CN113134076A - 一种利用转录因子再生具备功能的视网膜神经节细胞的方法 - Google Patents
一种利用转录因子再生具备功能的视网膜神经节细胞的方法 Download PDFInfo
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Abstract
本发明属于生物医药研究领域,具体提供转录因子在以下一项或多项中的用途:制备视网膜疾病治疗药物;制备视网膜神经节细胞再生产品;制备诱导成体细胞重编程为视网膜神经节细胞的产品;制备视网膜神经节细胞;所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。本发明所述的再生视网膜神经节细胞所使用的细胞材料为自身的内源细胞,与通过细胞移植的方式相比无免疫排斥和成瘤风险;利用病毒表达基因简便易行,易于推广。
Description
技术领域
本发明属于生物医药研究领域,具体涉及一种利用转录因子再生具备功能的视网膜神经节细胞的方法。
背景技术
视网膜神经节细胞(Retinal ganglion cells)是视网膜中唯一神向大脑传递神经信号的经元,它的病变和死亡是导致失明的主要原因之一。与视网膜神经节细胞病变相关的疾病在临床上非常普遍,主要包括青光眼、遗传性视神经病变等。
据最新统计,仅青光眼这一项疾病,全世界目前有7960万患者,其中1120万人最终可能发展为双眼盲。我国流行病学资料显示,中国40岁以上人群青光眼患病率为2.3%,致盲率约为30%。预计到2020年,我国将有2100万青光眼患者,因此而致盲的人数将达到630万,给患者家庭和社会造成沉重的经济负担。
再生视网膜神经节细胞重建视网膜和大脑之间的神经连接,是使因视网膜神经节细胞病变而失明的病人重新恢复视力的最理想的方法,也是目前全球范围内再生医学领域竞争的一个热点。再生视网膜节细胞的方法主要分为两类:一类是通过细胞移植的手段,将体外由胚胎干细胞等分化的视网膜节细胞移植到动物体内;另外一类是利用动物视网膜中的其他细胞再生节细胞。另外,还有一些研究试图使轴突退化但细胞体存活的视网膜节细胞重新生长轴突,建立视网膜和大脑的连接。
尽管人们在视网膜神经节细胞再生方面做了大量的工作,目前在成体哺乳动物中成功再生具备功能的视网膜神经节细胞的案例还没有,本发明是在该领域的首次突破。
发明内容
为了克服现有技术中所存在的问题,本发明的目的在于提供利用转录因子再生具备功能的视网膜神经节细胞的方法。
为了实现上述目的以及其他相关目的,本发明采用如下技术方案:
本发明的第一方面,提供转录因子在以下一项或多项中的用途,所述用途是非治疗目的的用途:
1)制备视网膜疾病治疗药物;
2)制备视网膜神经节细胞再生产品;
3)制备诱导成体细胞重编程为视网膜神经节细胞的产品;
4)制备视网膜神经节细胞;
所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
本发明第三方面提供一种视网膜神经节细胞的制备方法,至少包括以下步骤:将转录因子施用于成体细胞使其转化为视网膜神经节细胞;所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
本发明第四方面提供一种重组细胞,为在宿主细胞中引入能表达转录因子的多核苷酸或者核酸构建体后获得,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
本发明第五方面提供一种产品,其特征在于,所述产品包括转录因子,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个,所述产品适用于视网膜神经节细胞再生、诱导成体细胞重编程为视网膜神经节细胞或治疗视网膜疾病。
本发明第六方面提供一种治疗视网膜疾病的方法,至少包括以下步骤:向患有视网膜疾病的对象给予有效量的转录因子,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
与现有技术相比,本发明具有如下有益效果:
本发明所述的再生视网膜神经节细胞所使用的细胞材料为自身的内源细胞,与通过细胞移植的方式相比无免疫排斥和成瘤风险;利用病毒表达基因简便易行,易于推广。
本发明利用AAV病毒为基因递送载体,通过体内细胞命运重编程的方式,将一种神经细胞类型转化为另外一种神经细胞类型,实现神经细胞再生和神经环路重建,从而进行疾病治疗和功能恢复。具体而言,是将视网膜中的无长突细胞和错位无长突细胞重编程为视网膜中的唯一输出神经元—视网膜神经节细胞,重建视网膜和大脑之间的神经连接,使失明动物或病人重新恢复视力。
附图说明
图1,视网膜神经细胞具备再生能力。a,小鼠视网膜切片图像,Lgr5阳性细胞位于视网膜中间细胞层(内核层),是一类完全分化的无长突神经细胞(Amacrineinterneurons)。b,在Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato小鼠中Lgr5阳性无长突神经细胞转化成为双极细胞。c,在Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato小鼠中Lgr5阳性无长突神经细胞转化成为水平细胞。d,Lgr5阳性无长突神经细胞从内核层向视网膜节细胞层迁移。
图2,在成熟小鼠体内将Lgr5阳性无长突神经细胞重编程为视网膜神经节细胞。a,体内细胞重编程策略。b,重编程使用的病毒载体结构图。c,玻璃体腔注射对照病毒的小鼠的视网膜铺片,视网膜中没有神经节细胞再生。d,玻璃体腔注射对照病毒的小鼠的视神经切片,视神经中没有再生的节细胞的轴突。e,玻璃体腔中注射表达转录因子病毒的视网膜铺片图像。f,图像e中的局部放大,箭头标注成功再生的视网膜神经节细胞的轴突。g,一个再生的视网膜神经节细胞的局部放大图。
图3,由Lgr5阳性无长突神经细胞再生的视网膜神经节细胞。a,再生的视网膜神经节细胞位于视网膜的神经节细胞层并从胞体向外伸展轴突。b,再生的视网膜神经节细胞的轴突汇集于视乳突处并向视神经生长。c,再生的视网膜神经节细胞表达神经节细胞的特异性分子RBPMS。d,再生的视网膜神经节细胞表达神经节细胞的特异性分子Brn3A。e,再生的视网膜神经节细胞表达对明暗及方向有选择性的一类神经节细胞的特异性分子CART。
图4,再生的视网膜神经节细胞表达alpha类神经节细胞的特异性分子SMI-32。
图5,由Lgr5阳性无长突神经细胞向视网膜神经节细胞重编程促进这些细胞由内核层向神经节细胞层迁移。
图6,再生的视网膜神经节细胞向视神经及大脑的不同视觉核团伸展轴突。a,视神经(Optic nerve)。b,背外侧膝状核(Dorsal lateral geniculate nucleus)。c,腹外侧膝状核(Ventral lateral geniculate nucleus)。d,橄榄顶盖前核(Olivary pretectalnucleus)。e,上丘(Superior colliculus)。f,上丘的局部放大图。g-I,脑片上丘处PSD-95组织化学染色,显示再生的视网膜神经节细胞神经轴突末梢在上丘处与下游神经元形成突触结构。
图7,不同转录因子组合将Lgr5阳性无长突神经细胞重编程为视网膜神经节细胞的效率。
图8,Prokr2CreERT2敲入小鼠的构建及分析。a,小鼠构建策略图。b-d,Prokr2CreERT2;Rosa26-tdTomato小鼠视网膜切片及RBPMS染色图。Prokr2-tdTomato阳性细胞不表达神经节细胞特异性标记物RBPMS。e,Prokr2CreERT2;Rosa26-tdTomato小鼠大脑切片。f,RBPMS。e,Prokr2CreERT2;Rosa26-tdTomato小鼠大脑切片上丘局部图。g,Prokr2CreERT2;Rosa26-tdTomato小鼠视神经切片。
图9,Prokr2阳性错位无长突细胞向神经节细胞的重编程。a,Prokr2CreERT2;Rosa26-tdTomato小鼠视网膜铺片。b,a中的局部放大。c,视网膜铺片展示再生的视网膜神经节细胞。d,c中的局部放大。e,视神经切片。f-k,再生的视网膜神经节细胞向大脑不同区域的神经轴突投射。f,外侧膝状核。g,橄榄顶盖前核。h,上丘表层。I,h的局部放大。j,上丘底层。k,j局部放大。
图10,Prokr2阳性错位无长突细胞向神经节细胞的重编程的策略及效率。a,重编程策略。b,重编程使用的AAV表达质粒结构图。c,不同转录因子组合的重编程效率。
图11,再生的视网膜神经节细胞在体内具备生理功能。a-d,体内神经细胞钙成像结果。a-d,体内神经细胞钙成像结果。a,再生的视网膜神经节细胞轴突末端在小鼠大脑上丘处随视觉刺激改变而产生的钙信号变化。图中的三个神经末梢随视觉刺激而发放神经信号(on response)。b,图中展示的是三个off response的神经末梢。c,具备运动角度选择性的神经末梢。d,具备运动方向选择性的神经末梢。e-j,光遗传实验结果。e,再生的神经节细胞的轴突末梢在光刺激下释放的神经递质能够在下游的大脑上丘神经元中激发AMPA受体介导的兴奋性突触后电流(EPSC)。f,再生的神经节细胞的轴突末梢在光刺激下释放的神经递质能够在下游的大脑上丘神经元中激发NMDA受体介导的兴奋性突触后电流(EPSC)。g,再生的神经节细胞的轴突末梢在光刺激下释放的神经递质能够在下游的大脑上丘神经元中激发动作电位。h和i,上丘神经元AMPA受体介导的兴奋性突触后电流(EPSC)强度及峰的数量的统计。j,上丘神经元NMDA受体介导的兴奋性突触后电流(EPSC)强度的统计。
图12,利用钙成像技术在活体动物中对再生的视网膜神经节细胞的生理功能进行分析。a,体内钙成像模式图。b,再生的视网膜神经节细胞轴突末梢图像。
图13,在青光眼动物疾病模型中再生视网膜神经节细胞及功能分析。a,Lgr5EGFP -IRES-CreERT2;Rosa26-tdTomato正常小鼠视网膜切片。b,Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato小鼠视网膜在高眼压损伤7天之后切片。c,Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato小鼠视网膜在高眼压损伤后通过每天滴K-115眼药水保护视网膜神经细胞,7天后视网膜切片。d,Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato小鼠视网膜在高眼压损伤后通过每天滴K-115眼药水保护视网膜神经细胞,7天后注射AAV病毒对Lgr5阳性细胞进行重编程,病毒注射6周后视网膜切片。e,注射AAV-DIO-EGFP病毒的对照眼睛视神经中没有再生的神经节细胞轴突。f,注射表达转录因子的AAV的眼睛的视神经中含有再生的神经节细胞的轴突。g-k,Lgr5EGFP -IRES-CreERT2;Rosa26-tdTomato小鼠大脑切片图,小鼠的左眼注射了对照组AAV-DIO-EGFP病毒,有眼注射了表达转录因子的AAV。来自有眼的再生的视网膜神经节细胞的轴突向大脑的左侧投射。l-m,再生的节细胞向上丘中下游神经元传递神经信号。l,再生的节细胞释放的神经递质能够激活下游神经元的AMPA受体介导的EPSC。m,再生的节细胞释放的神经递质能够激活下游神经元的NMDA受体介导的EPSC。n,再生的节细胞释放的神经递质能够激活下游神经元产生动作电位。
图14,利用眼压升高损伤视网膜神经节细胞模型的建立以及在疾病模型动物中再生神经节细胞的策略图。a,b,损伤前和伤后神经节细胞轴突在视神经中的组织切片图。c,利用眼压升高损伤内源视网膜神经节细胞后将Lgr5阳性无长突细胞重编程为神经节细胞的策略示意图。
具体实施方式
本发明首次在成体哺乳动物(小鼠)体内再生了视网膜神经节细胞,证明再生的神经节细胞能够向大脑长距离投射轴突建立视网膜和大脑之间的神经连接,并具备生理功能:能够在活体动物中整合由上游神经元感受的视觉信号,并将视觉信号传递到大脑中的下游神经元。这为在临床上通过相同手段再生视网膜神经节细胞使失明患者重新获得视力提供了实验依据。
另外,本技术发明是建立在学术新发现的基础之上的,我们在世界上首次发现完全分化的神经细胞仍然具备再生潜能,在正常动物体内能够从一种神经类型向其他神经类型转变。尽管在正常生理状态下,这种神经细胞类型转变的频率很低,但是这种神经细胞的再生潜能能够被发掘出来,通过基因表达的方法促进神经类型之间的转变,实现神经再生和修复的目的。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或者按照各制造商所建议的条件。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
本文使用的术语“对象”或“受试对象”是指任意的动物(例如,哺乳动物、鸟类、爬行类、两栖类、鱼类),包括但不限于人、非人的灵长类、啮齿类等,其成为具体治疗的受体。通常,当在本文中指代对象时,术语“对象”和“患者”可互换使用。
当用于本文所述的化合物、生物制剂或药物组合物时,术语“有效量”表示产生需要的治疗结果所需的量。例如,有效量是治疗、治愈或缓解疾病的症状的有效水平,因为该疾病而给予所述治疗化合物、生物制剂或组合物。具体治疗目标的有效量的探求会取决于多种因素,包括治疗的疾病和其严重程度和/或发展/进程的阶段;生物可及性,以及使用的特定化合物、生物制剂或药物组合物的活性;给药的途径或方法以及对象上的引入位点;所述特定化合物或生物制剂的清除速率和其他药代动力学性质;治疗的持续时间;感染方案;与所述特定化合物、生物制剂或组合物组合或同时使用的药物;治疗对象的年龄、体重、性别、饮食、生理和总体健康情况;以及相关科学领域中技术人员熟知的类似因素。根据待治疗的对象的情况,在剂量上的会出现一些必要的变化,并且在任何情况下,医师或其他单独给予治疗会确定单独患者的合适剂量。
在本发明的文本中,术语“多肽”和“蛋白”是等价的并且可互换使用的。它们是指任意氨基酸链,并且包括对其的任意翻译后修饰(例如磷酸化或糖基化)。
本文使用的术语“对象”是指任意的动物(例如,哺乳动物、鸟类、爬行类、两栖类、鱼类),包括但不限于人、非人的灵长类、啮齿类等,其成为具体治疗的受体。通常,当在本文中指代对象时,术语“对象”和“患者”可互换使用。另外,在本发明的方法中使用转基因动物(例如,转基因大鼠和小鼠)。
本文使用的术语“给予”是指采用玻璃体内、眼内、眼睛、视网膜下、鞘内、静脉内、皮下、经皮、皮内、颅内、局部等给药方式向对象提供治疗有效量的化学或生物化合物或药物组合物。本发明的化学或生物化合物可单独给予,但也可根据选择的给药途径和标准药学实践与其他化合物、赋形剂、填料、粘结剂、运载体或其他载剂一起给予。给药可以是运载体或载剂的方式,如可注射溶液,包括无菌水性或非水性溶液,或盐水溶液;乳膏;洗剂;胶囊;片剂;颗粒;粒料;粉末;悬浮液,乳剂或微乳剂;贴片;胶束;脂质体;囊泡;植入物,包括微植入物;滴眼液;其他蛋白和肽;合成聚合物;微球;纳米颗粒等。
本文使用的“疾病”是指疾病、病症或状况,或其他与健康或正常生物学活性不同的情况,并且这些术语可互换使用。所述术语是指损害正常功能的任意状况。所述状况可由散发性或可遗传的遗传异常引起。所述状况也可由非遗传异常引起。所述状况也可用对象受到来自环境因素的伤害引起,例如但不限于切割、挤压、烧伤、刺穿、拉伸、剪切、注射或另外改变对象的细胞、组织、器官或系统等。
本文使用的“治疗”或“处理”是指阻止或抑制,或尝试阻止或抑制疾病的发展或进程,和/或产生,或尝试产生疾病和/或其症状的减少、抑制、回归或缓解。如本领域技术人员所理解,可以使用各种临床和科学方法和试验来评价疾病的发展或进程,并且相似地,可以使用各种临床和科学方法和试验来评价疾病或其症状的减少、回归或缓解。另外,可以向对象或细胞培养物中施用治疗。
本发明所述的技术方案均可以用于治疗或非治疗用途。
本发明一实施例提供转录因子在以下一项或多项中的用途:
1)制备视网膜疾病治疗药物;
2)制备视网膜神经节细胞再生产品;
3)制备诱导成体细胞重编程为视网膜神经节细胞的产品;
4)制备视网膜神经节细胞;
所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
进一步的,所述转录因子为人源或鼠源;
所述视网膜疾病可以是中枢神经系统的组织或细胞损伤的结果。治疗的视网膜疾病也可以是神经变性疾病(例如,视网膜色素变性)。损伤或患有神经变性疾病的组织或细胞可以是神经节细胞,如视网膜神经节细胞或感光细胞。在一些实施方式中,治疗的视网膜疾病包括神经节细胞退化。这样的神经节细胞退化可以由青光眼诱导。
即,可选的,所述视网膜疾病可选自视网膜色素变性、黄斑变性、青光眼或遗传学视神经病变。
考虑用本文所述的治疗方法治疗的神经变性疾病可以是在自然中遗传性的或散发性的(例如,分离的,非可遗传的情况下发生)。如本领域技术人员所理解,神经变性疾病也包括除了视网膜神经变性疾病以外的状况,并且本发明的方法、组合物和药物被考虑适用于其他这样的疾病。这样的疾病包括阿尔茨海默病、癫痫、亨廷顿病、帕金森病、肌萎缩侧索硬化、青光眼、年龄相关听力丧失、进行性核上性麻痹、轻度认知障碍、痴呆、脊髓性小脑萎缩症等。
所述视网膜神经节细胞再生产品是指能够诱导成体细胞重编程为视网膜神经节细胞的产品。
可选的,所述转录因子为DNA、mRNA或蛋白形式。
可选的,所述转录因子可以为多核苷酸构建体的形式,所述多多核苷酸构建体含有转录因子的基因片段。
所述的核酸构建体可以是将转录因子的基因片段克隆入已知载体获得。
所述核酸构建体可以为质粒、病毒载体或慢病毒载体。
优选为AAV病毒载体。
可选的,所述转录因子也可以为置于重组细胞中,所述重组细胞含有前述的核酸构建体。
所述重组细胞可以为在宿主细胞中引入能表达转录因子的多核苷酸或者核酸构建体后获得。
所述宿主细胞选自无长突神经细胞或错位无长突神经细胞或视网膜中的其他细胞中的一种或多种。
所述视网膜中的其他细胞是指视网膜中无长突神经细胞和错位无长突神经细胞以外的细胞。
所述Brn3B的cDNA序列如SEQ ID NO:1或SEQ ID NO:6所示;所述Sox4的cDNA序列如SEQ ID NO:2或SEQ ID NO:7所示;所述Atoh7的cDNA序列如SEQ ID NO:3或SEQ ID NO:8所示;所述Sox11的cDNA序列如SEQ ID NO:4或SEQ ID NO:9所示;所述Isl1的cDNA序列如SEQ ID NO:5或SEQ ID NO:10所示。
具体的,
鼠源DNA序列:
Brn3B,SEQ ID NO:1:
atgatgatgatgtccctgaacagcaagcaggcgttcagcatgcctcacgcaggcagcctgcacgtggagcccaagtactcggcgctacacagtgcctccccgggctcctctgcgcccgcggcgccctcggccagttcccctagcagctccagcaacgctggcggcggcggcggtggcggcggaggcggaggcggcggcggccggagcagcagttccagcagcagtggcagcggcggcagcggcggcggcgggggctcggaggcgatgcggagagcttgtcttccaaccccaccgagcaatatattcggcgggctggatgagagtctgctggcccgtgccgaggctctggccgccgtggacatcgtctcccagagtaagagccaccaccaccatccgccccaccacagccccttcaagccggacgccacttaccacaccatgaacaccatcccgtgcacgtcggcagcctcctcttcttctgtgcccatctcgcacccgtccgctctggctggcacccatcaccaccaccaccaccaccatcaccaccatcaccagccgcaccaggcgctggagggcgagctgcttgagcacctaagccccgggctggccctgggagctatggcgggccccgacggcacggtggtgtccactccggctcacgcaccacacatggccaccatgaaccccatgcaccaagcagccctgagcatggcccacgcacatgggctgccctcgcacatgggctgcatgagcgacgtggatgcagacccgcgggacctggaggcgttcgccgagcgtttcaagcagcgacgcatcaagctgggagtgacccaggcagatgtgggctcggcgctggccaacctcaagatcccgggcgtgggctcgctcagccagagcaccatctgcaggtttgagtctctcacgctgtcacacaacaacatgatcgcgctcaagcccatcctgcaggcgtggctggaggaagctgagaaatcccaccgcgagaagctcactaagccggagctcttcaatggcgcggagaagaagcgcaagcgcacgtccatcgcggcgccggagaagcgctctctggaagcctacttcgccatccagccaaggccctcctcggagaagatcgcggccatcgccgaaaagctggatctcaagaaaaatgtggtgcgcgtctggttctgcaaccagaggcagaaacagaagagaatgaaatactctgccggcatttag。
Sox4,SEQ ID NO:2:
atggtacaacagaccaacaacgcggagaacactgaggctctgctggccggggagagctcggactcgggcgccggcctggagctgggcatcgcgtcctccccgacgcctggctccaccgcgtcgacgggcggcaaggcggacgaccccagctggtgcaagacgcccagtggccacatcaagcggcccatgaacgcctttatggtgtggtcgcagatcgagcggcgcaagatcatggagcagtcgcccgacatgcacaacgccgagatctccaagcggctaggcaaacgctggaagctgctcaaggacagcgacaagattccgttcatccaggaggcggagcggctgcgcctcaagcacatggctgactaccctgactacaagtaccggccgcgaaagaaggtgaagtcgggcaacgcgggcgcgggatcggcggccacagccaagccaggggagaagggcgacaaggtcgcgggcagcagcggccacgcgggaagcagccacgcggggggtggcgcgggcggcagctccaagcccgcgcccaagaagagctgtggccccaaggtggcgggcagctcggtcggcaagccccacgctaagctggtcccggcgggcggcagcaaggcggctgcatcgttctctccagagcaagctgccctgctgcccctgggggagcccacggccgtctacaaggtgcggactcccagtgcggccactccggccgcctcctcctcgccgtccagtgcgctggccaccccagccaaacaccctgccgacaagaaagtgaagcgcgtctacctgtttggaagcctgggcgcttcggcgtctcccgtcgggggcctgggagcgagcgccgaccccagtgatccactggggttgtacgaagatggaggcccgggatgctcgcccgatggccggagtctgagcggccgcagcagcgcagcatcatcgccagccgccagccgatcgcccgctgaccaccgcggctacgccagcctacgcgcagcctcgcccgccccgtccagcgcgccctcgcacgcgtcctcctcgctctcctcgtcctcttcctcctcctcgggctcttcgtcgtccgacgacgagttcgaagacgacctgctcgacctgaaccccagctcaaactttgagagcatgtccctgggcagtttcagctcctcatcggcgctcgatcgggacctggattttaacttcgaacccggctcaggctcccacttcgaattcccggactattgcacgcccgaggtgagcgagatgatctcgggagattggctggagtccagcatctctaacctggtcttcacctactga。
Atoh7,SEQ ID NO:3:
atgaagtcggcctgcaaaccccacggccctccggcgggagctcgcggcgcgcccccgtgcgcgggcgcagccgagcgcgcggtctcgtgcgcggggcccgggcggctggagagcgcggcgcgcaggcgtctggcggccaacgcgcgcgagcggcgccgcatgcaggggctgaacacggcgttcgaccggctgcgcagggtggtgccgcagtggggccaggacaagaagctgtccaagtacgagacactgcagatggcgctcagctacatcatcgcgctcacccgcatcctagccgaagccgagcgggactgggtcgggctgcgctgcgagcagcggggccgcgatcacccctacctccctttcccgggtgctaggctccaggtagaccctgagccctatgggcagaggctcttcggcttccagccggagcccttccccatggccagctaa。
Sox11,SEQ ID NO:4:
ATGGTGCAGCAGGCCGAGAGCTCGGAAGCCGAGAGCAACCTGCCCCGGGACGCGCTGGACACCGAGGAGGGCGAGTTCATGGCGTGCAGCCCGGTGGCCCTGGACGAGAGCGACCCGGACTGGTGCAAGACGGCGTCGGGCCACATCAAACGGCCCATGAACGCCTTCATGGTGTGGTCCAAGATCGAGCGCAGGAAGATCATGGAGCAGTCGCCCGACATGCACAACGCCGAGATCTCCAAGAGGCTGGGCAAGCGCTGGAAGATGCTGAAGGACAGCGAGAAGATCCCGTTCATCAGGGAGGCGGAGCGCCTGCGCCTCAAGCACATGGCTGATTATCCCGACTACAAGTACCGGCCGCGCAAAAAGCCCAAGACGGACCCAGCGGCCAAGCCCAGCGCGGGCCAGAGCCCCGACAAGAGCGCGGCGGGCGCCAAGGCAGCCAAGGGCCCCGGCAAGAAGTGCGCCAAGCTCAAGGCGCCTGCGGGCAAGGCGGGCGCGGGCAAGGCGGCGCAGCCGGGGGACTGCGCCGCGGGCAAGGCAGCCAAGTGCGTCTTCCTGGACGACGACGATGAAGACGACGACGAAGATGACGAGCTGCAGCTACGGCCCAAGCCGGACGCTGACGACGACGACGACGAGCCCGCGCACTCGCACCTGCTGCCGCCGCCGACGCAGCAGCAACCCCCTCAGCTGCTGAGGCGCTACAGCGTGGCCAAGGTCCCCGCCAGCCCCACGCTCAGCAGTGCCGCCGAGTCCCCCGAGGGCGCGAGCCTGTACGACGAAGTGCGCGCGGGCGGCCGGCTCTACTACAGCTTCAAGAACATCACCAAGCAGCAGCCTCCGCCCGCGCCTCCCGCGCTGTCGCCCGCGTCCTCCCGCTGCGTGTCCACCTCCTCATCCAGCGGCAGCAGCAGCGGCAGCGGCGCCGAGGATGCAGACGACCTCATGTTCGACCTGAGCTTGAATTTCTCCCAGGGCGCGCACAGCGCCTGCGAGCAGCCACTGGGCGCGGGAGCGGCGGGGAACCTGTCCCTGTCGCTGGTGGATAAGGACCTGGATTCCTTCAGCGAGGGCAGCCTGGGTTCCCACTTCGAGTTCCCCGACTACTGCACGCCGGAGCTGAGCGAGATGATCGCGGGGGACTGGCTGGAGGCGAACTTCTCCGACCTGGTGTTCACGTATtga
Isl1,SEQ ID NO:5:
ATGGGAGACATGGGCGATCCACCAAAAAAAAAACGTCTGATTTCCCTGTGTGTTGGTTGCGGCAATCAAATTCACGACCAGTATATTCTGAGGGTTTCTCCGGATTTGGAGTGGCATGCAGCATGTTTGAAATGTGCGGAGTGTAATCAGTATTTGGACGAAAGCTGTACGTGCTTTGTTAGGGATGGGAAAACCTACTGTAAAAGAGATTATATCAGGTTGTACGGGATCAAATGCGCCAAGTGCAGCATAGGCTTCAGCAAGAACGACTTCGTGATGCGTGCCCGCTCTAAGGTGTACCACATCGAGTGTTTCCGCTGTGTAGCCTGCAGCCGACAGCTCATCCCGGGAGACGAATTCGCCCTGCGGGAGGATGGGCTTTTCTGCCGTGCAGACCACGATGTGGTGGAGAGAGCCAGCCTGGGAGCTGGAGACCCTCTCAGTCCCTTGCATCCAGCGCGGCCTCTGCAAATGGCAGCCGAACCCATCTCGGCTAGGCAGCCAGCTCTGCGGCCGCACGTCCACAAGCAGCCGGAGAAGACCACCCGAGTGCGGACTGTGCTCAACGAGAAGCAGCTGCACACCTTGCGGACCTGCTATGCCGCCAACCCTCGGCCAGATGCGCTCATGAAGGAGCAACTAGTGGAGATGACGGGCCTCAGTCCCAGAGTCATCCGAGTGTGGTTTCAAAACAAGCGGTGCAAGGACAAGAAACGCAGCATCATGATGAAGCAGCTCCAGCAGCAGCAACCCAACGACAAAACTAATATCCAGGGGATGACAGGAACTCCCATGGTGGCTGCTAGTCCGGAGAGACATGATGGTGGTTTACAGGCTAACCCAGTAGAGGTGCAAAGTTACCAGCCGCCCTGGAAAGTACTGAGTGACTTCGCCTTGCAAAGCGACATAGATCAGCCTGCTTTTCAGCAACTGGTCAATTTTTCAGAAGGAGGACCAGGCTCTAATTCTACTGGCAGTGAAGTAGCATCGATGTCCTCGCAGCTCCCAGATACACCCAACAGCATGGTAGCCAGTCCTATTGAGGCATGA。
人源DNA序列:
Brn3B,SEQ ID NO:6:
atgatgatgtccctgaacagcaagcaggcgtttagcatgccgcacggcggcagcctgcacgtggagcccaagtactcggcactgcacagcacctcgccgggctcctcggctcccatcgcgccctcggccagctcccccagcagctcgagcaacgctggtggtggcggcggcggcggcggcggcggcggcggcggcggaggccgaagcagcagctccagcagcagtggcagcagcggcggcgggggctcggaggctatgcggagagcctgtcttccaaccccaccgagcaatatattcggcgggctggatgagagtctgctggcccgcgccgaggctctggcagccgtggacatcgtctcccagagcaagagccaccaccaccatccaccccaccacagccccttcaaaccggacgccacctaccacactatgaataccatcccgtgcacgtcggccgcctcttcttcatcggtgcccatctcgcacccttccgcgttggcgggcacgcaccaccaccaccaccatcaccaccaccaccaccaccaaccgcaccaggcgctggagggcgagctgctggagcacctgagtcccgggctggccctgggcgctatggcgggccccgacggcgctgtggtgtccacgccggctcacgcgccgcacatggccaccatgaaccccatgcaccaagcagcgctcagcatggcccacgcgcacgggctgccgtcgcacatgggctgcatgagcgacgtggacgccgacccgcgggacctggaggcattcgccgagcgcttcaagcagcgacgcatcaagctgggggtgacccaggcagatgtgggctccgcgctggccaacctcaagatccccggcgtgggctcgcttagccagagcaccatctgcaggttcgagtccctcacactgtcccacaataatatgatcgcgctcaaacccatcctgcaggcatggctcgaggaggccgagaagtcccaccgcgagaagctcaccaagcctgaactcttcaatggcgcggagaagaagcgcaagcgcacgtccatcgctgcgccagagaagcgctcgctcgaagcctactttgccattcagcctcggccctcctctgaaaagatcgccgccatcgcggagaagctggacctgaagaaaaacgtggtgcgcgtctggttctgcaaccagaggcagaaacagaaaagaatgaaatattccgccggcatttag。
Sox4,SEQ ID NO:7:
atggtgcagcaaaccaacaatgccgagaacacggaagcgctgctggccggcgagagctcggactcgggcgccggcctcgagctgggaatcgcctcctcccccacgcccggctccaccgcctccacgggcggcaaggccgacgacccgagctggtgcaagaccccgagtgggcacatcaagcgacccatgaacgccttcatggtgtggtcgcagatcgagcggcgcaagatcatggagcagtcgcccgacatgcacaacgccgagatctccaagcggctgggcaaacgctggaagctgctcaaagacagcgacaagatccctttcattcgagaggcggagcggctgcgcctcaagcacatggctgactaccccgactacaagtaccggcccaggaagaaggtgaagtccggcaacgccaactccagctcctcggccgccgcctcctccaagccgggggagaagggagacaaggtcggtggcagtggcgggggcggccatgggggcggcggcggcggcgggagcagcaacgcggggggaggaggcggcggtgcgagtggcggcggcgccaactccaaaccggcgcagaaaaagagctgcggctccaaagtggcgggcggcgcgggcggtggggttagcaaaccgcacgccaagctcatcctggcaggcggcggcggcggcgggaaagcagcggctgccgccgccgcctccttcgccgccgaacaggcgggggccgccgccctgctgcccctgggcgccgccgccgaccaccactcgctgtacaaggcgcggactcccagcgcctcggcctccgcctcctcggcagcctcggcctccgcagcgctcgcggccccgggcaagcacctggcggagaagaaggtgaagcgcgtctacctgttcggcggcctgggcacgtcgtcgtcgcccgtgggcggcgtgggcgcgggagccgaccccagcgaccccctgggcctgtacgaggaggagggcgcgggctgctcgcccgacgcgcccagcctgagcggccgcagcagcgccgcctcgtcccccgccgccggccgctcgcccgccgaccaccgcggctacgccagcctgcgcgccgcctcgcccgccccgtccagcgcgccctcgcacgcgtcctcctcggcctcgtcccactcctcctcttcctcctcctcgggctcctcgtcctccgacgacgagttcgaagacgacctgctcgacctgaaccccagctcaaactttgagagcatgtccctgggcagcttcagttcgtcgtcggcgctcgaccgggacctggattttaacttcgagcccggctccggctcgcacttcgagttcccggactactgcacgcccgaggtgagcgagatgatctcgggagactggctcgagtccagcatctccaacctggttttcacctactga。
Atoh7,SEQ ID NO:8:
atgaagtcctgcaagcccagcggcccgccggcgggagcgcgcgttgcacccccgtgcgcgggcggcaccgagtgcgcgggcacgtgcgccggggccgggcggctggagagcgcggcgcgcaggcgcctggcggccaacgcgcgcgagcgccgccgcatgcaggggctcaacactgccttcgaccgcttacgcagggtggttccccagtggggccaggataaaaagctgtccaagtacgagaccctgcagatggccctgagctacatcatggctctgacccggatcctggccgaggccgagcgattcggctcggagcgggactgggtgggtctccactgtgagcacttcggccgcgaccactacctcccgttcccgggcgcgaagctgccgggcgagagcgagctgtacagccagagactcttcggcttccagcccgagcccttccagatggccacctag。
Sox11,SEQ ID NO:9:
atggtgcagcaggcggagagcttggaagcggagagcaacctgccccgggaggcgctggacacggaggagggcgaattcatggcttgcagcccggtggccctggacgagagcgacccagactggtgcaagacggcgtcgggccacatcaagcggccgatgaacgcgttcatggtatggtccaagatcgaacgcaggaagatcatggagcagtctccggacatgcacaacgccgagatctccaagaggctgggcaagcgctggaaaatgctgaaggacagcgagaagatcccgttcatccgggaggcggagcggctgcggctcaagcacatggccgactaccccgactacaagtaccggccccggaaaaagcccaaaatggacccctcggccaagcccagcgccagccagagcccagagaagagcgcggccggcggcggcggcgggagcgcgggcggaggcgcgggcggtgccaagacctccaagggctccagcaagaaatgcggcaagctcaaggcccccgcggccgcgggcgccaaggcgggcgcgggcaaggcggcccagtccggggactacgggggcgcgggcgacgactacgtgctgggcagcctgcgcgtgagcggctcgggcggcggcggcgcgggcaagacggtcaagtgcgtgtttctggatgaggacgacgacgacgacgacgacgacgacgagctgcagctgcagatcaaacaggagccggacgaggaggacgaggaaccaccgcaccagcagctcctgcagccgccggggcagcagccgtcgcagctgctgagacgctacaacgtcgccaaagtgcccgccagccctacgctgagcagctcggcggagtcccccgagggagcgagcctctacgacgaggtgcgggccggcgcgacctcgggcgccgggggcggcagccgcctctactacagcttcaagaacatcaccaagcagcacccgccgccgctcgcgcagcccgcgctgtcgcccgcgtcctcgcgctcggtgtccacctcctcgtccagcagcagcggcagcagcagcggcagcagcggcgaggacgccgacgacctgatgttcgacctgagcttgaatttctctcaaagcgcgcacagcgccagcgagcagcagctggggggcggcgcggcggccgggaacctgtccctgtcgctggtggataaggatttggattcgttcagcgagggcagcctgggctcccacttcgagttccccgactactgcacgccggagctgagcgagatgatcgcgggggactggctggaggcgaacttctccgacctggtgttcacatattga。
Isl1,SEQ ID NO:10:
atgggagacatgggagatccaccaaaaaaaaaacgtctgatttccctatgtgttggttgcggcaatcagattcacgatcagtatattctgagggtttctccggatttggaatggcatgcggcatgtttgaaatgtgcggagtgtaatcagtatttggacgagagctgtacatgctttgttagggatgggaaaacctactgtaaaagagattatatcaggttgtacgggatcaaatgcgccaagtgcagcatcggcttcagcaagaacgacttcgtgatgcgtgcccgctccaaggtgtatcacatcgagtgtttccgctgtgtggcctgcagccgccagctcatccctggggacgaatttgcgcttcgggaggacggtctcttctgccgagcagaccacgatgtggtggagagggccagtctaggcgctggcgacccgctcagtcccctgcatccagcgcggccactgcaaatggcagcggagcccatctccgccaggcagccagccctgcggccccacgtccacaagcagccggagaagaccacccgcgtgcggactgtgctgaacgagaagcagctgcacaccttgcggacctgctacgccgcaaacccgcggccagatgcgctcatgaaggagcaactggtagagatgacgggcctcagtccccgtgtgatccgggtctggtttcaaaacaagcggtgcaaggacaagaagcgaagcatcatgatgaagcaactccagcagcagcagcccaatgacaaaactaatatccaggggatgacaggaactcccatggtggctgccagtccagagagacacgacggtggcttacaggctaacccagtggaagtacaaagttaccagccaccttggaaagtactgagcgacttcgccttgcagagtgacatagatcagcctgcttttcagcaactggtcaatttttcagaaggaggaccgggctctaattccactggcagtgaagtagcatcaatgtcctctcaacttccagatacacctaacagcatggtagccagtcctattgaggcatga。
所述成体细胞选自无长突神经细胞或错位无长突神经细胞或视网膜中的其他细胞中的一种或多种。
所述产品可以为试剂盒、注射剂或药物。
所述药物还包括一种或多种常规的药学上可接受的辅料。
通常,所述药物中除了有效成分外,根据不同剂型的需要,还会包括一种或多种药学上可接受的载体或辅料。
“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时,它们不会产生不利的、过敏的或其它不良反应。
“药学上可接受的载体或辅料”应当与所述有效成分相容,即能与其共混而不会在通常情况下大幅度降低药物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类,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Tween;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。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
本发明一实施例的视网膜神经节细胞的制备方法,至少包括以下步骤:将转录因子施用于成体细胞将其转化为视网膜神经节细胞;所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
本发明中,除非特别说明,药物剂型并无特别限定,可以被制成针剂、口服液、片剂、胶囊、滴丸、喷剂等剂型,可通过常规方法进行制备。药物剂型的选择应与给药方式相匹配。
本发明的药物制剂的有效量应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
进一步的,所述成体细胞选自无长突神经细胞或错位无长突神经细胞或视网膜中的其他细胞中的一种或多种。
所述转录因子为DNA、mRNA或蛋白形式。
所述转录因子为人源或鼠源。
所述成体细胞来源对象可以为哺乳动物或非哺乳动物。例如可以为爬行动物、非人的灵长类动物、啮齿类动物、人类等。
所述Brn3B的cDNA序列如SEQ ID NO:1或SEQ ID NO:6所示;所述Sox4的cDNA序列如SEQ ID NO:2或SEQ ID NO:7所示;所述Atoh7的cDNA序列如SEQ ID NO:3或SEQ ID NO:8所示;所述Sox11的cDNA序列如SEQ ID NO:4或SEQ ID NO:9所示;所述Isl1的cDNA序列如SEQ ID NO:5或SEQ ID NO:10所示。
在一种实施方式中,所述视网膜神经节细胞的制备方法,至少包括以下步骤:将转录因子编码基因转染至成体细胞,得到视网膜神经节细胞。
所述转录因子编码基因通过重组载体转染至体细胞。
所述重组载体为将所述转录因子编码基因插入表达载体,得到的重组载体。
所述表达载体可以为质粒、病毒载体或慢病毒载体。
优选的,所述表达载体选自AAV病毒载体。
所述制备方法可以为体内制备或者体外制备。
所述体外制备可以为:获得成体细胞,将所述成体细胞中引入转录因子,将所述成体细胞在合适培养条件、合适的培养基内进行培养。所述培养基可以为DMEM培养基。
本发明一实施例的一种重组细胞,为在宿主细胞中引入能表达转录因子的多核苷酸或者核酸构建体后获得,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
所述宿主细胞为成体细胞。选自无长突神经细胞或错位无长突神经细胞或视网膜中的其他细胞中的一种或多种。
本发明一实施例的产品,包括转录因子,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个,所述产品适用于视网膜神经节细胞再生、诱导成体细胞重编程为视网膜神经节细胞或治疗视网膜疾病。
所述产品可以为试剂盒、注射剂或药物。
所述转录因子可以为DNA、mRNA或蛋白形式。
可选的,所述转录因子可以为多核苷酸构建体的形式,所述多多核苷酸构建体含有转录因子的基因片段。
所述的核酸构建体可以是将转录因子的基因片段克隆入已知载体获得。
所述核酸构建体可以为质粒、病毒载体或慢病毒载体。
优选为AAV病毒载体。
可选的,所述转录因子也可以为置于重组细胞中,所述重组细胞含有前述的核酸构建体。
所述重组细胞可以为在宿主细胞中引入能表达转录因子的多核苷酸或者核酸构建体后获得。
可选的,所述转录因子为人源或鼠源。
本发明的治疗视网膜疾病的方法,至少包括以下步骤:向患有视网膜疾病的对象给予有效量的转录因子,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
所述转录因子在药学上可接受的载剂中给予。在一些实施方式中,所述转录因子注射入需要的对象的眼部,其也可使用缓释载剂给予。
可选的,所述转录因子为DNA、mRNA或蛋白形式。
所述转录因子可以为人源或鼠源。
所述成体细胞来源于哺乳动物。
所述Brn3B的cDNA序列如SEQ ID NO:1或SEQ ID NO:6所示;所述Sox4的cDNA序列如SEQ ID NO:2或SEQ ID NO:7所示;所述Atoh7的cDNA序列如SEQ ID NO:3或SEQ ID NO:8所示;所述Sox11的cDNA序列如SEQ ID NO:4或SEQ ID NO:9所示;所述Isl1的cDNA序列如SEQ ID NO:5或SEQ ID NO:10所示。
所述转录因子用于给予受试对象的眼睛。
本发明各个主题中的专有名词或术语的解释以及实施例均可通用,不再重复赘述。
实施例1
1.1小鼠及饲养方式:
本发明中使用的Lgr5EGFP-IRES-CreERT2,PvalbCreERT2,和Rosa26-tdTomato小鼠品系是从Jackson laboratory购买的。Lgr5EGFP-IRES-CreERT2品系小鼠和PvalbCreERT2品系小鼠分别与Rosa26-tdTomato小鼠杂交获得Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato小鼠和PvalbCreERT2;Rosa26-tdTomato小鼠。
Prokr2CreERT2小鼠品系是利用CRISPR/Cas9技术,通过同源重组的方式,在上海科技大学实验室自己构建的,该小鼠在Prokr2基因ATG位点定点敲入了CreERT2-PolyA表达框。小鼠构建的简要过程如下:通过In-Fusion cloning的方法构建包含CreERT2-PolyA表达框以及同源重组臂的载体(donor vector)。将Cas9 mRNA、gRNA和donor vector显微注射到C57BL/6J小鼠的受精卵中,注射后的受精卵在培养3.5天后移植到假孕雌鼠中获得F0代小鼠。经长片段PCR鉴定,将含有正确同源重组的F0代小鼠与C57BL/6J小鼠交配获得阳性F1代Prokr2CreERT2小鼠。将Prokr2CreERT2小鼠与Rosa26-tdTomato小鼠杂交获得Prokr2CreERT2;Rosa26-tdTomato小鼠。
所有的小鼠都饲养在一个有12小时光照和12小时黑暗循环的SPF级动物设施中。通过灌胃他莫昔芬(Tamoxifen,体重0.1-0.2mg/g)激活CreERT2。实验中雄性和雌性小鼠同等比例使用,没有性别选择,所有的动物实验程序都得到了上海科技大学动物护理与使用委员会的批准。
1.2 AAV载体构建与病毒包装:
为了利用腺相关病毒AAV表达小鼠的Atoh7,Brn3B,Sox4,Sox11,Isl1基因以及EGFP,我们将这些基因以及EGFP的cDNA序列克隆到一个CAG启动子驱动的,Cre重组酶依赖性的表达载体中(Addgene#22222),替换掉原有载体上的Arch-GFP序列。当利用一个AAV载体同时表达一个小鼠基因和报告基因EGFP时,我们通过在这两个DNA片段之间放置一段P2A片段的形式将它们同时表达。
AAV病毒颗粒的制备:将包含编码基因序列的AAV表达质粒、pAAV7m8血清型质粒和pHelper质粒利用PEI共转染HEK293T细胞,48-72小时后收集细胞。用碘克沙醇密度梯度离心法纯化病毒颗粒,qPCR法测定病毒滴度。经鉴定,本实施例所构建的载体能够分别表达Atoh7+EGFP基因;Brn3B+EGFP基因;Sox4+EGFP基因,Sox11+EGFP基因,Isl1+EGFP基因。
AAV玻璃体下腔注射:
通过向腹腔注射氯胺酮(Ketamine,80mg/kg)和甲苯噻嗪(Xylazine,8mg/kg)的混合液麻醉小鼠。小鼠麻醉后,向眼睛表面滴加盐酸苯来弗林滴眼液(Phenylepherinehydrochloride ophthalmic solution,2.5%)使小鼠瞳孔放大,并使用0.5%盐酸丙卡因滴眼液(Proparacaine hydrochloride)对动物眼睛表面进行短暂麻醉,然后利用无菌针头在角膜边缘穿刺一个小孔释放房水降低眼压。使用34号针头将1.5ul AAV病毒颗粒悬浮液注射到玻璃体腔内。混合注射几种AAV病毒时,保证每个AAV终浓度为每毫升1x1012病毒颗粒。实验包括以下几组:
实验1组(对照组):注射AAV-DIO-EGFP病毒。
实验2组:注射AAV-DIO-Brn3B病毒。
实验3组:注射AAV-DIO-Sox4病毒。
实验4组:注射AAV-DIO-Brn3B+AAV-DIO-Sox4病毒。
实验5组:注射AAV-DIO-Atoh7+AAV-DIO-Brn3B+AAV-DIO-Sox4病毒。
实验6组:注射AAV-DIO-Atoh7+AAV-DIO-Brn3B+AAV-DIO-Sox4+AAV-DIO-Sox11+AAV-DIO-Isl1病毒。
1.4小鼠青光眼模型构建:
采用眼压升高诱导的缺血再灌注模型损伤小鼠视网膜神经节细胞,模拟急性闭角型青光眼。为了达到这个目的,用一根注射用针头扎入麻醉的小鼠眼睛的前房,注射针的另外一端通过一根软管连接到一个装有含有0.1%肝素(Heparine)的生理盐水的瓶子。通过改变盐水瓶的高度来改变眼睛内部的压力。实验使用的损伤条件为:生理盐水水面到眼球的高度为150cm,压力损伤的时间为60分钟。在这个压力条件下小鼠视网膜内血流停止(缺血),60分钟后拔出针头使血流恢复(再灌注)。该损伤条件很快导致视网膜神经节细胞轴突及胞体变性,同时也引起视网膜其他神经元的死亡。为防止视网膜其他神经元凋亡,我们在损伤后利用Rock inhibitor Ripasudil(0.4%,溶于PBS)溶液涂抹小鼠眼表,每日一次。
1.5免疫组化分析:
对深度麻醉的小鼠分别用生理盐水(0.9%NaCl)和4%PFA灌流,然后取小鼠眼睛、视神经和大脑,利用4%PFA固定24小时。组织进一步利用30%蔗糖脱水,OCT包埋,利用冰冻切片机将组织切成厚度10um(眼睛和视神经)和30μm(大脑)的冰冻切片。免疫组织化学分析按标准程序进行。使用的抗体包括:视网膜神经节细胞特异性抗体rabbit-anti-RBPMS(Abcam,1:400),mouse-anti-Brn3a(Santa Cruz Biotechnology,1:200),α-RGCs特异性抗体rabbit anti-SMI-32(Abcam,1:400),感光型RGCs特异性抗体rabbit anti-melanopsin(Abcam,1:500),方向选择性RGCs特异性抗体rabbit anti-CART(cocaine-andamphetamine-regulated transcript,Phoenix Peptide,1:2500),突触后膜特异性抗体mouse anti-PSD95(Abcam,1:400)。使用的荧光标记的第二抗体包括:Alexa Fluor647donkey anti-rabbit(IFKineTM,1:400),Alexa Fluor 647donkey anti-mouse(IFKineTM,1:400),Alexa Fluor 488donkey anti-rabbit(Abcam,1:400)。组织切片免疫荧光染色后使用蔡司LSM880共聚焦显微镜、尼康转盘(CSU W1 Sora)共聚焦显微镜和STEDsp8显微镜显微成像。
1.6体内钙成像:
利用乌拉坦(urethane)(1.5g/kg)麻醉小鼠,并将麻醉后的小鼠固定在立体定位装置中,用药用眼药膏涂抹眼睛表面防止水分蒸发和白内障的形成。利用牙科水泥将一个定制的钛制金属板固定在颅骨上(牙科水泥中加入Fe3O4用来遮光),钛制金属板大致以lambda区域为中心并与小鼠的身体长轴平行。在上丘和下丘交界处的脑颅上做一个直径约3毫米的开口,然后将直径为3毫米的盖破片轻轻按压在硬脑膜上,并用黑色的牙科水泥封闭以便于利用双光子显微镜对上丘处视网膜神经节细胞的轴突末端进行成像。在利用双光子显微镜成像过程中,在固定小鼠头部的金属板上方放置一块遮光布,防止来自提供视觉刺激的显示器的光线对成像的干扰。
使用Matlab(Mathworks)的Psychtoolbox工具生成视觉刺激信号,并将其通过一个LCD显示器(Dell,1280×1024像素,75Hz刷新频率)呈现在小鼠手术部位对侧的眼睛前面,LCD显示器距离小鼠眼睛15cm。刺激为全屏幕正弦波移动明暗光栅,背景灰度均匀(空间频率:0.05cycles/°,时间频率:2hz)。每次刺激持续1秒钟,每一种刺激方式重复5次,两次刺激之间的间隔时间为1-2秒。显示器上移动光栅设置了4个角度,每个角度有两个移动方向(共8个移动方向)。相近角度的光栅之间相差45度。
利用LotosScan显微镜(LotosScan,苏州生物医学工程与技术研究所)与Ti:Sa激光器(Chameleon VISION-S,Coherent)进行轴突末端的双光子荧光成像。激发波长固定在920nm,成像使用40X,0.8NA的物镜(尼康)。光束尺寸大到足以填满40倍物镜的后孔径,以50赫兹的帧率获取图像。
使用Matlab(Mathworks)和ImageJ(美国国立卫生研究院)对图像进行分析。针对成像过程中图像的横向漂移,使用基于刚体变化的逐帧对齐原理的图像处理软件TurboR(ImageJ插件)对图像进行了处理。根据突触末端的大小、形状和亮度,人为选择轴突末端。通过对轴突末端每帧图像荧光强度的平均值,确定单个轴突末端成像的时间进程。如果在成像过程中大脑搏动明显,则这些数据不被使用。成像过程中由神经纤维网造成的背景信号通过之前报道的方法被清除掉。在对图像进行校正之后,对每个视觉刺激表现的反应(Ft,轴突末端的荧光强度)利用紧接刺激开始前0.2秒时轴突末端的荧光强度(F0)进行标准化。对于每一种视觉刺激,平均荧光强度的变化(ΔF/F)是通过计算同一刺激条件下所有刺激引起的荧光强度变化的平均值。具有视觉反应的细胞是通过比较在没有刺激和刺激条件下轴突末端荧光强度的方差分析而定义的。
1.7体外全细胞记录:
对小鼠进行深度麻醉,然后利用冰冷并且富含氧气(利用95%,O2,5%CO2气体充气)的脑片切片液对小鼠进行心脏灌流。脑片切片液的组成为:92mM氯化胆碱,2.5mMKCl,1.2mM NaH2PO4,30mM NaHCO3,10mM MgSO4,0.5mM CaCl2,25mM葡萄糖,5mM抗坏血酸钠,3mM丙酮酸钠,2mM硫脲。通过加入浓盐酸,将切割溶液的pH值调整为7.3-7.4,并将渗透压调整为310-315mOsm。从脑颅中取出脑组织后,用振动刀片切片机将包含上丘脑区的脑组织在切割溶液中冠状切成300-500μm厚的切片。切割后的脑片在切割溶液中在31-32℃条件下孵育15分钟,然后转移到室温富含氧的保存液中保存。保存液的组成为:92mM NaCl,30mMNaHCO3,1.25mM NaH2PO4,2.5mM KCl,2mM MgSO4,2mM CaCl2,25mM葡萄糖,20mM HEPES,5mM抗坏血酸钠,3mM丙酮酸钠,和2mM硫脲,pH值为7.3-7.4,渗透压为310-315mOsm。在保存液中保存一小时后,将切片转移到通氧的记录液中(119mM NaCl,24mM NaHCO3,1.25mMNaH2PO4,2.5mM KCl,2mM MgSO4,2mM CaCl2,12.5mM葡萄糖)。一只小鼠通常可以制备3-5个含有上丘的脑片,选取中间的脑片进行记录。
使用2-4MΩ玻璃电极,通过全细胞膜片钳技术记录脑片上神经元的突触反应。电极内溶液的组成为:25mM的葡萄糖酸钾,20mM的mM KCl,0.5mM EGTA,10mM HEPES-NaOH,10mM p-肌酸,4mM ATP-Mg,0.3mM GTP,pH 7.3。利用LED(Thorlabs,35mW/mm2)光源产生470nm的蓝光,并通过一个40倍的物镜(OLYMPUS)对脑片局部区域进行刺激。实验发现,5毫秒的刺激时间可以使所记录的突触后神经元反应饱和,神经元的输入电阻在1到5GΩ之间,串联电阻小于20MΩ。全细胞膜片钳技术记录的条件为:首次将膜电位保持在-70mV,给予蓝光刺激,并记录AMPA受体介导的突触后电流(在此膜电位下,NMDA受体活性被溶液中的镁离子阻断)。然后将膜电位切换到+55mv,记录AMPA和NMDA受体共同介导的混合电流。对多个脑片神经元进行记录,生成细胞总数N。然后在记录液中分别加入AMPA受体拮抗剂CNQX和NMDA受体拮抗剂D-APV再次进行记录。所有的记录信号均使用Axopatch700B进行放大信号,并使用Digidata1440模拟数字板进行数字化处理。使用pClamp软件在50khz频率下进行刺激和数据采集。所有的仪器设备和软件都有Axon Instruments/Molecular Devices(MolecularDevices,CA)公司购买。
1.8实验结果:
如图1所示,在正常的生理状态下在成体哺乳动物的视网膜中完全分化的神经细胞仍然具备再生潜能,可以从一种神经细胞类型向其他神经细胞类型转变,并可以进行位置迁移。
如图2所示,通过表达一种或几种转录因子的组合,可以在成年小鼠的视网膜中使高度分化的无长突神经元重编程为视网膜神经节细胞。再生的视网膜神经节细胞具备神经节细胞典型的特征,它们向视乳头伸展轴突。
如图3所示,再生的视网膜神经节细胞位于视网膜的最内层,向视神经伸展轴突,并表达节细胞特异性的标记性分子(RBPMS和Brn3A)以及节细胞亚型特异性的分子CART。
如图4所示,一些再生的视网膜神经节细胞表达alpha类型节细胞特异性的标记物SMI-32。
如图5所示,在视网膜中间细胞层的Lgr5阳性无长突中间神经元中表达节细胞发育过程中起重要调控作用的转录因子之后,一些Lgr5阳性无长突中间神经元从中间细胞层向节细胞层迁移。
如图6所示,再生的视网膜神经节细胞向视神经及大脑中负责视觉的不同区域投射轴突,并且在轴突末端形成典型的突触前结构。
如图7所示,单一转录因子(Brn3B或Sox4)能够将Lgr5阳性无长突细胞重编程为视网膜神经节细胞,Brn3B和Sox4组合大大提高重编程的效率。
如图8所示,本发明通过CRISP-Cas9介导的基因组编辑技术,在小鼠基因组的Prokr2基因位点引入了一个CreERT2-polyA序列。这种基因工程的小鼠在视网膜神经节细胞成的错位无长突细胞中特异性地表达CreERT2转录酶。这一基因组改造使我们能够对这些细胞进行调控,检验是否能够利用这一类神经元再生视网膜神经节细胞。
如图9所示,位于视网膜神经节细胞层的错位无长突细胞能够被重编成为视网膜神经节细胞。新生成的视网膜神经节细胞向视神经及大脑投射轴突建立视网膜和大脑的神经连接。
如图10所示,两种(Brn3B+Sox4)或三种(Atoh7+Brn3B+Sox4)转录因子的组合能够有效地将Prokr2阳性错位无长突神经元重编程为视网膜神经节细胞。
如图11所示,再生的视网膜神经节细胞能够整合视觉刺激产生的神经信号并将神经信号传递到大脑中负责视觉的神经元核团。再生的视网膜神经节细胞能够将视觉电信号传递到大脑中的下游神经元。
如图12所示,利用活体动物神经活性钙成像技术,我们发现再生的视网膜神经节细胞能够在动物体内将视觉神经电信号从视网膜传递到大脑。
如图13所示,本发明发现在动物体内原来的视网膜神经节细胞完全被损伤的情况下,新生的视网膜神经节细胞仍然能够长距离向大脑中负责视觉的神经核团投射轴突,并能将神经电信号传递到下游神经元。
如图14所示,本发明使用的由眼压升高引起的视网膜节细胞轴突损伤的模型能够完全损伤所以视网膜神经节细胞的轴突。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员,在不脱离本发明的精神和范围的情况下,当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化,均为本发明的等效实施例;同时,凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变,均仍属于本发明的技术方案的范围内。
序列表
<110> 上海科技大学
<120> 一种利用转录因子再生具备功能的视网膜神经节细胞的方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1236
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgatgatga tgtccctgaa cagcaagcag gcgttcagca tgcctcacgc aggcagcctg 60
cacgtggagc ccaagtactc ggcgctacac agtgcctccc cgggctcctc tgcgcccgcg 120
gcgccctcgg ccagttcccc tagcagctcc agcaacgctg gcggcggcgg cggtggcggc 180
ggaggcggag gcggcggcgg ccggagcagc agttccagca gcagtggcag cggcggcagc 240
ggcggcggcg ggggctcgga ggcgatgcgg agagcttgtc ttccaacccc accgagcaat 300
atattcggcg ggctggatga gagtctgctg gcccgtgccg aggctctggc cgccgtggac 360
atcgtctccc agagtaagag ccaccaccac catccgcccc accacagccc cttcaagccg 420
gacgccactt accacaccat gaacaccatc ccgtgcacgt cggcagcctc ctcttcttct 480
gtgcccatct cgcacccgtc cgctctggct ggcacccatc accaccacca ccaccaccat 540
caccaccatc accagccgca ccaggcgctg gagggcgagc tgcttgagca cctaagcccc 600
gggctggccc tgggagctat ggcgggcccc gacggcacgg tggtgtccac tccggctcac 660
gcaccacaca tggccaccat gaaccccatg caccaagcag ccctgagcat ggcccacgca 720
catgggctgc cctcgcacat gggctgcatg agcgacgtgg atgcagaccc gcgggacctg 780
gaggcgttcg ccgagcgttt caagcagcga cgcatcaagc tgggagtgac ccaggcagat 840
gtgggctcgg cgctggccaa cctcaagatc ccgggcgtgg gctcgctcag ccagagcacc 900
atctgcaggt ttgagtctct cacgctgtca cacaacaaca tgatcgcgct caagcccatc 960
ctgcaggcgt ggctggagga agctgagaaa tcccaccgcg agaagctcac taagccggag 1020
ctcttcaatg gcgcggagaa gaagcgcaag cgcacgtcca tcgcggcgcc ggagaagcgc 1080
tctctggaag cctacttcgc catccagcca aggccctcct cggagaagat cgcggccatc 1140
gccgaaaagc tggatctcaa gaaaaatgtg gtgcgcgtct ggttctgcaa ccagaggcag 1200
aaacagaaga gaatgaaata ctctgccggc atttag 1236
<210> 2
<211> 1323
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggtacaac agaccaacaa cgcggagaac actgaggctc tgctggccgg ggagagctcg 60
gactcgggcg ccggcctgga gctgggcatc gcgtcctccc cgacgcctgg ctccaccgcg 120
tcgacgggcg gcaaggcgga cgaccccagc tggtgcaaga cgcccagtgg ccacatcaag 180
cggcccatga acgcctttat ggtgtggtcg cagatcgagc ggcgcaagat catggagcag 240
tcgcccgaca tgcacaacgc cgagatctcc aagcggctag gcaaacgctg gaagctgctc 300
aaggacagcg acaagattcc gttcatccag gaggcggagc ggctgcgcct caagcacatg 360
gctgactacc ctgactacaa gtaccggccg cgaaagaagg tgaagtcggg caacgcgggc 420
gcgggatcgg cggccacagc caagccaggg gagaagggcg acaaggtcgc gggcagcagc 480
ggccacgcgg gaagcagcca cgcggggggt ggcgcgggcg gcagctccaa gcccgcgccc 540
aagaagagct gtggccccaa ggtggcgggc agctcggtcg gcaagcccca cgctaagctg 600
gtcccggcgg gcggcagcaa ggcggctgca tcgttctctc cagagcaagc tgccctgctg 660
cccctggggg agcccacggc cgtctacaag gtgcggactc ccagtgcggc cactccggcc 720
gcctcctcct cgccgtccag tgcgctggcc accccagcca aacaccctgc cgacaagaaa 780
gtgaagcgcg tctacctgtt tggaagcctg ggcgcttcgg cgtctcccgt cgggggcctg 840
ggagcgagcg ccgaccccag tgatccactg gggttgtacg aagatggagg cccgggatgc 900
tcgcccgatg gccggagtct gagcggccgc agcagcgcag catcatcgcc agccgccagc 960
cgatcgcccg ctgaccaccg cggctacgcc agcctacgcg cagcctcgcc cgccccgtcc 1020
agcgcgccct cgcacgcgtc ctcctcgctc tcctcgtcct cttcctcctc ctcgggctct 1080
tcgtcgtccg acgacgagtt cgaagacgac ctgctcgacc tgaaccccag ctcaaacttt 1140
gagagcatgt ccctgggcag tttcagctcc tcatcggcgc tcgatcggga cctggatttt 1200
aacttcgaac ccggctcagg ctcccacttc gaattcccgg actattgcac gcccgaggtg 1260
agcgagatga tctcgggaga ttggctggag tccagcatct ctaacctggt cttcacctac 1320
tga 1323
<210> 3
<211> 450
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaagtcgg cctgcaaacc ccacggccct ccggcgggag ctcgcggcgc gcccccgtgc 60
gcgggcgcag ccgagcgcgc ggtctcgtgc gcggggcccg ggcggctgga gagcgcggcg 120
cgcaggcgtc tggcggccaa cgcgcgcgag cggcgccgca tgcaggggct gaacacggcg 180
ttcgaccggc tgcgcagggt ggtgccgcag tggggccagg acaagaagct gtccaagtac 240
gagacactgc agatggcgct cagctacatc atcgcgctca cccgcatcct agccgaagcc 300
gagcgggact gggtcgggct gcgctgcgag cagcggggcc gcgatcaccc ctacctccct 360
ttcccgggtg ctaggctcca ggtagaccct gagccctatg ggcagaggct cttcggcttc 420
cagccggagc ccttccccat ggccagctaa 450
<210> 4
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggtgcagc aggccgagag ctcggaagcc gagagcaacc tgccccggga cgcgctggac 60
accgaggagg gcgagttcat ggcgtgcagc ccggtggccc tggacgagag cgacccggac 120
tggtgcaaga cggcgtcggg ccacatcaaa cggcccatga acgccttcat ggtgtggtcc 180
aagatcgagc gcaggaagat catggagcag tcgcccgaca tgcacaacgc cgagatctcc 240
aagaggctgg gcaagcgctg gaagatgctg aaggacagcg agaagatccc gttcatcagg 300
gaggcggagc gcctgcgcct caagcacatg gctgattatc ccgactacaa gtaccggccg 360
cgcaaaaagc ccaagacgga cccagcggcc aagcccagcg cgggccagag ccccgacaag 420
agcgcggcgg gcgccaaggc agccaagggc cccggcaaga agtgcgccaa gctcaaggcg 480
cctgcgggca aggcgggcgc gggcaaggcg gcgcagccgg gggactgcgc cgcgggcaag 540
gcagccaagt gcgtcttcct ggacgacgac gatgaagacg acgacgaaga tgacgagctg 600
cagctacggc ccaagccgga cgctgacgac gacgacgacg agcccgcgca ctcgcacctg 660
ctgccgccgc cgacgcagca gcaaccccct cagctgctga ggcgctacag cgtggccaag 720
gtccccgcca gccccacgct cagcagtgcc gccgagtccc ccgagggcgc gagcctgtac 780
gacgaagtgc gcgcgggcgg ccggctctac tacagcttca agaacatcac caagcagcag 840
cctccgcccg cgcctcccgc gctgtcgccc gcgtcctccc gctgcgtgtc cacctcctca 900
tccagcggca gcagcagcgg cagcggcgcc gaggatgcag acgacctcat gttcgacctg 960
agcttgaatt tctcccaggg cgcgcacagc gcctgcgagc agccactggg cgcgggagcg 1020
gcggggaacc tgtccctgtc gctggtggat aaggacctgg attccttcag cgagggcagc 1080
ctgggttccc acttcgagtt ccccgactac tgcacgccgg agctgagcga gatgatcgcg 1140
ggggactggc tggaggcgaa cttctccgac ctggtgttca cgtattga 1188
<210> 5
<211> 1050
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgggagaca tgggcgatcc accaaaaaaa aaacgtctga tttccctgtg tgttggttgc 60
ggcaatcaaa ttcacgacca gtatattctg agggtttctc cggatttgga gtggcatgca 120
gcatgtttga aatgtgcgga gtgtaatcag tatttggacg aaagctgtac gtgctttgtt 180
agggatggga aaacctactg taaaagagat tatatcaggt tgtacgggat caaatgcgcc 240
aagtgcagca taggcttcag caagaacgac ttcgtgatgc gtgcccgctc taaggtgtac 300
cacatcgagt gtttccgctg tgtagcctgc agccgacagc tcatcccggg agacgaattc 360
gccctgcggg aggatgggct tttctgccgt gcagaccacg atgtggtgga gagagccagc 420
ctgggagctg gagaccctct cagtcccttg catccagcgc ggcctctgca aatggcagcc 480
gaacccatct cggctaggca gccagctctg cggccgcacg tccacaagca gccggagaag 540
accacccgag tgcggactgt gctcaacgag aagcagctgc acaccttgcg gacctgctat 600
gccgccaacc ctcggccaga tgcgctcatg aaggagcaac tagtggagat gacgggcctc 660
agtcccagag tcatccgagt gtggtttcaa aacaagcggt gcaaggacaa gaaacgcagc 720
atcatgatga agcagctcca gcagcagcaa cccaacgaca aaactaatat ccaggggatg 780
acaggaactc ccatggtggc tgctagtccg gagagacatg atggtggttt acaggctaac 840
ccagtagagg tgcaaagtta ccagccgccc tggaaagtac tgagtgactt cgccttgcaa 900
agcgacatag atcagcctgc ttttcagcaa ctggtcaatt tttcagaagg aggaccaggc 960
tctaattcta ctggcagtga agtagcatcg atgtcctcgc agctcccaga tacacccaac 1020
agcatggtag ccagtcctat tgaggcatga 1050
<210> 6
<211> 1227
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgatgatgt ccctgaacag caagcaggcg tttagcatgc cgcacggcgg cagcctgcac 60
gtggagccca agtactcggc actgcacagc acctcgccgg gctcctcggc tcccatcgcg 120
ccctcggcca gctcccccag cagctcgagc aacgctggtg gtggcggcgg cggcggcggc 180
ggcggcggcg gcggcggagg ccgaagcagc agctccagca gcagtggcag cagcggcggc 240
gggggctcgg aggctatgcg gagagcctgt cttccaaccc caccgagcaa tatattcggc 300
gggctggatg agagtctgct ggcccgcgcc gaggctctgg cagccgtgga catcgtctcc 360
cagagcaaga gccaccacca ccatccaccc caccacagcc ccttcaaacc ggacgccacc 420
taccacacta tgaataccat cccgtgcacg tcggccgcct cttcttcatc ggtgcccatc 480
tcgcaccctt ccgcgttggc gggcacgcac caccaccacc accatcacca ccaccaccac 540
caccaaccgc accaggcgct ggagggcgag ctgctggagc acctgagtcc cgggctggcc 600
ctgggcgcta tggcgggccc cgacggcgct gtggtgtcca cgccggctca cgcgccgcac 660
atggccacca tgaaccccat gcaccaagca gcgctcagca tggcccacgc gcacgggctg 720
ccgtcgcaca tgggctgcat gagcgacgtg gacgccgacc cgcgggacct ggaggcattc 780
gccgagcgct tcaagcagcg acgcatcaag ctgggggtga cccaggcaga tgtgggctcc 840
gcgctggcca acctcaagat ccccggcgtg ggctcgctta gccagagcac catctgcagg 900
ttcgagtccc tcacactgtc ccacaataat atgatcgcgc tcaaacccat cctgcaggca 960
tggctcgagg aggccgagaa gtcccaccgc gagaagctca ccaagcctga actcttcaat 1020
ggcgcggaga agaagcgcaa gcgcacgtcc atcgctgcgc cagagaagcg ctcgctcgaa 1080
gcctactttg ccattcagcc tcggccctcc tctgaaaaga tcgccgccat cgcggagaag 1140
ctggacctga agaaaaacgt ggtgcgcgtc tggttctgca accagaggca gaaacagaaa 1200
agaatgaaat attccgccgg catttag 1227
<210> 7
<211> 1425
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggtgcagc aaaccaacaa tgccgagaac acggaagcgc tgctggccgg cgagagctcg 60
gactcgggcg ccggcctcga gctgggaatc gcctcctccc ccacgcccgg ctccaccgcc 120
tccacgggcg gcaaggccga cgacccgagc tggtgcaaga ccccgagtgg gcacatcaag 180
cgacccatga acgccttcat ggtgtggtcg cagatcgagc ggcgcaagat catggagcag 240
tcgcccgaca tgcacaacgc cgagatctcc aagcggctgg gcaaacgctg gaagctgctc 300
aaagacagcg acaagatccc tttcattcga gaggcggagc ggctgcgcct caagcacatg 360
gctgactacc ccgactacaa gtaccggccc aggaagaagg tgaagtccgg caacgccaac 420
tccagctcct cggccgccgc ctcctccaag ccgggggaga agggagacaa ggtcggtggc 480
agtggcgggg gcggccatgg gggcggcggc ggcggcggga gcagcaacgc ggggggagga 540
ggcggcggtg cgagtggcgg cggcgccaac tccaaaccgg cgcagaaaaa gagctgcggc 600
tccaaagtgg cgggcggcgc gggcggtggg gttagcaaac cgcacgccaa gctcatcctg 660
gcaggcggcg gcggcggcgg gaaagcagcg gctgccgccg ccgcctcctt cgccgccgaa 720
caggcggggg ccgccgccct gctgcccctg ggcgccgccg ccgaccacca ctcgctgtac 780
aaggcgcgga ctcccagcgc ctcggcctcc gcctcctcgg cagcctcggc ctccgcagcg 840
ctcgcggccc cgggcaagca cctggcggag aagaaggtga agcgcgtcta cctgttcggc 900
ggcctgggca cgtcgtcgtc gcccgtgggc ggcgtgggcg cgggagccga ccccagcgac 960
cccctgggcc tgtacgagga ggagggcgcg ggctgctcgc ccgacgcgcc cagcctgagc 1020
ggccgcagca gcgccgcctc gtcccccgcc gccggccgct cgcccgccga ccaccgcggc 1080
tacgccagcc tgcgcgccgc ctcgcccgcc ccgtccagcg cgccctcgca cgcgtcctcc 1140
tcggcctcgt cccactcctc ctcttcctcc tcctcgggct cctcgtcctc cgacgacgag 1200
ttcgaagacg acctgctcga cctgaacccc agctcaaact ttgagagcat gtccctgggc 1260
agcttcagtt cgtcgtcggc gctcgaccgg gacctggatt ttaacttcga gcccggctcc 1320
ggctcgcact tcgagttccc ggactactgc acgcccgagg tgagcgagat gatctcggga 1380
gactggctcg agtccagcat ctccaacctg gttttcacct actga 1425
<210> 8
<211> 459
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgaagtcct gcaagcccag cggcccgccg gcgggagcgc gcgttgcacc cccgtgcgcg 60
ggcggcaccg agtgcgcggg cacgtgcgcc ggggccgggc ggctggagag cgcggcgcgc 120
aggcgcctgg cggccaacgc gcgcgagcgc cgccgcatgc aggggctcaa cactgccttc 180
gaccgcttac gcagggtggt tccccagtgg ggccaggata aaaagctgtc caagtacgag 240
accctgcaga tggccctgag ctacatcatg gctctgaccc ggatcctggc cgaggccgag 300
cgattcggct cggagcggga ctgggtgggt ctccactgtg agcacttcgg ccgcgaccac 360
tacctcccgt tcccgggcgc gaagctgccg ggcgagagcg agctgtacag ccagagactc 420
ttcggcttcc agcccgagcc cttccagatg gccacctag 459
<210> 9
<211> 1326
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggtgcagc aggcggagag cttggaagcg gagagcaacc tgccccggga ggcgctggac 60
acggaggagg gcgaattcat ggcttgcagc ccggtggccc tggacgagag cgacccagac 120
tggtgcaaga cggcgtcggg ccacatcaag cggccgatga acgcgttcat ggtatggtcc 180
aagatcgaac gcaggaagat catggagcag tctccggaca tgcacaacgc cgagatctcc 240
aagaggctgg gcaagcgctg gaaaatgctg aaggacagcg agaagatccc gttcatccgg 300
gaggcggagc ggctgcggct caagcacatg gccgactacc ccgactacaa gtaccggccc 360
cggaaaaagc ccaaaatgga cccctcggcc aagcccagcg ccagccagag cccagagaag 420
agcgcggccg gcggcggcgg cgggagcgcg ggcggaggcg cgggcggtgc caagacctcc 480
aagggctcca gcaagaaatg cggcaagctc aaggcccccg cggccgcggg cgccaaggcg 540
ggcgcgggca aggcggccca gtccggggac tacgggggcg cgggcgacga ctacgtgctg 600
ggcagcctgc gcgtgagcgg ctcgggcggc ggcggcgcgg gcaagacggt caagtgcgtg 660
tttctggatg aggacgacga cgacgacgac gacgacgacg agctgcagct gcagatcaaa 720
caggagccgg acgaggagga cgaggaacca ccgcaccagc agctcctgca gccgccgggg 780
cagcagccgt cgcagctgct gagacgctac aacgtcgcca aagtgcccgc cagccctacg 840
ctgagcagct cggcggagtc ccccgaggga gcgagcctct acgacgaggt gcgggccggc 900
gcgacctcgg gcgccggggg cggcagccgc ctctactaca gcttcaagaa catcaccaag 960
cagcacccgc cgccgctcgc gcagcccgcg ctgtcgcccg cgtcctcgcg ctcggtgtcc 1020
acctcctcgt ccagcagcag cggcagcagc agcggcagca gcggcgagga cgccgacgac 1080
ctgatgttcg acctgagctt gaatttctct caaagcgcgc acagcgccag cgagcagcag 1140
ctggggggcg gcgcggcggc cgggaacctg tccctgtcgc tggtggataa ggatttggat 1200
tcgttcagcg agggcagcct gggctcccac ttcgagttcc ccgactactg cacgccggag 1260
ctgagcgaga tgatcgcggg ggactggctg gaggcgaact tctccgacct ggtgttcaca 1320
tattga 1326
<210> 10
<211> 1050
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgggagaca tgggagatcc accaaaaaaa aaacgtctga tttccctatg tgttggttgc 60
ggcaatcaga ttcacgatca gtatattctg agggtttctc cggatttgga atggcatgcg 120
gcatgtttga aatgtgcgga gtgtaatcag tatttggacg agagctgtac atgctttgtt 180
agggatggga aaacctactg taaaagagat tatatcaggt tgtacgggat caaatgcgcc 240
aagtgcagca tcggcttcag caagaacgac ttcgtgatgc gtgcccgctc caaggtgtat 300
cacatcgagt gtttccgctg tgtggcctgc agccgccagc tcatccctgg ggacgaattt 360
gcgcttcggg aggacggtct cttctgccga gcagaccacg atgtggtgga gagggccagt 420
ctaggcgctg gcgacccgct cagtcccctg catccagcgc ggccactgca aatggcagcg 480
gagcccatct ccgccaggca gccagccctg cggccccacg tccacaagca gccggagaag 540
accacccgcg tgcggactgt gctgaacgag aagcagctgc acaccttgcg gacctgctac 600
gccgcaaacc cgcggccaga tgcgctcatg aaggagcaac tggtagagat gacgggcctc 660
agtccccgtg tgatccgggt ctggtttcaa aacaagcggt gcaaggacaa gaagcgaagc 720
atcatgatga agcaactcca gcagcagcag cccaatgaca aaactaatat ccaggggatg 780
acaggaactc ccatggtggc tgccagtcca gagagacacg acggtggctt acaggctaac 840
ccagtggaag tacaaagtta ccagccacct tggaaagtac tgagcgactt cgccttgcag 900
agtgacatag atcagcctgc ttttcagcaa ctggtcaatt tttcagaagg aggaccgggc 960
tctaattcca ctggcagtga agtagcatca atgtcctctc aacttccaga tacacctaac 1020
agcatggtag ccagtcctat tgaggcatga 1050
Claims (13)
1.转录因子在以下一项或多项中的用途:
1)制备视网膜疾病治疗药物;
2)制备视网膜神经节细胞再生产品;
3)制备诱导成体细胞重编程为视网膜神经节细胞的产品;
4)制备视网膜神经节细胞;
所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
2.如权利要求1所述的用途,其特征在于,还包括以下特征中的一项或多项:
a.所述转录因子为人源或鼠源;
b.所述视网膜疾病选自视网膜色素变性、黄斑变性、青光眼或遗传学视神经病变;
c.所述转录因子为DNA、mRNA或蛋白形式;
d.所述Brn3B的cDNA序列如SEQ ID NO:1或SEQ ID NO:6所示;所述Sox4的cDNA序列如SEQ ID NO:2或SEQ ID NO:7所示;所述Atoh7的cDNA序列如SEQ ID NO:3或SEQ ID NO:8所示;所述Sox11的cDNA序列如SEQ ID NO:4或SEQ ID NO:9所示;所述Isl1的cDNA序列如SEQID NO:5或SEQ ID NO:10所示;
e.所述视网膜疾病治疗药物或视网膜神经节细胞再生产品用于给予受试对象的眼睛;
f.所述产品为试剂盒、注射剂或药物。
3.如权利要求1或2所述的用途,其特征在于,所述成体细胞选自无长突神经细胞或错位无长突神经细胞或视网膜中的其他细胞中的一种或多种。
4.一种视网膜神经节细胞的制备方法,至少包括以下步骤:将转录因子施用于成体细胞使其转化为视网膜神经节细胞;所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
5.如权利要求4所述的视网膜神经节细胞的制备方法,其特征在于,还包括以下特征中的一项或多项:
a.所述成体细胞选自无长突神经细胞或错位无长突神经细胞或视网膜中的其他细胞中的一种或多种;
b.所述转录因子为DNA、mRNA或蛋白形式;
c.所述转录因子为人源或鼠源;
d.所述成体细胞来源于哺乳动物;
e.所述Brn3B的cDNA序列如SEQ ID NO:1或SEQ ID NO:6所示;所述Sox4的cDNA序列如SEQ ID NO:2或SEQ ID NO:7所示;所述Atoh7的cDNA序列如SEQ ID NO:3或SEQ ID NO:8所示;所述Sox11的cDNA序列如SEQ ID NO:4或SEQ ID NO:9所示;所述Isl1的cDNA序列如SEQID NO:5或SEQ ID NO:10所示。
6.如权利要求4所述的视网膜神经节细胞的制备方法,至少包括以下步骤:将转录因子编码基因转染至成体细胞使其转化为视网膜神经节细胞。
7.如权利要求6所述的视网膜神经节细胞的制备方法,其特征在于,所述转录因子编码基因通过重组载体转染至成体细胞,所述重组载体为将所述转录因子编码基因插入表达载体,得到的重组载体。
8.如权利要求7所述的视网膜神经节细胞的制备方法,所述表达载体选自AAV病毒载体。
9.一种重组细胞,为在宿主细胞中引入能表达转录因子的多核苷酸或者核酸构建体后获得,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
10.一种产品,其特征在于,所述产品包括转录因子,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个,所述产品适用于视网膜神经节细胞再生、诱导成体细胞重编程为视网膜神经节细胞或治疗视网膜疾病。
11.如权利要求10所述的产品,其特征在于,还包括以下特征中的一项或多项:
a.所述转录因子为DNA、mRNA或蛋白形式;
b.所述转录因子为人源或鼠源。
12.一种治疗视网膜疾病的方法,至少包括以下步骤:向患有视网膜疾病的对象给予有效量的转录因子,所述转录因子选自Brn3B、Sox4、Atoh7、Sox11和Isl1中任意一个或多个。
13.如权利要求12所述的治疗视网膜疾病的方法,其特征在于,还包括以下特征的一项或多项:
a.所述转录因子为DNA、mRNA或蛋白形式;
b.所述转录因子为人源或鼠源;
c.所述成体细胞来源于哺乳动物;
d.所述Brn3B的cDNA序列如SEQ ID NO:1或SEQ ID NO:6所示;所述Sox4的cDNA序列如SEQ ID NO:2或SEQ ID NO:7所示;所述Atoh7的cDNA序列如SEQ ID NO:3或SEQ ID NO:8所示;所述Sox11的cDNA序列如SEQ ID NO:4或SEQ ID NO:9所示;所述Isl1的cDNA序列如SEQID NO:5或SEQ ID NO:10所示;
e.所述转录因子用于给予受试对象的眼睛。
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CN202010047628.2A CN113134076A (zh) | 2020-01-16 | 2020-01-16 | 一种利用转录因子再生具备功能的视网膜神经节细胞的方法 |
KR1020227027252A KR20220130150A (ko) | 2020-01-16 | 2021-01-15 | 망막 신경절 세포의 재생 |
EP21741592.6A EP4090751A4 (en) | 2020-01-16 | 2021-01-15 | REGENERATION OF RETINAL NODE CELLS |
AU2021208775A AU2021208775A1 (en) | 2020-01-16 | 2021-01-15 | Regeneration of retinal ganglion cells |
CA3165427A CA3165427A1 (en) | 2020-01-16 | 2021-01-15 | Regeneration of retinal ganglion cells |
CN202180009064.6A CN115943213A (zh) | 2020-01-16 | 2021-01-15 | 视网膜神经节细胞的再生 |
IL294358A IL294358A (en) | 2020-01-16 | 2021-01-15 | Regeneration of retinal ganglion cells |
PCT/CN2021/072108 WO2021143827A1 (en) | 2020-01-16 | 2021-01-15 | Regeneration of retinal ganglion cells |
JP2022543585A JP2023512473A (ja) | 2020-01-16 | 2021-01-15 | 網膜神経節細胞の再生 |
US17/812,381 US20220347320A1 (en) | 2020-01-16 | 2022-07-13 | Regeneration of retinal ganglion cells |
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CN115058396A (zh) * | 2022-07-29 | 2022-09-16 | 西南医科大学 | 一种逆行标记小鼠视网膜节细胞的方法 |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012061045A2 (en) * | 2010-11-01 | 2012-05-10 | Massachusetts Eye And Ear Infirmary | Methods and compositions for preserving retinal ganglion cells |
WO2017176810A1 (en) * | 2016-04-04 | 2017-10-12 | Biotime, Inc. | Pluripotent stem cell-derived 3d retinal tissue and uses thereof |
CN110305846A (zh) * | 2018-03-20 | 2019-10-08 | 中山大学中山眼科中心 | 视网膜节细胞的制备方法 |
CN110302398A (zh) * | 2019-07-08 | 2019-10-08 | 向孟清 | 一种含有Atoh7和/或Pou4f的组合物、其制备方法及医药用途 |
-
2020
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-
2022
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012061045A2 (en) * | 2010-11-01 | 2012-05-10 | Massachusetts Eye And Ear Infirmary | Methods and compositions for preserving retinal ganglion cells |
WO2017176810A1 (en) * | 2016-04-04 | 2017-10-12 | Biotime, Inc. | Pluripotent stem cell-derived 3d retinal tissue and uses thereof |
CN110305846A (zh) * | 2018-03-20 | 2019-10-08 | 中山大学中山眼科中心 | 视网膜节细胞的制备方法 |
CN110302398A (zh) * | 2019-07-08 | 2019-10-08 | 向孟清 | 一种含有Atoh7和/或Pou4f的组合物、其制备方法及医药用途 |
Non-Patent Citations (6)
Title |
---|
CHAI-AN MAO 等: "Reprogramming amacrine and photoreceptor progenitors into retinal ganglion cells by replaceing Neurod1 with Atoh7", 《DEVELOPMENT》 * |
FUGUO WU 等: "Two transcription factors, Pou4f2 and Isl1, are sufficient to specify the retinal ganglion cell fate", 《PNAS》 * |
GERVASIO MARTÍN-PARTIDO 等: "The role of Islet-1 in cell specification, differentiation, and maintenance of phenotypes in the vertebrate neural retina", 《NEURAL REGENERATION RESEARCH》 * |
MENGFEI CHEN 等: "Lgr5+ amacrine cells possess regenerative potential in the retina of adult mice", 《AGING CELL》 * |
YING JIANG 等: "Transcription Factors SOX4 and SOX11 Function Redundantly to Regulate the Development of Mouse Retinal Ganglion Cells", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
吴维霖 等: "Brn3b 基因对视神经损伤条件下视网膜神经节细胞的保护作用", 《眼科新进展》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115058396A (zh) * | 2022-07-29 | 2022-09-16 | 西南医科大学 | 一种逆行标记小鼠视网膜节细胞的方法 |
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CN115943213A (zh) | 2023-04-07 |
KR20220130150A (ko) | 2022-09-26 |
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