CN116966202A - iPS诱导定向分化成的神经干细胞联合药物用于治疗阿尔茨海默症的用途 - Google Patents
iPS诱导定向分化成的神经干细胞联合药物用于治疗阿尔茨海默症的用途 Download PDFInfo
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Abstract
本发明涉及iPS诱导定向分化成的神经干细胞联合药物用于治疗阿尔茨海默症的用途。本发明提供了一种用于治疗阿尔茨海默症的药物组合物，其中所述药物组合物含有本发明特异性制备的神经干细胞和斯皮诺素。神经干细胞与斯皮诺素一起治疗AD能够从根本上通过分化为功能性神经元，促进海马突触素的表达，抑制细胞凋亡，抑制神经元纤维缠结，进而修复神经功能，具有较好的治疗前景。
Description
技术领域
本申请涉及生物领域，具体的涉及iPS诱导定向分化成的神经干细胞联合药物用于治疗阿尔茨海默症的用途。
背景技术
阿尔茨海默病(AD)是一个多病因的神经退行性疾病，炎症与氧化应激，代谢障碍，钙离子通道受损，线粒体障碍，神经营养因子(NT)缺乏等都与AD的发生密切相关，这些病因导致了AD的治疗缺乏特异性。神经元的损伤和丢失是大多数神经退行性疾病的共同的最后通路，AD复杂的病理改变，最终导致胆碱能神经元损伤和丢失，引起学习记忆障碍。目前临床上AD的治疗如药物治疗、基因治疗、康复训练等只能改善症状，并不能阻止疾病的进展。近年来，神经再生理论的发展和干细胞体外分离及培养的成功，为神经退行性疾病的治疗提供了一个崭新的视野，尤其是神经干细胞(NSCs)在AD的治疗上发挥了关键的作用。
iPSCs是将已分化的体细胞经过特殊基因重编程恢复到未分化原始水平，使其具有干细胞的基本特性。iPSCs具有自我更新和多向分化的潜能，可从患者自身取材，不存在免疫排斥、伦理等问题，为干细胞治疗带来了新的希望。近十年来，iPSCs在各领域研究颇多，由iPSCs来源的细胞在神经科学领域如AD，PD及ALS等疾病的治疗方面取得了较满意的成绩。人iPSCs体外诱导分化为胆碱能表型的神经元细胞，将此种细胞移植PDAPP小鼠(一种年龄依赖性Aβ沉积和渐进性认知障碍的转基因鼠)双侧海马，结果显示移植的细胞存活，并能分化为胆碱乙酰转移酶(ChAT)阳性神经元及γ-氨基丁酸(GABA)能神经元，细胞移植组小鼠的空间记忆能力明显提高。除了细胞移植外，iPSCs在人类疾病模型和药物监测方面也具有极大的发挥空间。
NSCs 是具有自我更新和多向分化潜能的原始细胞 ，可分化为神经元 、星形胶质细胞及少突胶质细胞等多种类型的神经细胞 ，且具有高迁移 、高播散及低免疫原性的特点。实验证实，将外源性的NSCs移植到体内后，NSCs能向病灶迁移，并分化为特定部位的相应细胞，其分化方向既与NSCs的内在特性有关，也与所处微环境密切相关。例如过量表达淀粉样蛋白前体将会导致移植的NSCs产生更多的星形胶质细胞而不是神经元。在体外通过细胞因子将NSCs诱导为特定细胞后移植到损伤部位，或者是直接将NSCs移植到损伤部位后同时注入相关的细胞因子，这两种方法都可以改变NSCs局部微环境，进而促进其增殖分化。但到目前为止尚没有发现利用某些细胞因子能将NSCs全部诱导为所需功能的神经细胞。将NSCs移植到海马区后，多种细胞因子产生增多，如N‐甲基‐D‐天冬氨酸2B单位、突触素、蛋白激酶Cζ亚型、酪氨酸受体激酶B及脑源性神经营养因子等，且长时程增强效应增强，AD小鼠的空间学习和记忆能力得到改善。研究发现，将NSCs移植到12个月大小的转基因AD小鼠的海马区后，与未移植NSCs的对照组比较，在水迷宫实验中实验组小鼠的空间记忆和学习能力明显优于对照组，冰冻切片结果也显示实验组的神经元细胞明显多于对照组，提示NSCs移植到海马区后能够分化为神经元，进而改善了AD小鼠的学习和记忆能力。有研究发现，雪旺细胞及嗅鞘细胞分别与NSCs联合移植，NSCs的存活率均较单独移植NSCs高，且分化为胆碱能神经元的数量也增多，AD大鼠的学习记忆能力改善也更明显。到目前为止，尽管许多基础研究表明，移植NSCs在AD治疗中效果明显。
内源性神经再生损害或神经再生不足导致功能性神经元减少，最终引起AD认知功能受损。NSCs移植缓解AD模型鼠的学习记忆障碍，一方面移植的NSCs存活、迁移、并分化为神经元，补充丢失的神经细胞;另一方面移植的NSCs分泌NT，保护移植细胞的存活，并部分改善AD脑内的病理微环境，促进内源性NSCs激活。尽管干细胞治疗在哺乳动物中有较多的证据，但是干细胞治疗AD的临床试验寥寥可数。细胞移植治疗可以从根本上解决AD中神经细胞丢失的问题，其可能的机制为:替换损伤和丢失的神经细胞，干细胞可分化为胆碱能神经元，与宿主整合，修复神经通路，直接替代丢失的神经元，是能够治愈神经退行性疾病的根本原因;分泌营养因子，干细胞可分泌神经营养因子如NGF、脑源性神经营养因子(BDNF)、NT-3等促进细胞存活，增加突触连接，改善认知功能;抗淀粉样蛋白生成，干细胞移植减少Aβ水平，减轻Aβ毒性反应，有利于移植细胞存活及认知恢复；抗炎症反应，干细胞移植减少促炎因子白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α、诱导型一氧化氮合酶(iNOS)表达，发挥神经保护作用；促进内源性NSCs激活，外源性NSCs移植改善脑内微环境，有利于内源性NSCs存活并刺激其激活；提高脑内神经元代谢活动，干细胞移植增加神经元之间连接及代谢，改善认知功能。大量研究表明，将NSCs移植到脑内，移植的NSCs可在体内存活、迁移并分化为功能性神经元，整合入宿主神经回路，修复神经功能。但是目前，采用ips诱导的NSC细胞用于AD的治疗研究还不够多，特别是联合其他药物一起治疗的形式还不够多，有待于进一步的研究。
发明内容
本发明针对现有技术的不足，提供了一种诱导性神经干细胞联合其他药物用于治疗AD的方法。
具体的所述的诱导性神经干细胞为申请人在先专利申请（申请号：CN202311119527.1）中明确记载的方法制备获得的。
具体的，将红细祖细胞重编程制备并获得相应的ips细胞，将红细祖细胞重编程制备并获得相应的ips细胞是通过将红细祖细胞通过电转化 Epi5TM Episomal iPSC 重编程试剂盒中的重编程因子后在mTeSRTM培养基培养获得相应的ips细胞。将所述ips细胞在诱导分化培养基中培养后获得神经干细胞，所述的诱导分化培养基组成为DMEM/F12中添加2%B27 Supplement、1% N2 Supplement，20 ng/ml bFGF，10-500μg/mL Caveolin1-4B7单抗组成；其中，所述Caveolin1-4B7单抗的重链可变区序列如SEQ ID NO：1所示，轻链可变区序列如SEQ ID NO:2所示。
具体的，所述Caveolin1-4B7单抗为特异性针对窖蛋白1的鼠抗单克隆抗体。其是通过表位活性肽：TVSKLLEKVRKVSVNVKTVRGSL免疫小鼠后通过杂交瘤技术制备获得的。如在前在专利申请号为CN202311119527.1中明确记载的那样，该单抗小鼠腹水的效价为6.4×106。单抗Caveolin1-4B7能够有效的抑制细胞中的窖蛋白1的表达，并且具有剂量依赖性的抑制效果，在200μg/mL的浓度条件下，窖蛋白1相对表达量不到0.08，抑制效果较好。同时，采用，通过亲和力测定，本发明的单克隆抗体Caveolin1-4B7与表位肽的亲和力为1.53nM，亲和特性较好。
更进一步的，本发明还提供了一种用于治疗阿尔茨海默症的药物组合物，其中所述药物组合物含有本发明特异性制备的神经干细胞和斯皮诺素。
斯皮诺素通过抑制 COX-2蛋白过表达，减轻炎症，减少细胞凋亡，改善AD症状。
本发明申请人通过众多的组合实验，鉴定获得了将神经干细胞与斯皮诺素一起治疗AD能够从根本上通过分化为功能性神经元，抑制细胞凋亡，进而修复神经功能，具有较好的治疗前景。
进一步的，所述红细胞祖细胞可以是商业购买的，也可以是直接从血液中分离制备获得的。
所述分离，是采用RosetteSep™ Human Progenitor Cell Basic Pre-Enrichm祖细胞预富集混合物后分离制备的。
进一步的，所述红细胞祖细胞通过电转化 Epi5TM Episomal iPSC 重编程试剂盒中的重编程因子后在干细胞培养基中培养获得相应的ips细胞。
进一步的，重编程因子是本领域熟知的“有助于将靶细胞重编程为诱导性多能干细胞的因子”，具体的是能够帮助诱导靶细胞重编程为诱导性多能干细胞的因子，其中所述因子选自Oct3/4和属于Myc、Klf和Sox因子家族的因子，这类重编程因子包括例如，Oct3/4、Sox2、Sox1、Sox3、c-Myc、n-Myc、l-Myc、Klf1、Klf2、Klf4和Klf5等因子，或这些因子的具有保留的重编程能力的突变体。所述对重编程的帮助可为如下形式：例如将细胞的甲基化模式变为与干细胞类似的甲基化模式，将细胞的表达模式转换为干细胞的表达模式，或者通过将组蛋白的结合调节为与干细胞中所观察到的相类似来影响聚合核DNA的构象，其中，通过适合的重编程因子，上述的每种形式都可单独或以组合的方式得以实现。除上述的因子之外，技术人员还了解鉴定其他适合的重编程因子的方法，诸如亚硫酸氢盐基因组测序、RT-PCR、实时PCR、微列阵分析、染色体组型分析、畸胎瘤形成、碱性磷酸酶染色等方法。
在一些方面，重编程载体中将包括编码Sox和Oct(特别是Oct3/4)的核酸。例如，一个或多个重编程载体可以包含编码Sox2、Oct4、Nanog和任选Lin28的表达盒，或编码Sox2、Oct4、Klf4和任选c-Myc的表达盒，或编码Sox2、Oct4和任选Esrrb的表达盒，或编码Sox2、Oct4、Nanog、Lin-28、Klf4、c-Myc和任选SV40大的T抗原的表达盒。编码这些重编程因子的核酸可以包含在同一表达盒中、不同表达盒中、同一重编程载体中，或不同重编程载体中。Oct4和Sox基因家族的一些成员(Sox1、Sox2、Sox3和Sox15)已经被鉴定为参与诱导过程的关键性转录调节子，其缺失会使诱导不能进行。另一方面，另外的基因，包括Klf家族的一些成员(Klf1、Klf2、Klf4和Klf5)、Myc家族的一些成员(c-Myc、L-Myc和N-Myc)、Nanog和Lin28已经被鉴定为增强诱导效率。Oct4(Pou5f1)是八聚合体(octamer，″Oct″)家族转录因子之一，在维持多能+性方面具有关键作用。Oct4 细胞例如卵裂球和胚胎干细胞中Oct4的缺失会导致自发的滋养层分化，因此Oct4的存在产生胚胎干细胞的多能性和分化潜力。″Oct″家族中的多个其它基因，包括Oct4的近亲Oct1和Oct6，不能引发诱导，因此证明了Oct-4在诱导过程中的专有性。与Oct4类似，Sox基因家族与维持多能性相关，虽然其与多潜能(multipotent)和单能干细胞相关，而Oct4与此相反，Oct4专有性地在多能干细胞中表达。虽然Sox2是用于重编程诱导的最初基因，但是发现Sox家族中的其它基因也在诱导过程中同样起作用。Sox1以类似于Sox2的效率产生iPS细胞，基因Sox3、Sox15和Sox18也产生iPS细胞。
具体的，所述ips分化为神经干细胞是通过在添加有窖蛋白1抑制剂的诱导分化培养基中实现的。
具体的，所述的窖蛋白1抑制剂是特异性针对窖蛋白1的单克隆抗体Caveolin1-4B7。
该单克隆抗体Caveolin1-4B7通过测序，鉴定获得其重链可变区序列如SEQ IDNO：1所示，轻链可变区序列如SEQ ID NO:2所示。
具体的，通过亲和力测定，本发明的单克隆抗体Caveolin1-4B7与表位肽的亲和力为1.53nM，亲和特性较好。
在一些实施方案中，本发明的重链可变区包含与选自 SEQ ID NO :1的氨基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
( i ）包含选自 SEQIDNO :1的氨基酸序列或由所述氨基酸序列组成：或者
( ii ）包含与选自 SEQ ID NO :1的氨基酸序列相比具有1个或多个（优选不超过10个，更优选不超过5、4、3、2、1个）的氨基酸改变（优选氨基酸置换，更优选氨基酸保守置换）的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，本发明的轻链可变区
（ i ）包含与选自 SEQ ID NO :2的氨基酸序列具有至少90%、91%、92%、93%、94%、95%、96%、97%、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
（ ii )包含选自 SEQ ID NO :2的氨基酸序列或由所述氨基酸序列组成；或者
（ iii ）包含与选自 SEQ ID NO :2的氨基酸序列相比具有1个或多个（优选不超过10个，更优选不超过5、4、3、2、1个）的氨基酸改变（优选氨基酸置换，更优选氨基酸保守置换）的氨基酸序列或由所述氨基酸序列组成。
保守性氨基酸置换是优选的，即，例如，作为极性酸性氨基酸的天冬氨酸/谷氨酸；作为极性碱性氨基酸的赖氨酸/精氨酸/组氨酸；作为非极性或疏水性氨基酸的亮氨酸/异亮氨酸/甲硫氨酸/缬氨酸/丙氨酸/甘氨酸/脯氨酸；作为极性或不带电的亲水性氨基酸的丝氨酸/苏氨酸。保守性氨基酸置换还包括基于侧链的分组。例如，具有脂肪族侧链的一组氨基酸是甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；具有脂肪族-羟基侧链的一组氨基酸是丝氨酸和苏氨酸；具有含酰胺侧链的一组氨基酸是天冬酰胺和谷氨酰胺；具有芳香族侧链的一组氨基酸是苯丙氨酸、酪氨酸和色氨酸；具有碱性侧链的一组氨基酸是赖氨酸、精氨酸和组氨酸；具有含硫侧链的一组氨基酸是半胱氨酸和甲硫氨酸。例如，可以合理预期：将亮氨酸替换为异亮氨酸或缬氨酸、将天冬氨酸替换为谷氨酸、将苏氨酸替换为丝氨酸、或类似地将氨基酸替换为结构上相关的氨基酸将不会对得到的多肽的性质产生大的影响。可以通过测定多肽的比活性容易地确定氨基酸替换是否产生功能性抗体。
为了iPS细胞的临床施用，方法还可以包括使iPS细胞分化为分化的细胞，例如，心肌细胞、造血细胞、肌细胞、神经元、成纤维细胞、胰腺细胞、肝细胞或上皮细胞。在另一个方面，可以公开从如上文所述的iPS细胞群体分化而来的分化的细胞、组织或器官。组织可以包括神经、骨、肠、上皮、肌肉、软骨或心脏组织；器官可以包括脑、脊髓、心脏、肝、肾、胃、肠或胰腺。
本发明的培养基通常还包括至少必需氨基酸(即His、Ile、Leu、Lys、Met、Phe、Thr、Try、Val)以及某些非必需氨基酸。如果细胞系不能合成氨基酸或细胞系不能产生足够量的氨基酸以支持最大生长，则非必需氨基酸通常包括在细胞培养基中。此外，哺乳动物细胞也可以使用谷氨酰胺作为主要能量来源。包含的谷氨酰胺的浓度通常高于其他氨基酸(2-8mM)。 本发明的培养基还可包含血清。血清是凝固血液的上清液。血清组分包括附着因子、微量营养物质(例如微量元素)、生长因子(例如激素、蛋白酶)和保护性元素(例如抗毒素、抗氧化剂、抗蛋白酶)。血清可从多种动物来源获得，包括人、牛或马血清。当包含在根据本发明的细胞培养基中时，通常以5-10％(体积)的浓度添加血清。优选的细胞培养基是无血清的。细胞培养基可任选地包含一种或多种缓冲剂。合适的缓冲剂包括但不限于N-[2-羟乙基]-哌嗪-N'-[2-乙磺酸](HEPES)、MOPS、MES、磷酸盐、碳酸氢盐和适用于细胞培养应用的其它缓冲剂。合适的缓冲剂是提供缓冲能力而对培养的细胞没有实质细胞毒性的缓冲剂。合适的缓冲剂的选择在细胞培养领域的普通技术范围内。
优选的，本发明的培养基是DMEM/F12中添加2% B27 Supplement、1% N2Supplement，20 ng/ml bFGF，10-500μg/mL Caveolin1-4B7单抗组成。
有益效果：本发明提供了一种用于治疗阿尔茨海默症的药物组合物，其中所述药物组合物含有本发明特异性制备的神经干细胞和斯皮诺素。神经干细胞与斯皮诺素一起治疗AD能够从根本上通过分化为功能性神经元，促进海马突触素的表达，抑制细胞凋亡，抑制神经元纤维缠结，进而修复神经功能，具有较好的治疗前景。
附图说明
图1 单抗对ips细胞向神经干细胞的分化影响
实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被 这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解 本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
尽管本文使用了特定的术语，但它们仅用于一般性和描述性的意义，而 不是为了限制的目的。除非另有定义，否则本文使用的所有技术和科学术语 具有与本公开描述的主题所属领域的普通技术人员通常理解的相同的含义。
实施例1 红细祖细胞的分离
采集外周血20ml，加入100μl RosetteSep™ Human Progenitor Cell BasicPre-Enrichm祖细胞预富集混合物，温育10min，转移至含有30ml人淋巴细胞分离液的SepMate TM管中1200r/min离心12min。用Stem Span TM无血清培养基重悬细胞，上述细胞计数后按5×105个细胞/孔接种于6孔板中，置于37℃、5％CO2培养箱中培养7d后，取少量细胞，采用瑞氏-吉姆萨染色液对分离培养7天后获得的红系祖细胞进行染色，并于显微镜下观察发现红系祖细胞呈圆形，大小基本抑制，并且呈现紫红色，表明分离得到了红细祖细胞。
实施例2 红细祖细胞重编程为ips细胞及诱导分化为神经干细胞
将实施例1制备分离培养的红细祖细胞调整细胞数为1×106个，100μl电转缓冲液中加入 2μl Epi5TM Episomal iPSC 重编程试剂盒中的重编程因子，采用常规电转染细胞按2ml/孔接种于基质胶包被的6孔板中，置于培养箱中培养。并使用ReproTeSRTM重编程培养基隔天换液，转染后第20天，观察iPSCs可见典型的克隆，采用试剂盒检测干细胞多能性基因发现OCT4、SOX2、NANOG、KLF4和LIN28均阳性表达，表明制备获得了ips细胞。将ips克隆重新接种于基质胶包被的6孔板中，置于培养箱中培养，使用mTeSRTM培养基继续培养，备用。
单抗处理组：当前述ips细胞集落密度达到70-80%汇合时，使用胰蛋白酶和EDTA分离ips细胞。用DMEM/F12培养基洗涤细胞一次后，将细胞更换到用80µg/mL Matregel和10mg/mL聚-L-鸟氨酸包被的培养皿中，细胞的密度为106个细胞/孔，培养基为DMEM/F12中添加2% B27 Supplement、1% N2 Supplement，20 ng/ml bFGF，200μg/mL Caveolin1-4B7单抗在37℃和5%CO2条件下培养。培养基隔日更换，当达到85%汇合时，细胞以1:3传代。
自然分化组：当前述ips细胞集落密度达到70-80%汇合时，使用胰蛋白酶和EDTA分离ips细胞。用DMEM/F12培养基洗涤细胞一次后，培养基为DMEM/F12中在37℃和5%CO2条件下培养。培养基隔日更换，当达到85%汇合时，细胞以1:3传代。
无单抗对照组：当前述ips细胞集落密度达到70-80%汇合时，使用胰蛋白酶和EDTA分离ips细胞。用DMEM/F12培养基洗涤细胞一次后，将细胞更换到用80µg/mL Matregel和10mg/mL聚-L-鸟氨酸包被的培养皿中，细胞的密度为106个细胞/孔，培养基为DMEM/F12中添加2% B27 Supplement、1% N2 Supplement，20 ng/ml bFGF在37℃和5%CO2条件下培养。培养基隔日更换，当达到85%汇合时，细胞以1:3传代。
诱导一周后，将二组培养的细胞进行神经干细胞标志物Nestin染色， 随机选取10个200倍视野进行观察，分别计算Nestin阳性细胞总数占视野中细胞总数的比例， Nestin阳性细胞率（%）＝阳性细胞数/总细胞数×100%。 以上实验每次设3个平行组， 作统计分析，结果如图1所示。
从图1可以看出，自然分化组相比于无单抗对照组和单抗处理组，Nestin阳性细胞率较低，差异及其显著（P<0.01）。另外，相对于没有采用单抗处理的诱导方式，添加了单抗处理后，由于窖蛋白1在干细胞向神经细胞分化中起负向调控作用，因此，能够显著的抑制窖蛋白1的表达，进而促进ips细胞向神经干细胞的分化， Nestin阳性细胞率达到（96.31±4.83）%，显著的高于没有采用单抗的对照组。
实施例3 大鼠艾尔茨海默症模型的制备
健康雄性SD大鼠，SPF级，7-9月龄，体重250-280g；
大鼠用10％水合氯醛腹腔注射麻醉（0.3ml/100g），麻醉后固定于大鼠脑立体定位仪上，双侧海马区进针，坐标为：AP-4mm(前囟后)，MR-2.8mm(中线右侧)，H-3mm(自脑膜起的深度)。定位后钻开颅骨，微量注射器垂直进针3mm，以0.2uL·min-1的速度缓慢注入2μl Aβ25-35(10μg)，留针5min。
分组：正常对照组、模型组(注射Aβ蛋白+无血清DMEM)、干细胞组A（注射实施例2单抗处理组制备的干细胞（用无血清DMEM稀释））、干细胞组B（注射实施例2无单抗对照组制备的干细胞（用无血清DMEM稀释））、斯皮诺素联合干细胞组A（斯皮诺素+实施例2单抗处理组制备的干细胞（用无血清DMEM稀释））、斯皮诺素联合干细胞组B（斯皮诺素+实施例2无单抗对照组制备的干细胞（用无血清DMEM稀释））。每组10只大鼠。
制模后1周，开始给药治疗。（干细胞和/或斯皮诺素只注射一次，注射部位同前）。具体的给药为用戊巴比妥钠将大鼠麻醉，用微量进样器给药，注射速度为0.5μl／min。两侧移植点各前述的干细胞悬液5μl(约含5×104个细胞)。斯皮诺素注射量为5μl(最终给药量为5mg/kg)与干细胞给药间隔3h。模型组：注射5μl生理盐水。正常对照组：不作任何处理。给药后骨蜡封闭颅骨，缝合头皮。单笼饲养至大鼠完全清醒，肌肉注射青霉素，密切观察实验动物4周。细胞移植后第4周开始，用 Morris水迷宫测试各组大鼠学习记忆能力。 结果如表1所示。
表1 各组大鼠Morris水迷宫实验结果比较
| 组别 | 潜伏期（s） | 过平台次数 |
| 正常对照组 | 21.80±1.88 | 4.52±1.27 |
| 模型组 | 52.49±4.59 | 0.86±0.84 |
| 干细胞组A | 29.21±2.01# | 2.57±0.76# |
| 干细胞组B | 33.94±2.57# | 2.03±0.68# |
| 斯皮诺素联合干细胞组A | 21.98±1.94* | 4.25±1.02* |
| 斯皮诺素联合干细胞组B | 25.33±1.03* | 3.48±0.93* |
#与模型组相比，P<0.05；*与干细胞组相比，P<0.05；
细胞移植后第4周，与模型组比较，干细胞组A和B组潜伏期明显缩短、通过平台次数明显增加(P<0.05)；与干细胞组A和B组比较，斯皮诺素联合干细胞组的潜伏期、通过平台次数有显著性差异(P<0．05)，接近正常对照组。这也说明，斯皮诺素联合干细胞组A能够有效的促进AD的治疗及正常功能的恢复,从结果也可以看出，斯皮诺素联合干细胞组A的效果要比斯皮诺素联合干细胞组B的效果要有大幅度的提高，这说明，采用单抗处理后的干细胞较没有单抗诱导的干细胞活性要有大幅度的提高。
移植到体内的NSCs分化的神经元要发挥治疗作用必须与宿主神经元形成突触连接，建立功能性神经回路，释放神经递质，产生神经营养因子或保护因子，改变脑组织对损伤的反应，帮助受损神经元修复。而海马突触素的表达是检验神经元发挥作用的重要指标。通过针对各组的海马突触素的表达情况采用ELISA试剂盒进行检测，结果如表2所示。
表2 各组大鼠海马突触素的表达情况
| 组别 | 突触素的表达情况 |
| 正常对照组 | 0.422±0.113 |
| 模型组 | 0.280±0.086 |
| 干细胞组A | 0.391±0.097# |
| 干细胞组B | 0.351±0.084# |
| 斯皮诺素联合干细胞组A | 0.418±0.104* |
| 斯皮诺素联合干细胞组B | 0.402±0.092* |
#与模型组相比，P<0.05；*与干细胞组相比，P<0.05；
从表2可以看出，干细胞组较模型组突触素明显增多，P<0.05，干细胞和斯皮诺素联合使用组与干细胞组相比突触素也明显增多，P<0.05。斯皮诺素联合干细胞组A要比斯皮诺素联合干细胞组B具有更强的促进突触素表达的效果。
另外，AD是中枢神经系统一种常见的进行性神经退行性疾病。其临床表现为进行性记忆力减退，认知能力下降等。其主要病理改变是神经细胞之间存在大量的老年斑以及神经细胞内存在大量的神经元纤维缠结。采用传统Bielshowsky镀银法检测神经元纤维缠绕结形成情况。结果显示，模型组大鼠在海马可见大量的神经元纤维缠结形成，对照组大鼠未见神经元纤维缠结形成，而干细胞组A和组B仅有少量的神经元纤维缠结形成，斯皮诺素联合干细胞组A几乎没有神经元纤维缠结形成。这也说明斯皮诺素能够抑制Aβ25-35诱导产生的炎症因子和细胞凋亡同时抑制神经元纤维缠结形成。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (5)
1.一种用于治疗阿尔茨海默症的药物组合物，其特征在于由神经干细胞和斯皮诺素组成；其中，神经干细胞由红细祖细胞重编程获得的ips细胞通过诱导分化制备的；红细祖细胞重编程制备的ips细胞是通过将红细祖细胞通过电转化 Epi5TM Episomal iPSC 重编程试剂盒中的重编程因子后在mTeSRTM培养基培养获得相应的ips细胞；将所述ips细胞在诱导分化培养基中培养后获得神经干细胞，所述的诱导分化培养基组成为DMEM/F12中添加2%B27 Supplement、1% N2 Supplement，20 ng/ml bFGF，10-500μg/mL Caveolin1-4B7单抗组成；其中，所述Caveolin1-4B7单抗的重链可变区序列如SEQ ID NO：1所示，轻链可变区序列如SEQ ID NO:2所示。
2.神经干细胞和斯皮诺素的组合在制备用于治疗阿尔茨海默症的药物组合物中的用途；其中，神经干细胞由红细祖细胞重编程获得的ips细胞通过诱导分化制备的；红细祖细胞重编程制备的ips细胞是通过将红细祖细胞通过电转化 Epi5TM Episomal iPSC 重编程试剂盒中的重编程因子后在mTeSRTM培养基培养获得相应的ips细胞；将所述ips细胞在诱导分化培养基中培养后获得神经干细胞，所述的诱导分化培养基组成为DMEM/F12中添加2%B27 Supplement、1% N2 Supplement，20 ng/ml bFGF，10-500μg/mL Caveolin1-4B7单抗组成；其中，所述Caveolin1-4B7单抗的重链可变区序列如SEQ ID NO：1所示，轻链可变区序列如SEQ ID NO:2所示。
3.神经干细胞和斯皮诺素的组合在制备用于促进大鼠与阿尔茨海默症相关的认知能力恢复的药物组合物中的用途；其中，神经干细胞由红细祖细胞重编程获得的ips细胞通过诱导分化制备的；红细祖细胞重编程制备的ips细胞是通过将红细祖细胞通过电转化Epi5TM Episomal iPSC 重编程试剂盒中的重编程因子后在mTeSRTM培养基培养获得相应的ips细胞；将所述ips细胞在诱导分化培养基中培养后获得神经干细胞，所述的诱导分化培养基组成为DMEM/F12中添加2% B27 Supplement、1% N2 Supplement，20 ng/ml bFGF，10-500μg/mL Caveolin1-4B7单抗组成；其中，所述Caveolin1-4B7单抗的重链可变区序列如SEQ ID NO：1所示，轻链可变区序列如SEQ ID NO:2所示。
4.如权利要求2或3所述的用途，其特征在于所述的药物组合物为注射剂的剂型。
5.如权利要求3所述的用途，其特征在于神经干细胞胞每次给药5μl约含5×104个干细胞；斯皮诺素给药量为5μl，最终给药量为5mg/kg；斯皮诺素与干细胞给药间隔3h。
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