标记的二抗室温孵育PVDF膜2小时;
(8)向PVDF膜滴加适量ECL发光液,Bio-Rad凝胶成像系统采集数据。
实施例9,hESC-RPE细胞HLA抗原表达的细胞免疫荧光鉴定
(1)参照实例5药物处理hESC-RPE细胞后收集细胞爬片,依次以4%PFA(Boster公司)室温固定15分钟、3%BSA(Solarbio公司)室温封闭60分钟,并用BSA抗原封闭液分别稀释PE标记的HLA-ABC抗体(1:100)、APC标记的HLA-DR抗体(1:100)、APC标记的HLA-G抗体(1:100)和PE标记的HLA-E抗体(1:100,均为BD公司),孵育hESC-RPE细胞爬片4℃过夜;
(2)次日DAPI(Beyotime公司)室温孵育细胞爬片样本5分钟;抗荧光淬灭封片剂(Beyotime公司)封片后以激光共聚焦显微镜(ZEISS公司)采集图像。
实施例10,hESC-RPE细胞与免疫细胞的共培养实验。
(1)磁珠分选法获取淋巴细胞。取健康人外周血,Ficoll(1.077,GE公司)法提取血液中单个核细胞(hPBMCs),分别用人CD4+T淋巴细胞磁珠分选试剂盒、CD8+T淋巴细胞磁珠分选试剂盒及NK细胞分选试剂盒(均为Miltenyi公司)从hPBMCs中分选获取相应淋巴细胞;
(2)将GFP慢病毒转染24小时的hESC-RPE细胞接种于培养皿或细胞爬片,参照实例5用Ruxolitinib或DMSO预处理hESC-RPE细胞后,以淋巴细胞:hESC-RPE细胞=10:1的比例将磁珠分选收获的淋巴细胞加入hESC-RPE细胞的培养体系中,共培养时间为12小时;
(3)细胞免疫荧光观察趋化作用:收集细胞爬片进行免疫细胞化学染色,用PE标记的抗人CD4+T细胞、抗人CD8+T细胞抗体或抗人CD56+细胞抗体(1:200稀释,均为BD公司)过夜孵育细胞爬片;DAPI室温孵育细胞爬片样本5分钟;抗荧光淬灭封片剂封片后以激光共聚焦显微镜采集图像。
实施例11,ELISA法定量检测共培养上清中IFN-γ浓度。
(1)参照实例10进行hESC-RPE细胞与CD4+T淋巴细胞共培养实验,收集上清样本,用BCA试剂盒(康为世纪公司)测定样本中蛋白浓度;
(2)按照人细胞因子IFN-γ试剂盒(Thermofisher公司)说明书,提前准备好1×Wash buffer、1×Assay buffer、Biotin-Conjugate试剂以及Streptavidin-HRP试剂;
(3)向预制的ELISA 96孔板中加入400μL/孔的Wash buffer,轻晃孔板10秒以洗涤板底;将孔板倒置于吸水纸上以清空孔中的Wash buffer,随后依次向孔内加入样品稀释液、梯度稀释的标准品和各组样品,其中各组样品按照10μg总蛋白等量上样,用样品稀释液补足体积至50μL;
(4)每孔加入50μL Biotin-Conjugate试剂,盖上密封膜后放置于摇床上,400rpm室温反应2小时;
(5)同上方法清空孔板中反应液和样品,并用Wash buffer清洗孔板3次,每孔加入100μL Streptavidin-HRP试剂,盖上密封膜后放置于摇床上,400rpm室温反应1小时;
(6)同上方法清空孔板中反应液,并用Wash buffer清洗孔板3次,每孔加入100μLTMB反应底物,室温避光孵育10min;
(7)直接向每孔中加入100μL反应终止液,并立即用Thermofiher酶标仪检测(设置检测波长:450μM,参考波长:620μM)。
实施例12,LDH实验检测细胞毒性。
(1)参照实例10进行hESC-RPE细胞与CD8+T淋巴细胞或NK细胞的共培养实验;该部分共培养实验于96孔板中进行,应按照LDH试剂盒(Roche公司)要求设置试验(共培养)孔、自然释放孔和阳性对照孔;
(2)将新鲜配制的LDH反应底物加入96孔板中,每孔100μL,室温避光孵育30分钟;
(3)孵育完毕后,每孔加入50μL终止液,随即用Thermofiher酶标仪检测(检测波长:450μM,参考波长:620μM)。
(4)CD8+T淋巴细胞及NK细胞细胞对hESC-RPE细胞的毒性作用按照以下公式计算(吸光度值):
实施例13,感光细胞外节吞噬实验。
(1)感光细胞外节(POS)的制备。实验前一晚将LE大鼠转移至暗室(至少12h),次日在无直接光照条件下处死大鼠LE并摘取眼球,置于0.01M PBS中,并环形剪开去除角膜、晶体、玻璃体;在20mM Tris-HCl缓冲液中取出视网膜新鲜组织(3mL),将视网膜剪成2mm×2mm的组织碎片;将视网膜组织用移液器移入5mL离心管,轻轻振荡1分钟后以转移到4℃预冷离心机中,1000rpm离心5分钟;将上清液转入5mL离心管,然后以500uL Tris-HCl缓冲液轻轻洗沉淀,取清液与离心后上清液合并,转移到4℃预冷离心机中,4000rpm离心5分钟,该步骤重复一次;将离心后上清液转入1.5mL离心管内,在4℃预冷离心机中以15000rpm离心30分钟;吸弃上清后离心管底部沉淀即为感光细胞外节POS;0.01MPBS重悬POS并计数,按2x109/L浓度储存备用。
(2)参照实例5用Ruxolitinib或IFN-γ及其对照组溶剂处理hESC-RPE细胞后以6孔板每孔1×107/2mL诱导培养基浓度将POS加入hESC-RPE细胞培养体系中,细胞培养箱中孵育过夜;
(3)次日吸弃培养上清后用0.01M PBS清洗细胞3次以洗涤细胞表面未被吞噬的POS,Tryple法消化细胞并收集单细胞悬液;
(4)以4%PFA和0.3%Triton X-100室温处理细胞15分钟,以固定细胞并破膜;0.3%Triton X-100+3%BSA的破膜封闭混合液稀释Rhodopsin抗体(1:100,Abcam公司)并于4℃孵育细胞30分钟;AF488标记的山羊抗小鼠二抗(1:500,Thermofisher公司)4℃避光孵育细胞30分钟;用0.01M PBS平衡盐溶液重悬细胞浓度至1×106/mL后流式细胞流式细胞仪检测。
实施例14,划痕实验检测细胞迁移能力。
(1)将hESC-RPE细胞传代种植于Vitronectin基质胶包被的6孔板中。当P3代次第7天左右hESC-RPE细胞已铺满孔板底部时,用100μL无菌移液枪头沿孔板底直径,在细胞片层中间制造一条界线清晰的完整划痕;
(2)以0.01M PBS浸润并轻晃6孔板以漂洗脱落的细胞;
(3)参照实例5用含Ruxolitinib或IFN-γ的无血清诱导培养基处理hESC-RPE细胞,并将细胞放回培养箱中继续生长;
(4)每间隔12小时将6孔板取出于显微镜下观察拍照;ImageJ软件(2.0.0版本)测量计算各组划痕愈合情况。
实施例15,人源化免疫系统小鼠模型的构建。
(1)在医学伦理允许的情况下获取胎龄13-22周的胎儿,并于冰浴、无菌条件下取出胎儿肝脏、胸腺;
(2)将其中一部分胎肝切碎、碾磨为细碎组织后以1mg/mL胶原酶+1mg/mL透明质酸酶+2U/mL DNA酶混合试剂于37℃消化20分钟;经过滤、离心(1500rpm)后收集胎肝细胞沉淀,用含10%胎牛血清的RPMI1640培养基(Gibco)重悬调整细胞浓度至1×106/mL,并以Ficoll(1.077)法收集其中的单个核细胞(PBMC);
(3)CD34+造血干细胞磁珠分选试剂盒(Miltenyi公司)分选胎肝PBMC中CD34+造血干细胞,并以流式细胞术鉴定分选后细胞纯度;
(4)造模前一天,选取出生后约8周大小的雄性NPG小鼠(NOD.Cg-PrkdcscidIl2rgtm1Vst/Vst,维通达公司),以150cGy的亚致死剂量辐照小鼠;
(5)造模移植手术在消毒后的生物安全柜中进行,固定鼠尾,以胰岛素针吸取约2×105/200μL分选后的人CD34+造血干细胞行尾静脉注射;
(6)配制麻醉试剂100mg/mL氯氨酮+100mg/mL甲苯噻嗪,按照15μL/g小鼠体重行腹腔注射麻醉;
(7)确认麻醉效果后,小鼠腹部备皮、消毒,调整小鼠体位为右侧卧、左腹朝上,找到小鼠脾脏位置,该侧肾脏位于脾脏背侧约5mm,镊子控制此处皮肤并沿脾脏长轴方向行15mm切口,同时切开皮下的肌肉及腹膜,轻压腹部使该侧肾脏突出于腹腔外;
(8)用注射器借助基质胶(索莱宝公司)及0.01M PBS溶液通过16G穿刺针头将2mm直径大小的胎肝、胸腺组织移植于小鼠肾脏包膜下;
(9)将小鼠肾脏轻柔放回腹腔,缝合腹壁术口,小鼠放置于加热板观察至麻醉苏醒,确认无异常迹象后将小鼠饲养于经灭菌处理、单独加压通风笼盒,饲料及饮水应经辐照或高压灭菌;
(10)每隔2周在无菌条件下从小鼠眼眶取血约200μL,裂解红细胞后用PE标记的抗人CD4+T细胞、抗人CD8+T细胞抗体或抗人CD56+细胞抗体染色,流式细胞术检测外周血中人免疫细胞比例。
实施例16,人源化免疫系统小鼠皮下细胞移植和活体成像实验。
(1)用装载GFP及Luciferase报告基因的慢病毒以MOI(慢病毒:细胞数)=10转染P3代次hESC-RPE细胞;
(2)参照实例5用Ruxolitinib或其溶剂DMSO预处理hESC-RPE细胞48小时,后用Tryple法收集单细胞悬液;生理盐水调整各组的细胞浓度为2×105/100μL;
(3)将人源化免疫系统小鼠右后肢毛发剔除,用胰岛素针吸取各组细胞悬液200μL注射于小鼠右后肢背侧皮下,注射完成后在注射位点涂抹抗生素软膏,并继续将小鼠饲养于无菌条件下;
(4)分别于移植后第2、4、8、12天用小动物活体成像系统(Perkin Elmer公司)拍照采集人源化免疫系统小鼠皮下hESC-RPE细胞存活状态,操作过程通过小动物麻醉机以2%异氟烷气体麻醉小鼠。
实施例17,小鼠皮下组织的免疫荧光和流式细胞学检测。
(1)行皮下移植后第4天,断颈处死小鼠并取右后肢移植区皮肤及皮下组织,样本大小约3×3×3mm3,以4%PFA室温固定2小时后置于质量分数为30%的蔗糖溶液中,4℃冰箱中过夜以使组织块脱水;次日将脱水后的皮下组织用OCT包埋剂包埋,经冷冻固定后用冰冻切片机以15μm厚度连续切片,切片保存于-20℃备用;0.3%Triton X-100+3%BSA的破膜封闭混合液分别稀释兔抗人CD45、兔抗人CD3及兔抗人CD56抗体(1:200,Abcam公司),4℃过夜孵育组织切片;次日以AF568或AF647偶联的羊抗兔二抗(1:1000,Thermofisher公司)室温孵育切片2小时,再以DAPI室温孵育5分钟;抗荧光淬灭封片剂封片后以激光共聚焦显微镜采集图像。
(2)同法收集移植区皮下组织,经剪碎、碾磨后以0.1%胰蛋白酶+0.02%EDTA的混合酶解液于37℃恒温摇床消化30分钟,经过滤、离心后获得组织单细胞悬液;流式染色试剂Staining buffer重悬细胞至100μL/106浓度用PE偶联的抗人CD45+细胞抗体、抗人CD3+T细胞抗体及抗人CD56+细胞抗体(1:200稀释,均为BD公司)孵育细胞样本,再以Perm/Washbuffer(BD公司)洗涤并按100μL/106细胞浓度重悬细胞;流式细胞仪上机检测组织细胞样本。实施例18,小鼠视网膜色素上皮损伤模型的构建。
(1)配制碘酸钠溶液至浓度5mg/mL,现配现用;
(2)选取成功构建的人源化免疫系统小鼠模型(约12周,雄性),腹部消毒后按照40mg/kg体重行腹腔注射。
实施例19,小鼠视网膜下腔移植hESC-RPE细胞。
(1)用装载GFP报告基因的慢病毒转染P3代次hESC-RPE细胞,2天后参照实例5用Ruxolitinib(hESC-RPE+Ruxo组)或其溶剂DMSO(hESC-RPE组)预处理hESC-RPE细胞48小时,后用Tryple法收集单细胞悬液;生理盐水调整各组的细胞浓度为5×105/1μL,置于4℃冰箱以保存细胞活力;另设置单独注射生理盐水不含细胞的伪移植组(CTRL组);
(2)选取实例18中造模3天后的小鼠,配制麻醉剂100mg/mL氯氨酮+100mg/mL甲苯噻嗪,按照15μL/g小鼠体重行腹腔注射麻醉;
(3)将鼠眼置于手术显微镜下,眼周皮肤碘伏消毒3次,助手分开小鼠眼睑使眼球充分暴露,先后以托吡卡胺滴眼液散瞳及盐酸奥布卡因行眼球表面麻醉;
(4)在外眦部距离角巩膜缘约1mm处分离球结膜使下方巩膜暴露;在此处用胰岛素针头在巩膜上造一小穿刺口,随后用10uL微量注射器吸取1uL提前准备好的hESC-RPE细胞悬液或生理盐水,沿暴露巩膜穿刺口处进针至视网膜下,缓慢推注细胞悬液并保持10秒后缓慢退出针头;
(5)术眼涂典必殊眼膏(Alcon公司),小鼠放置于加热板观察至麻醉苏醒,后送还至无菌条件下继续饲养观察。
实施例20,小鼠闪光视网膜电图fERG检测。
(1)小鼠视网膜下腔移植后第1、2、3、4、6、12周进行该项检测;
(2)检测前需提前暗适应至少12小时,检测时全程保持于暗室中操作;通过小动物麻醉机以2%异氟烷气体麻醉小鼠
(3)麻醉完成后在暗红光照明下将小鼠俯卧位固定于动物实验平台,双眼以托吡卡胺滴眼液散瞳;
(4)于鼠尾钳夹接地电极,后颈皮下插入负极,双眼角膜接触金环电极不同通道;
(5)用视觉电生理系统(Diagnosis公司)记录各组小鼠暗适应3.0cd*s/m2闪光刺激下的波形;
(6)双眼涂典必殊眼膏(Alcon公司),小鼠放置于加热板观察至麻醉苏醒,后送还至无菌条件下继续饲养观察。
实施例21,小鼠视网膜冰冻切片及组织免疫荧光检测。
(1)小鼠视网膜下腔移植后2周进行该项检测;
(2)断颈处死小鼠,摘取眼球并立即将其置于质量分数4%多聚甲醛中室温固定1.5小时,于显微镜下去除角膜、晶体及虹膜等前段结构,随后转移至质量分数30%蔗糖溶液,4℃冰箱中过夜以使视网膜组织脱水;
(3)将脱水后的眼球用OCT包埋剂(Sakura公司)包埋,经-20℃冷冻固定后用冰冻切片机经视网膜后极部和锯齿缘行10μm厚度连续切片,切片保存于-20℃备用;
(4)选取各组形态结构完整冰冻切片,0.01M PBS漂洗3遍后用0.1%Triton X-100溶液室温破膜15分钟,再以3%BSA室温封闭1小时;
(5)用0.1%Triton X-100+3%BSA抗原稀释液配制以下2组抗体:(a)羊抗ZO1(1:500,Abcam公司)+兔抗人CD45(1:200,Abcam公司);(b)兔抗RPE65(1:500,Abcam);孵育小鼠视网膜冰冻切片样本4℃过夜;
(6)次日根据上述一抗来源用AF 568偶联的羊抗兔或驴抗羊IgG二抗(1∶1000)室温孵育2小时,
(7)为观察小鼠视网膜细胞及移植后hESC-RPE细胞的凋亡情况,另取各组冰冻切片用Tunel试剂盒(Roche)进行原位细胞凋亡染色;
(8)DAPI室温孵育5分钟,并以抗荧光淬灭封片剂封片;激光共聚焦系统拍照采集视网膜冰冻切片图像。
实施例22,小鼠视网膜组织流式细胞学检测。
(1)小鼠视网膜下腔移植后2周进行该项检测;
(2)断颈处死小鼠,摘取眼球并立即将其置于质量分数4%多聚甲醛中室温固定0.5小时,于显微镜取出完整视网膜及色素膜组织;
(3)以1.2U/mL中性蛋白酶+1mg/mL胶原酶+1mg/mLⅡ型胶原酶的混合酶解液消化视网膜及色素膜组织,并经过滤、离心后收集单细胞悬液;
(4)用0.1%Triton X-100+3%BSA破膜封闭液配制PE标记的抗人CD45(1:200,Abcam公司)流式抗体,室温孵育细胞样本30分钟;
(5)另取各组视网膜细胞悬液,参照实例3中方法利用凋亡试剂盒对细胞样本进行染色标记;
(6)0.01M PBS漂洗并重悬调整细胞浓度至1×106/mL后用流式细胞仪上机检测。
结果显示:
(1)根据附图1和附图2,本发明采取诱导策略所获得的hESC-RPE细胞具有经典的RPE细胞形态、较高的纯度及稳定的功能性抗原表达;
(2)根据附图3和附图4,正常体外培养条件下hESC和hESC-RPE细胞的人类白细胞HLA-A,HLA-DR,HLA-E和HLA-G抗原都为低表达,但IFN-γ刺激后hESC-RPE细胞的HLA抗原表达显著上调,尤其是HLA-DR;
(3)根据附图5,IFN-γ的刺激使hESC-RPE细胞上“抗原处理和提呈”、“免疫排斥反应”、“适应性免疫应答”等通路被激活,并加速了“细胞凋亡”,其中上调的基因包括:(a)HLA相关基因,如HLA-DRB1、HLA-DRB5、HLA-E、HLA-DQA1、HLA-F、TAP1及CD74等;(b)IFN信号通路下游分子,如IFIT1、IFIT3级IFIT6等;(c)淋巴细胞趋化因子,如CXCL10;表明IFN-γ使hESC-RPE细胞的免疫原性明显上调;
(4)根据附图6,Ruxolitinib的预处理使hESC-RPE细胞上“干扰素信号通路”、“MHC-Ⅱ类复合体组装”、“抗原处理和提呈”、“T细胞激活调节”等信号通路被下调,表明Ruxolitinib的预处理通过阻断MHC复合体(HLA抗原)的表达,可有效抑制IFN-γ引起的hESC-RPE细胞免疫原性上调、特别是T淋巴细胞激活依赖的抗原识别;
(5)根据附图7,Ruxolitinib的预处理使得IFN-γ作用下的JAK-STAT信号通路磷酸化过程被抑制,从而阻断IFN-γ的细胞生物学效应;
(6)根据附图8,分别从转录水平和蛋白水平证实了Ruxolitinib的预处理阻断了IFN-γ引起的HLA-ABC、HLA-DR、HLA-E和HLA-G的表达上调;
(7)根据附图9,共培养实验说明Ruxolitinib对hESC-RPE的免疫原性调节作用能减少其对CD4/CD8 T淋巴细胞及NK细胞的趋化作用,抑制其对CD4淋巴细胞的激活,降低CD8T淋巴细胞及NK细胞的细胞毒性效应;
(8)根据附图10,IFN-γ的刺激使hESC-RPE细胞的“伤口愈合”、“细胞生长”、“吞噬杯形成”等信号通路被下调,从而损害了细胞发育、迁移、吞噬等重要功能;
(9)根据附图11,Ruxolitinib的预处理可有效挽救IFN-γ引起的hESC-RPE细胞吞噬功能损伤,且实验剂量(4μM)下Ruxolitinib本身不会影响细胞吞噬功能;
(10)根据附图12,IFN-γ作用下hESC-RPE细胞迁移功能受损,而Ruxolitinib的预处理可减少这种效应,且实验剂量(4μM)下Ruxolitinib本身不会影响细胞迁移功能;
(11)根据附图13,人源化小鼠皮下移植实验证明Ruxolitinib的预处理可减少的人CD45、CD3、CD56免疫细胞对hESC-RPE的攻击,从而显著延长移植细胞在人源化模型动物体内的存活时间;
(12)根据附图14,视网膜下腔移植实验证实Ruxolitinib的预处理可抑制微环境中人免疫细胞对hESC-RPE细胞的毒性作用,并改善hESC-RPE细胞在变性视网膜的整合和功能,从而有效挽救RPE损伤模型动物的视力。
综上所述,本发明中Ruxolitinib的预处理可通过抑制JAK-STAT磷酸化,阻断IFN-γ引起的hESC-RPE细胞HLA抗原表达从而降低淋巴细胞的识别和攻击,显著延长hESC-RPE细胞在人源化模型动物体内的存活,促进hESC-RPE细胞在变性视网膜的整合和功能。
以上只是本发明的优选实施方式,并不用以限制本发明,应当指出,基于本发明技术原理做出的改进或变型也视为本发明专利的保护范围。
Claims (10)
1.一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,包括在移植前体外条件下,通过Ruxolitinib的预处理,阻断hESC-RPE细胞的JAK1磷酸化过程,从而抑制细胞JAK1-STAT通路的信号转导,以调控移植后hESC-RPE细胞对依赖JAK-STAT信号通路的细胞因子的应答,其特征在于,Ruxolitinib的预处理在抑制细胞因子作用、降低免疫原性的同时,本身不会对hESC-RPE细胞的存活和功能产生明显副作用。
2.根据权利要求1所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,所述JAK/STAT信号通路为JAK1/STAT1信号通路。
3.根据权利要求2所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述JAK1/STAT1信号通路为IFN-γ作用下细胞激活的JAK1/STAT1信号通路。
4.根据权利要求1所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述JAK1信号通路抑制剂为小分子药物鲁索替尼(Ruxolitinib)。其使用浓度为4-10μM,预处理时间为48-72小时。
5.根据权利要求1所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述干细胞产品为人胚胎干细胞来源的视网膜色素上皮细胞(hESC-RPE细胞)。
6.根据权利要求1所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述JAK/STAT信号通路抑制剂制备的hESC-RPE细胞有较低的免疫原性和完整的视网膜色素上皮细胞功能。
7.根据权利要求6所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述免疫原性指的是表达的抗原可致敏T淋巴细胞、自然杀伤细胞(NK细胞),表现为免疫细胞的活化、增殖,并产生对hESC-RPE细胞的毒性作用。
8.根据权利要求6所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述细胞功能指的是细胞存活、增殖功能、吞噬功能、紧密连接、迁移功能。
9.根据权利要求1所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述视网膜变性疾病包括但不限于年龄相关性黄斑变性(AMD),视网膜色素变性(RP)及Stargardt’s综合征(SD)。
10.根据权利要求9所述的一种JAK/STAT信号通路抑制剂预处理hESC-RPE细胞的方法,其特征在于,所述视网膜变性疾病的治疗方法为经玻璃体腔注射或视网膜下腔注射干细胞产品的细胞替代疗法。
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