CN108795987A - 一种FerritinH、Bcl2及EGFP基因联合修饰的神经干细胞制备方法及应用 - Google Patents
一种FerritinH、Bcl2及EGFP基因联合修饰的神经干细胞制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种FerritinH、Bcl2及EGFP基因联合修饰的神经干细胞(NSCs)制备方法及应用。本发明构建的过表达慢病毒能安全有效地对NSCs进行三基因联合修饰,修饰后的干细胞过表达Bcl2,提升了移植后NSCs的抗凋亡及生存能力,增加有效存活干细胞数量,改善了脑梗死治疗的效果;通过FerritinH及EGFP报告基因成像,在活体水平实现对NSCs的磁共振及荧光成像双模态示踪,实现对移植后NSCs的实时、动态、无创、系统监测,为促进干细胞治疗临床转化提供实验依据,为深入阐明神经干细胞作用机制提供新的手段,在神经系统病变的干细胞替代治疗或转基因治疗方面均具有重要的科学意义和临床应用前景。
Description
技术领域
本发明涉及基因工程与生物医学工程技术领域,更具体地,涉及一种 FerritinH、Bcl2及EGFP基因联合修饰的神经干细胞制备方法及应用。
背景技术
脑梗死又称缺血性卒中,中医称之为卒中或中风。本病由各种原因所致的局 部脑组织区域血液供应障碍,导致脑组织缺血缺氧性病变坏死,进而产生临床上 对应的神经功能缺失表现。脑梗死依据发病机制的不同分为脑血栓形成、脑栓塞 和腔隙性脑梗死等主要类型。其中脑血栓形成是脑梗死最常见的类型,约占全部 脑梗死的60%,因而通常所说的‘脑梗死’实际上指的是脑血栓形成。本病的病 死率约为10%,致残率可达50%以上。存活者的复发率高达40%,脑梗死复发 可严重削弱患者的日常生活和社会功能,而且可明显增加死亡率。
基因工程,又称基因拼接技术和DNA重组技术,在体外将分离到的或合成 的目的基因,通过与质粒、病毒等载体重组连接,然后将其导入不含该基因的受 体细胞,使受体细胞产生新的基因产物或获得新的遗传特性。基于基因工程的基 因治疗,能取代机体致病的突变基因,有目的地抑制异常基因表达或重新开启己 关闭的基因,是遗传病、肿瘤、癌症等重大疾病的一种理想的治疗模式。
慢病毒(Lentivirus)载体是以HIV-I(人类免疫缺陷I型病毒)为基础发展 起来的载体,它能相对定点整合到染色体的特定位置,转导的目的基因能整合至 靶细胞基因组,具有长期表达、免疫反应小等优点,在分裂和非分裂细胞中都能 高效表达,慢病毒载体安全性高,在干细胞基因工程中具有优势。
基于干细胞的细胞治疗或基因治疗是再生医学的新模式。神经干细胞 (NSCs)移植是神经系统损伤和退行性疾病的极具前景的新治疗策略。动物实 验表明NSCs移植治疗脑梗死具有确切、明显的促神经功能修复效果,但初步临 床实验中干细胞治疗效果并不确切,部分实验中患者神经功能恢复效果并不显 著。其最直接原因是移植后干细胞所处微环境诱导干细胞大量凋亡,干细胞的低 存活率限制了其再生修复能力。此外,在活体水平对移植入体内的干细胞进行示 踪,动态监测干细胞体内存活、分布、迁移及转归,是干细胞治疗临床转化的必 然需求,但是却一直未能有突破性进展。
发明内容
本发明的目的是为了克服现有技术的不足,利用基因工程技术,成功构建FerritinH-T2A-Bcl2-EGFP过表达慢病毒,转染大鼠神经干细胞(NSCs)后获得 基因工程化干细胞FerritinH-Bcl2-EGFP-NSCs。FerritinH-T2A-Bcl2-EGFP过表达 慢病毒能安全、有效地对NSCs进行FerritinH、Bcl2及EGFP三基因联合修饰, 获得的基因工程化干细胞FerritinH-Bcl2-EGFP-NSCs不仅通过过表达Bcl2提升 了移植后NSCs的抗凋亡及生存能力,增加有效存活干细胞数量,改善治疗脑梗 死的效果;而且通过FerritinH及EGFP报告基因成像,在活体水平实现对NSCs 的磁共振及荧光成像双模态示踪,实现对移植后NSCs的实时、动态、无创、系 统监测。
本发明的第一个目的是提供一种基因修饰的可示踪神经干细胞的制备方法。
本发明的第二个目的是提供以上任一所述制备方法制备得到的基因工程化 的可示踪神经干细胞FerritinH-Bcl2-EGFP-NSCs
本发明的第三个目的是提供所述的融合基因FerritinH-T2A-Bcl2、重组质粒、FerritinH-T2A-Bcl2-EGFP过表达慢病毒和/或基因工程化的可示踪神经干细胞FerritinH-Bcl2-EGFP-NSCs在制备治疗脑梗死的医学上可接受的制剂中的应用。
为了实现上述目的,本发明是通过以下技术方案予以实现的:
本发明采用基因工程技术,制备Bcl2抗凋亡基因、FerritinH MR报告基因 及EGFP基因过表达慢病毒载体,对NSCs进行FerritinH-T2A-Bcl2-EGFP基因 修饰后移植于大鼠脑梗死模型,增强NSCs自身抗凋亡能力,利用FerritinH基因 实现干细胞MR可视化,利用EGFP基因实现干细胞荧光成像,对NSCs移植后 的存活、定居、迁移等生物学行为进行MRI及光学双模态成像活体示踪,探讨 通过抗凋亡修饰提高NSCs移植治疗效果的可行性,以及MRI报告基因及光学 双模态成像示踪NSCs移植后生物学行为的可行性,并通过与组织病理学对照, 揭示了NSCs促进脑梗死修复的潜在机制。
因此,本发明要求保护一种基因工程化的可示踪神经干细胞的制备方法,包 括以下步骤:
S1.合成融合基因FerritinH-T2A-Bcl2,其核苷酸序列如SEQ ID NO:3所示 的;
S2.将融合基因FerritinH-T2A-Bcl2插入含有EGFP基因的慢病毒表达载体, 得到重组质粒;
S3.将重组质粒转染宿主细胞,制备FerritinH-T2A-Bcl2-EGFP过表达慢病 毒;
S4.将FerritinH-T2A-Bcl2-EGFP过表达慢病毒转染神经干细胞,得到基因 工程化的可示踪神经干细胞。
所述神经干细胞可以是从已经建立细胞系的诱导性多能干细胞进一步诱导 分化得到的,或者直接采用已经建立的成熟的商品化的神经干细胞。
优选地,慢病毒表达载体为GV218载体。
优选地,宿主细胞为293T细胞。
优选地,步骤S2中,线性化载体DNA与融合基因FerritinH-T2A-Bcl2DNA 的摩尔比为1:(3~9)。
优选地,步骤S4中,MOI指数为10。
优选地,步骤S4中,转染时间为24h。
最优选地,所述制备方法包括以下步骤:
S1.以化学合成方法合成融合基因FerritinH-T2A-Bcl2,其核苷酸序列如SEQ IDNO:1所示;
S2.以步骤S1中合成的将融合基因FerritinH-T2A-Bcl2为模板,以SEQ ID NO:2~3所示核苷酸序列为引物,进行PCR扩增,扩增的到的片段经过回收纯 化后与已经线性化的慢病毒表达载体GV218进行连接反应,将融合基因 FerritinH-T2A-Bcl2插入慢病毒表达载体GV218得到重组质粒,其中,插入位点 位于Ubi和EGFP之间的多克隆位点中,线性化载体DNA与融合基因 FerritinH-T2A-Bcl2DNA的摩尔比为1:(3~9);
S3.将重组质粒转染宿主细胞293T细胞,制备FerritinH-T2A-Bcl2-EGFP过 表达慢病毒;
S4.将FerritinH-T2A-Bcl2-EGFP过表达慢病毒转染神经干细胞,得到基因 工程化的可示踪神经干细胞FerritinH-Bcl2-EGFP-NSCs,其中,MOI指数为10, 转染时间为24h。
以上任一所述制备方法制备得到的基因工程化的可示踪神经干细胞。
以上所述基因修饰的可示踪神经干细胞,该神经干细胞同时过表达FerritinH、Bcl2及EGFP基因。
以上所述的融合基因FerritinH-T2A-Bcl2、重组质粒、 FerritinH-T2A-Bcl2-EGFP过表达慢病毒和/或基因工程化的可示踪神经干细胞在 制备治疗脑梗死的医学上可接受的制剂中的应用,均属于本发明的保护范围。
与现有技术相比,本发明具有如下有益效果:
FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体构建成功,能将FerritinH、Bcl2 及EGFP基因转入293T细胞,进一步将其转导干细胞。FerritinH-T2A-Bcl2-EGFP 过表达慢病毒能安全、有效地对NSCs进行FerritinH、Bcl2及EGFP三基因联 合修饰,获得的基因工程化干细胞FerritinH-Bcl2-EGFP-NSCs不仅通过过表达 Bcl2提升了移植后NSCs的抗凋亡及生存能力,增加有效存活干细胞数量,改善 治疗脑梗死的效果;而且通过FerritinH及EGFP报告基因成像,在活体水平实 现对NSCs的磁共振及荧光成像双模态示踪,实现对移植后NSCs的实时、动态、 无创、系统监测,为促进干细胞治疗的临床转化提供实验依据,为深入阐明神经 干细胞作用机制提供新的手段,在神经系统病变的干细胞替代治疗或转基因治疗 方面均具有重要的科学意义和临床应用前景。
附图说明
图1为慢病毒载体GV218及辅助质粒示意图;其中,a为GV218转移载体 图谱;b为pHelper1.0载体图谱;c为pHelper2.0载体图谱。
图2为FerritinH-T2A-Bcl2-EGFP目的质粒荧光及Western Blot鉴定;a-b: 目的质粒转染293T细胞后荧光显微镜EGFP荧光表达情况(200×);c:目的质 粒转染293T细胞后Western Blot检测目的基因融合蛋白大小情况。
图3为FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体对NSCs最佳转染条件 检测;a:不同MOI FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体转染NSCs后, 荧光显微镜示MOI=10时EGFP绿色荧光强度最强;标尺为40μm;b:最佳MOI FerritinH-T2A-Bcl2-EGFP慢病毒载体转染NSCs后,于不同时间观察荧光显微镜 观察EGFP绿色荧光强度,显示24h时绿色荧光强度最强;标尺=40μm。
图4为FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体转染NSCs后流式细胞 仪检测转染细胞EGFP荧光表达率。
图5为NSCs不同转染组FerritinH、Bcl2的RNA及蛋白表达检测;a: FerritinH-T2A-Bcl2-EGFP、FerritinH、空病毒三组NSCs转染后FerritinH、Bcl2 的RNA琼脂糖凝胶电泳,28S、18S、5S带型清晰;b:NSCs转染后FerritinH、 Bcl2RNA表达水平;c-d:NSCs转染后FerritinH、Bcl2蛋白半定量条带及灰度 分析值。
图6为转染NSCs体外MRI及原子吸收光谱检测;a:FerritinH-Bcl2-EGFP -NSCs随着培养时间延长,细胞在T2WI、T1WI、T2FFE及T2-map序列上信号 逐渐减低;b:FerritinH-Bcl2-EGFP-NSCs细胞的T2值随培养时间逐渐减低;c: 原子吸收光谱检测示FerritinH-Bcl2-EGFP-NSCs细胞随培养时间延长,单细胞铁 含量逐渐增加。
图7为转染NSCs及电镜检测;a:FerritinH-Bcl2-EGFP-NSCs普鲁士蓝染色 细胞内见较多蓝染颗粒;b:对照组NSCs内未见明显的蓝染颗粒;c:透射电镜 示:FerritinH-Bcl2-EGFP-NSCs胞浆内见大量高密度铁颗粒(5800×)。
图8为转染NSCs凋亡及增殖活力检测;a-d:FerritinH-Bcl2-EGFP-NSCs组、FerritinH-NSCs组、Mock-NSCs组流式细胞仪检测;e:CCK8检测 FerritinH-Bcl2-EGFP-NSCs组、FerritinH-EGFP-NSCs组、Mock-NSCs组细胞增 殖活力。
图9为FerritinH-Bcl2-EGFP-NSCs成神经球及多向分化能力鉴定; FerritinH-Bcl2-EGFP-NSCs继续培养能形成神经球,Nestin表达阳性(图9a)。 FerritinH-Bcl2-EGFP-NSCs体外诱导分化,分化细胞GFAP(b)、NeuN(c)、 O4(d)免疫荧光染色:红色为阳性蛋白荧光,绿色为EGFP荧光,标尺=5μm。
图10为干细胞移植后系列MRI T2FFE序列成像及生物荧光活体成像;a:FerritinH-Bcl2-EGFP-NSCs移植组的大鼠脑T2FFE成像;b: FerritinH-Bcl2-EGFP-NSCs移植组大鼠脑生物荧光成像;c:FerritinH-EGFP-NSCs 移植组的大鼠脑T2FFE成像;d:FerritinH-EGFP-NSCs移植组的大鼠脑生物荧 光成像;e:Mock-NSCs移植组的大鼠脑T2FFE成像;f:Mock-NSCs移植组的 大鼠脑生物荧光成像。
图11为干细胞移植后系列MRI T2WI序列成像;a: FerritinH-Bcl2-EGFP-NSCs移植组;b:FerritinH-EGFP-NSCs移植组;c: Mock-NSCs移植组。
图12为不同转染组NSCs移植后8周移植区脑组织学检测;a: FerritinH-Bcl2-EGFP-NSCs移植组;b:FerritinH-EGFP-NSCs移植组;c:Mock-NSCs移植组;标尺=10μm。
图13为不同转染组NSCs移植后8周胼胝体区组织学检测;a: FerritinH-Bcl2-EGFP-NSCs移植组;b:FerritinH-EGFP-NSCs移植组;c: Mock-NSCs移植组;标尺=10μm。
图14为不同转染组NSCs移植后8周脑普鲁士蓝染色及免疫组化检测;a:FerritinH-Bcl2-EGFP-NSCs移植组脑普鲁士蓝染色;b:FerritinH-EGFP-NSCs移 植组脑普鲁士蓝染色;c:Mock-NSCs移植组脑普鲁士蓝染色;e: FerritinH-Bcl2-EGFP-NSCs移植组脑Bcl2免疫组化;f:FerritinH-EGFP-NSCs移 植组脑Bcl2免疫组化;g:Mock-NSCs移植组脑Bcl2免疫组化;标尺=200μm。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述,所述实 施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试 验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明, 为可从商业途径得到的试剂和材料。
1、细胞株
采用293T作为慢病毒的包装细胞。293T为贴壁依赖型成上皮样细胞,采用 含10%FBS的DMEM作为生长培养基。
2、菌株
大肠杆菌菌株DH5α,用于扩增慢病毒载体和辅助包装载体质粒。
3、病毒载体
慢病毒包装系统,包括三个质粒:①携带目的基因的工具载体质粒:GV218, 元件顺序:Ubi-MCS-EGFP-IRES-puromycin,克隆位点:AgeI/NheI;②病毒包 装辅助质粒(Helper 1.0);③病毒包膜辅助质粒(Helper 2.0),均由上海吉凯公司提 供,载体图谱见图1
4、实验动物
出生后1天的新生健康Sprague-Dawley大鼠12只,雄性,SPF级;健康成 体Sprague-Dawley大鼠90只,250-300g,雄性,SPF级(均由广州中医药大学 动物中心提供)。
5、统计学分析
计量资料以均数±标准差表示。细胞增殖活力、细胞凋亡率用平均百分 率表示。不同组NSCs的T2值、铁浓度、细胞增殖活力及凋亡率, FerritinH-Bcl2-EGFP-NSCs组、FerritinH-EGFP-NSCs组与Mock-NSCs组的 FerritinH、Bcl2的RNA及蛋白表达采用方差分析,组间比较采用LSD检验。统 计学分析使用SPSS 13.0软件,检验水准为P<0.05。
实施例1FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体的构建及鉴定
1、目的基因获取
根据GenBank中大鼠FerritinH(NM_012848)、Bcl2(NM_016993)基因序 列结合自裂解多肽T2A序列,使用化学合成的方法合成大鼠FerritinH-T2A-Bcl2 基因。设计特异性引物如下:
FerritinH-T2A-Bcl2上游引物(SEQ ID NO:1):FerritinH+Bcl2-P1
GAGGATCCCCGGGTACCGGTCGCCACCATGACCACCGCGTCTCCCTC
FerritinH-T2A-Bcl2下游引物(SEQ ID NO:2):FerritinH+Bcl2-P2
TCACCATGGTGGCGACCGGCTTGTGGCCCAGGTATGCAC
引物用于PCR扩增FerritinH-T2A-IFNβ序列。PCR产物在琼脂糖凝胶电泳 后,切下分子量约1Kb的目的条带备后用。
2、重组质粒构建及测序
回收PCR产物交换连接入酶切慢病毒表达载体(GV218载体,AgeI/NheI 酶切,元件顺序Ubi-MCS-EGFP-IRES-puromycin),连接后转化新鲜的大肠杆菌 感受态细胞,对生长出的阳性克隆行PCR鉴定,琼脂糖凝胶电泳中显示目的条 带位于1kb-1.5kb之间,而目的基因FerritinH-T2A-Bcl2PCR产物大小1354bp。 随后,提取连接产物载体质粒FerritinH-T2A-Bcl2-LV,使用ABI 3730xl自动测 序仪进行测序,Chromas测序结果读取软件进行读取分析。
所述重组质粒DNA的Chromas测序结果:FerritinH-T2A-Bcl2重组质粒测 序与GenBank中FerritinH(NM_012848)、Bcl2(NM_016993)基因序列比较, 与目标序列基本一致(只有一处GAA同义突变为GAG),提示FerritinH-T2A-Bcl2 慢病毒载体构建成功,其核苷酸序列如SEQ ID NO:3所示,其编码的蛋白的序 列如SEQ ID NO:4所示。
3、FerritinH-T2A-Bcl2-EGFP重组质粒表达检测
将处于对数生长期的293T细胞胰酶消化制成细胞悬液,5×104个293T细胞 接种于24孔培养板。在细胞恒温培养箱内培养至细胞汇合率约70%~80%时, 根据InvitrogenLipofectamine 3000转染试剂使用说明书,采用质粒DNA和 Lipofectamine 3000共转染293T细胞,培养6h后,更换成新鲜的完全培养基(含 10%血清)。转染24h后荧光显微镜观察质粒eGFP表达情况以判断感染效率,转 染36h后收集细胞。采用Western Blot检测目的基因表达。
检测表明:目的质粒FerritinH-T2A-Bcl2-EGFP转染293T细胞24h后,荧光 显微镜下293T细胞内可观察到明显的荧光(图2a、2b,200×),说明目的质粒 转染正常、目的质粒荧光标记基因表达正常。目的基因融合蛋白大小74KD,经 Western Blot检测显示55KD附近处有特征条带,其大小和目的基因融合蛋白相 比略小。用EGFP抗体检测表达,WesternBlot检测出Bcl2-EGFP,表明剪切完 全(图2c)。
4、FerritinH-T2A-Bcl2-EGFP过表达慢病毒包装、收获、浓缩及滴度测定
将293T细胞接种于10cm2细胞培养皿,于细胞恒温培养箱内培养。待细胞 密度达70%~80%时采用FerritinH-T2A-Bcl2-EGFP载体质粒、包装质粒pHelper 1.0载体、包膜质粒pHelper 2.0载体与Lipofectamine 3000对293T细胞进行共转 染。培养6h更换为完全培养基(含10%血清)。继续培养48h~72h,收集富含 慢病毒颗粒的293T细胞上清液,对其过滤、离心、浓缩后获得高滴度病毒浓缩 液,分装于50μL病毒管中,-80℃保存。使用ELISA法、荧光法及药筛法测定 病毒滴度。测定结果表明,病毒滴度为2×108TU/mL。
实施例2FerritinH-T2A-Bcl2-EGFP过表达慢病毒对NSCs最佳转染条件测定
1、NSCs分离、培养
新生1d的SD大鼠,引颈处死,无菌条件下分离双侧侧脑室周围组织,剪 碎脑组织、过滤、离心、弃上清,重悬后接种至25cm2培养瓶中,37℃、5%CO2、 饱和湿度下培养至神经球形成,当神经球透光度明显减低时,以1:2传代培养。
2、FerritinH-T2A-Bcl2-EGFP过表达慢病毒对NSCs最佳转染条件测定
按1×105个NSCs细胞/孔种于12孔培养板中,每孔加入1mL完全培养基。 当细胞融合度约50%时移除培养基,再用PBS洗涤后转染。为了确定最佳MOI 及转染时间,MOI(MOI=病毒液体积×病毒滴度/转染细胞数)设置为1、10、100, 每孔相应加入1μL、10μL、100μL病毒液,以及0.5uLpolybrene,再加入新鲜的 完全培养基使每孔最终液体量为1mL。置于恒温细胞培养箱内培养,转染时间 设置为12h、24h、36h,达到预定时间后,观察细胞状态,更换新鲜的完全培养 基继续培养。待12孔板内细胞融合度达70%以上(约为转染后第3d),采用荧 光显微镜观察细胞荧光强度及荧光表达率。采用携带FerritinH基因的慢病毒载 体以同样步骤转染NSCs获得FerritinH-EGFP-NSCs;采用不携带目的基因的空 病毒以同样步骤转染NSCs获得Mock-NSCs;培养、传代作为后续实验的对照组。
通过对比转染细胞荧光强度及荧光表达率(见图3),确定 FerritinH-T2A-Bcl2-EGFP慢病毒转染MSCs的最佳转染条件为:MOI=10,转染 时间为24h。
实施例3FerritinH-T2A-Bcl2-EGFP过表达慢病毒转染NSCs的体外有效性检测
1.流式细胞仪检测FTH-Bcl2-EGFP-NSCs荧光表达率
NSCs按2×105细胞/孔接种于6孔板,按上述最佳转染条件进行慢病毒转染。 于24h后除去含有病毒的培养液,移除培养基,PBS洗涤,胰酶消化,吹打成 细胞悬液、计数。取5×105个FTH-Bcl2-NSCs后采用流式细胞仪检测转染后细胞 eGFP的表达率。
结果表明:流式细胞仪测定EGFP荧光表达情况见图4,转染后EGFP表达 率稳定在78.62±0.29%左右。
2.Real-time PCR检测转染后NSCs的FerritinH、Bcl2的mRNA水平
NSCs按2×105细胞/孔接种于6孔板,分别采用FerritinH-T2A-Bcl2-EGFP 慢病毒、FerritinH-EGFP慢病毒、空病毒进行慢病毒转染,获得 FerritinH-Bcl2-EGFP-NSCs、FerritinH-EGFP-NSCs与Mock-NSCs。采用常规方法 提取RNA样品。取1μL RNA样品50倍稀释,测定OD值,OD260/OD280>1.8, 说明提取的RNA较纯,无蛋白质污染。取RNA样品1μL,1%琼脂糖凝胶电泳 80V×20min,用凝胶成像系统观察总RNA的5s rRNA、18s rRNA和28s rRNA条带,三条条带均完整,证明总RNA抽提比较完整。逆转录cDNA合成采用 TaKaRa公司的逆转录试剂盒PrimeScript RT reagent Kit,总反应体系20μL。PCR 引物设计采用Primer 5.0软件,选取β-actin作为内参。按Real-time PCR试剂盒 (SYBR Green qPCR SuperMix)说明书配制反应体系,反应在ABI7500 型实时定量PCR仪进行。
Real-time PCR结果表明:FerritinH-Bcl2-EGFP-NSCs组、 FerritinH-EGFP-NSCs组的FerritinH mRNA水平均高于空病毒组,差异均具有统 计学意义(P<0.05);FerritinH-Bcl2-EGFP-NSCs组与FerritinH-EGFP-NSCs组的FerritinH mRNA水平无统计学差异(P>0.05)。FerritinH-Bcl2-EGFP-NSCs组的 Bcl2mRNA高于FerritinH-EGFP-NSCs组与空病毒组,差异有统计学意义 (P<0.05);FerritinH-EGFP-NSCs组与空病毒组的Bcl2mRNA水平无统计学差 异(P>0.05)(见图5a,b)。
3.Western Blot检测转染后NSCs的FerritinH、Bcl2蛋白表达
NSCs按2×105细胞/孔接种于6孔板,分别采用FerritinH-T2A-Bcl2-EGFP 慢病毒、FerritinH-EGFP慢病毒、空病毒进行慢病毒转染,获得 FerritinH-Bcl2-EGFP-NSCs、FerritinH-EGFP-NSCs与Mock-NSCs。消化、离心后 获得细胞沉淀加入适量新配单去污裂解液(含蛋白磷酸酶抑制剂PMSF),振荡 器上震荡10min,12000r/min,4℃离心20min,取上清;提取好的蛋白溶液和 5×上样缓冲液按4:1混合,煮沸10min,缓慢恢复至室温后,稍离心,放于-20℃ 保存。每个样本设3个复孔,FerritinH、Bcl2蛋白表达水平采用GAPDH作为内参,通过BandScan5.0软件分析目的条带进行灰度扫描、半定量,并根据灰度计 算值比率=目的蛋白/GAPDH比较各组蛋白表达差异。
Western Blot检测结果表明:FerritinH-Bcl2-EGFP-NSCs组、 FerritinH-EGFP-NSCs组的FerritinH蛋白表达均高于空病毒组,差异均具有统计 学意义(P<0.05);FerritinH-Bcl2-EGFP-NSCs组与FerritinH-EGFP-NSCs组的 FerritinH蛋白表达无统计学差异(P>0.05)。FerritinH-Bcl2-EGFP-NSCs组的Bcl2 蛋白表达高于FerritinH-EGFP-NSCs组与空病毒组,差异有统计学意义(P<0.05); FerritinH-EGFP-NSCs组与空病毒组的Bcl2蛋白表达无统计学差异(P>0.05)(见 图5c,d)。
4.MRI检测
1×106个NSCs接种于24孔板,每孔分别加入50μl FerritinH-T2A-Bcl2-EGFP 病毒液(按MOI=10稀释后),置于37℃、5%CO2饱和湿度培养箱内培养24h 后除去含有病毒的培养液,加入枸橼酸铁(含Fe3+),使培养液铁浓度达到500 μmol/L,继续培养1、2、3、5、7d后PBS液洗涤3次,每次2min,将转染后 细胞转移至96孔板,设对照为单纯NSCs组、PBS组,每组均设置5个复孔, 加入300μl 4%明胶溶液,明胶凝固后,进行MRI检测。MRI成像序列为:SE序列T1WI、T2WI、FFE及T2-map序列。主要参数如下:T1WI:TR/TE为500/15 ms,层厚/层距为1.5/0mm,视野110×110mm,矩阵:256×256,NSA:3次;T2WI:TR/TE为2600/100ms,层厚/层距为1.5/0mm,视野110×110mm,矩阵 256×256,NSA为3次。FFE:TR/TE为261/14ms,层厚/层距为1.5/0mm,视 野110×110mm,矩阵:256×256,NSA:3次,翻转角13.81ms;T2-map使用 单层面多SE序列测量T2驰豫时间,TR=2000ms,TE=20–160ms,8个回波, 层厚1.5mm,视野70mm,矩阵256×256,NSA为3次。在工作站上,利用感 兴趣区技术测量细胞的信号强度以及T2驰豫时间。
MRI检测结果表明:FerritinH-Bcl2-EGFP-NSCs在MRI上信号随时间逐渐 降低,至7d时降低明显(图6a),T2值明显缩短(图6b)。
5.原子吸收光谱检测
1×106个NSCs接种于24孔板,每孔分别加入50μl FerritinH-T2A-Bcl2-EGFP 病毒液(按MOI=10稀释后),每孔的最终液体量为500μl,置于37℃、5%CO2饱和湿度培养箱内培养24h后除去含有病毒的培养液,加入枸橼酸铁(含Fe3+), 使培养液铁浓度达到500μmol/L,继续培养1、2、3、5、7d后PBS液重新悬浮 NSCs,设对照组为单纯NSCs,每组均设置5个复孔,取悬浮液200μl加入1.4ml 浓盐酸中放置2-3d后转移到容量瓶中,去离子水定容摇匀。采用标准曲线法, 用火焰原子吸收光谱法测定标记后NSCs的平均铁含量。
原子吸收光谱检测表明:FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体转染 NSCs24h后,单个细胞内铁含量为3.5378±0.0985pg(图6c),对照组NSCs单 个细胞内铁含量为0.168±0.02305pg,两组差异具有统计学意义(P<0.05)。
6.普鲁士蓝染色
1×106个NSCs接种于24孔板,每孔分别加入50μl FerritinH-T2A-Bcl2-EGFP 病毒液(按MOI=10稀释后),每孔的最终液体量为500μl,细胞培养液中加入枸 橼酸铁(含Fe3 +),使培养液铁浓度达到500μmol/L,设对照组为单纯NSCs,每 组均设置5个复孔,置于37℃、5%CO2饱和湿度培养箱内培养,于24h后除 去含有病毒的培养液,PBS洗涤3次,10%多聚赖氨酸贴壁10min、4%多聚甲 醛固定20min,PBS液洗涤3次,Perl’s反应液(10%亚铁氰化钾溶液+37%盐 酸等体积混合)作用30min,PBS液洗涤3次,每次2min,显微镜下观察细胞内铁分布情况。
普鲁士蓝染色表明:FerritinH-Bcl2-EGFP-NSCs胞浆内见蓝染颗粒(图7a), 而对照组NSCs未见蓝染颗粒(图7b)。
7.透射电镜检测
1×106个NSCs接种于24孔板,每孔分别加入50μl FerritinH-T2A-Bcl2-EGFP 病毒液(按MOI=10稀释后),每孔的最终液体量为500μl,细胞培养液中加入 枸橼酸铁(含Fe3 +),使培养液铁浓度达到500μmol/L,设对照组为单纯NSCs, 每组均设置5个复孔,置于37℃、5%CO2饱和湿度培养箱内培养,于24h后 除去含有病毒的培养液,PBS液洗涤3次,每次2min,加入PBS重新悬浮后以 4000rpm离心10min,使细胞在离心管尖沉淀结块。前固定液(2%戊二醛、2.5% 多聚甲醛)4℃固定过夜。0.01mol/L二甲胂酸钠缓冲液洗涤。1%锇酸4℃后 固定1h。梯度丙酮脱水,环氧树脂Epon812包埋,制成50nm的超薄切片,饱 和醋酸铀及柠檬酸铅双染色,电镜下观察细胞超微结构及细胞内含铁颗粒的分 布。
电镜检测显示:FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体转染NSCs后, 细胞超微结构未见明显变化,铁颗粒多位于细胞浆内,少数位于核膜及胞膜(图 7c)。
实施例4FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体NSCs转染体外安全性检 测
1、流式检测转染后NSCs凋亡情况
按1×106个NSCs接种于24孔板,分为3组:FerritinH-Bcl2-EGFP-NSCs组、FerritinH-EGFP-NSCs组与Mock-NSCs组,每孔分别加入50μl相应的病毒液(按 MOI=10稀释后),每孔的最终液体量为500μl,每组均设置5个复孔,置于37℃、 5%CO2饱和湿度培养箱内培养24h后除去含有病毒的培养液,PBS液洗涤3次, 每次2min,每组取5×105个已标记的NSCs,1000rpm离心5min,去上清液, 将沉淀用PBS液洗涤2次,流式细胞仪检测细胞凋亡率。
流式细胞仪检测显示:FerritinH-Bcl2-EGFP-NSCs组、FerritinH-EGFP-NSCs 组与Mock-NSCs组的细胞凋亡率差异无统计学意义(图8a-d)(P>0.05)。
2、CCK8检测转染后NSCs增殖活力
1×106个NSCs接种于24孔板,分为3组:FerritinH-Bcl2-EGFP-NSCs组、FerritinH-EGFP-NSCs组与Mock-NSCs组,每孔分别加入50μl FerritinH-T2A-Bcl2-EGFP过表达慢病毒,FerritinH-EGFP过表达慢病毒、空病毒 液(按MOI=10稀释后),每孔的最终液体量为500μl,每组均设置5个复孔, 置于37℃、5%CO2饱和湿度培养箱内培养24h后除去含有病毒的培养液后 PBS液洗涤3次,每次2min,按5×103/孔接种于96孔板内,并配平每孔的总液 量为200μl,继续培养24h后,每孔加入10μl的CCK8液后4h,使用酶标仪在 450nm波长处测定每孔的OD值,计算细胞活力(%)=A(标记细胞)-A(空 白)/A(未标记细胞)-A(空白),反映细胞的增殖活力。以上实验重复3次,每次 每组设5个复孔。
CCK8检测表明:FerritinH-Bcl2-EGFP-NSCs组、FerritinH-EGFP-NSCs组与 Mock-NSCs组的细胞增殖活力差异无统计学意义(图8e)(P>0.05)。
3、FerritinH-Bcl2-EGFP-NSCs成神经球及多方向分化能力鉴定
1)FerritinH-Bcl2-EGFP-NSCs成神经球能力检测:取1×104个 FerritinH-Bcl2-EGFP-NSCs,10%多聚赖氨酸贴壁、4%多聚甲醛固定、0.3%的 Triton X-100穿膜、血清封闭液封闭后,加入一抗Nestin(1:200)及对应的二 抗IgG(1:100),DAPI染核,PBS洗涤,激光共聚焦显微镜观察标志物Nestin 的表达情况。
结果表明:FerritinH-Bcl2-EGFP-NSCs继续培养能形成神经球,Nestin表达 阳性(图9a)。
2)FerritinH-Bcl2-EGFP-NSCs多向分化鉴定:取1×104个 FerritinH-Bcl2-EGFP-NSCs,取第2代细胞,加入含B27的Neurobasal培养基, 诱导分化7d后,4%多聚甲醛固定、3%Triton X-100穿膜、血清封闭液封闭, 加入一抗(NeuN)(1:200)及对应的二抗IgG(1:100),DAPI染核,PBS洗 涤,激光共聚焦显微镜观察神经元标志物NeuN的表达情况。类似的方法采用含 FBS、N2的DMEM培养基诱导培养FerritinH-Bcl2-EGFP-NSCs,采用激光共聚焦显微镜观察星形胶质细胞标志物GFAP的表达情况。类似的方法采用含B27、 T3的Neurobasal培养基诱导培养FerritinH-Bcl2-EGFP-NSCs,激光共聚焦显微镜 观察少突胶质细胞标志物O4表达情况。
结果表明:FerritinH-Bcl2-EGFP-NSCs体外诱导分化后,可出现GFAP阳性 (图9b),NeuN阳性(图9c),O4阳性神经细胞(图9d)。
实施例5FerritinH-Bcl2-EGFP-NSCs治疗脑梗死的MRI及光学双模态成像示踪
一、实验步骤
1、大鼠MCAO模型制作
采用栓线法制作大鼠左侧大脑中动脉缺血模型:10%水合氯醛(剂量:0.4 mL/100g)腹腔注射麻醉大鼠后,固定大鼠四肢,颈前皮肤消毒后,分离、暴露 左侧大脑中动脉,参照文献[57]方法,采用栓线栓塞大鼠左侧大脑中动脉2h制 作模型。模型制作后1d,利用专用的动物线圈行大鼠头部MRI扫描证实模型制 作成功。
2、干细胞移植
取模型制作成功SD大鼠90只,随机分FerritinH-Bcl2-EGFP-NSCs组、 FerritinH-EGFP-NSCs组与Mock-NSCs组,每组30只,分别注射1×106个经 FerritinH-T2A-Bcl2-EGFP过表达慢病毒载体、FerritinH-EGFP过表达慢病毒载 体、空病毒转染的NSCs于脑梗死侧对侧纹状体区,NSCs悬浮于3μl培养液中, 26S Hamilton微量注射针,立体定位仪定位:前囟前0.5mm,中线右侧3.0mm, 头皮下6.0mm。
3、双模态成像
在神经干细胞移植前(基线)、移植后1、2、3、4、5、6、7、8w对各组大 鼠进行活体脑部MRI检测及小动物光学成像检测。
(1)活体MRI检测
大鼠1%戊巴比妥钠,0.35mL/100g腹腔注射麻醉后,俯卧位,3.0T MRI, 大鼠专用线圈。成像序列包括:冠状位T2WI:TR/TE 1600/80ms,层厚/层距1.0/0 mm,FOV 40×40mm,矩阵256×256,NSA=3;冠状位T2FFE:TR/TE/FA 600 ms/18.3ms/25°,层厚/层距1.0/0mm,FOV 40×40mm,矩阵:256×256,NSA=3。
MRI图像传入专用工作站进行处理,观察NSCs在脑内的信号的演变、NSCs 的迁移情况、梗死区的信号及大小的演变等情况。应用ImageJ软件测量每层脑 梗死面积,按照公式[59]计算脑梗死区大小:梗死体积百分比=梗死体积/总体 积×100%=(S1+S2+S3+..SN)H/(S1总+S2总+S3总+..SN总)H,S1-SN 表示每层的梗死面积,S1总-SN总表示每层的脑组织面积,H表示层厚。
(2)活体荧光成像
大鼠1%戊巴比妥钠,0.35mL/100g麻醉后,仰卧位,小动物活体荧光成像 仪对各组大鼠进行脑部活体荧光成像,采用488nm激发光激发,发射波长选择 516nm,调节相关曝光参数,FOV选择80×80mm,分别进行白光成像、荧光成 像及X线成像。
4、动物行为学检测
各组大鼠在相应时间点(与MRI及荧光活体成像相对应)采用神经功能缺 损量表(neurologic severity scores,NSS)对大鼠进行神经损害严重程度评分。NSS 评分:0分:无神经功能障碍;1分:(未提尾时左前肢屈曲)轻度神经功能缺损; 2分:(行走时向左侧转圈)中度神经功能缺损;3分:(向左侧倾斜)有重度神 经功能缺损;4分:无自发行走,意识减退;5分:与意识缺陷有关的死亡。
5、组织病理学检测
干细胞移植后8w,MRI及光学成像完成后,大鼠10%水合氯醛按0.35 mL/100g麻醉,4%多聚甲醛左心室灌注后取脑,10%多聚甲醛固定,15%、30% 及50%蔗糖溶液梯度脱水,OCT包埋,制作连续冠状位冰冻切片,切片层厚12 um,进行普鲁士蓝及Nestin、NeuN、GFAP、O4、CD11b、Bcl2免疫荧光染色。
(1)普鲁士蓝染色
取脑冰冻切片与Perl’s反应液(10%亚铁氰化钾溶液、37%盐酸等体积混合) 作用30min,PBS液洗涤2次,核固红复染5min,PBS液洗涤2次,酒精梯度 脱水(80%,1min;90%,1min×2;95%,1min×2),封片,显微镜下观察。
(2)Bcl2免疫组化染色
取脑冰冻切片,0.3%的Triton X-100穿膜,血清封闭液封闭后,加入一抗 (Bcl2)(1:200)及对应的二抗IgG(1:100),苏木素复染5min,PBS液洗涤 2次,酒精梯度脱水(80%,1min;90%,1min×2;95%,1min×2),封片,显 微镜下观察。
(3)NSCs、神经元、星形胶质细胞及小胶质细胞免疫荧光染色
取脑冰冻切片,0.3%的Triton X-100穿膜(除CD11b外),血清封闭液封 闭后,加入一抗(Nestin、NeuN、GFAP、O4及CD11b)(1:200)及对应的二抗 IgG(1:100),DAPI染核,PBS洗涤后,用激光共聚焦显微镜观察NSCs、神 经元、星形胶质细胞、小胶质细胞、巨噬细胞标志物及EGFP荧光染色情况。
二、实验结果
1、双模态成像
干细胞移植后1、2、3、4、5、6、7、8w进行系列活体MRI及活体荧光成 像,结果表明:FerritinH-Bcl2-EGFP-NSCs在移植后8周内,在MRI T2FFE序 列表现为局部低信号,信号范围及强度变化不明显(图10a);生物荧光成像移 植区可见局灶性红色荧光,与MRI位置一致,荧光信号可被监测至移植后8周, 但信号强度及范围逐渐减低(图10b)。FerritinH-EGFP-NSCs移植后8周内,在 MRI T2FFE序列表现为局部低信号,但于移植5周后信号范围及强度逐渐减低 (图10c);生物荧光成像移植区可见局灶性红色荧光,但持续至4周后信号消失(图10d)。Mock-NSCs移植后在MRI及荧光成像均未见显示(图10e,f)。
实验结果表明,FerritinH-Bcl2-EGFP-NSCs移植后可被MRI及荧光成像示踪 至移植后8周,FerritinH-Bcl2-EGFP-NSCs较FerritinH-NSCs存活时间更长。
2、NSCs移植后脑梗死面积及信号演变
NSCs移植后系列MR检测T2WI测量脑梗死体积百分比见表1。 FerritinH-Bcl2-EGFP-NSCs组,细胞移植后脑梗死灶于移植后4周开始脑梗死灶 体积逐渐减小、信号逐渐减低(图11a);FerritinH-EGFP-NSCs组移植后5周, 梗死灶体积开始逐渐减小、信号逐渐减低(图11b);Mock-EGFP-NSCs组,细 胞移植后大鼠脑梗死灶体积、信号强度始终无明显变化(图11c)。
实验结果表明FerritinH-Bcl2-EGFP-NSCs对脑梗死有显著治疗效果。
表1不同转染组NSCs移植后脑梗死体积百分比(%)
4、动物行为学检测
NSCs移植8周后神经功能评分见表2。FerritinH-Bcl2-EGFP-NSCs组功能评 分最低,与FerritinH-EGFP-NSCs组、Mock-NSCs组的差异有统计学意义(P<0.05);FerritinH-EGFP-NSCs组与Mock-NSCs组神经功能评分差异无统计 学意义(P>0.05)。
实验结果表明FerritinH-Bcl2-EGFP-NSCs治疗脑梗死神经功能恢复效果最 好。
表2NSCs移植后8周大鼠神经功能评分
5、组织病理学
NSCs移植后8周,FerritinH-Bcl2-EGFP-NSCs组,梗死灶对侧注射区及胼 胝体区可见较多EGFP+细胞,其中少数为EGFP+Nestin+细胞,较多EGFP+CD11b+细胞及EGFP+GFAP+细胞,未见EGFP+NeuN+及EGFP+O4+细胞(图12a,13a), 与此对应的注射区及胼胝体内见较多普鲁士蓝蓝染颗粒(图14a)及Bcl2+细胞 (图14e)。FerritinH-EGFP-NSCs组,移植区及胼胝体区可见少量EGFP+细胞, 其中较多为EGFP+CD11b+、EGFP+GFAP+细胞(图12b,13b),未见EGFP+Nestin+、 EGFP+NeuN+及EGFP+O4+细胞,普鲁士蓝染色显示移植区见大量蓝染颗粒(图 14b)及少量Bcl2+细胞(图14f),胼胝体内见少许普鲁士蓝蓝染颗粒,未见Bcl2+细胞。Mock-NSCs组,移植区及胼胝体区未见EGFP+细胞,可见少量CD11b+、 GFAP+细胞,未见Nestin+、NeuN+及O4+细胞(图12c,13c),普鲁士蓝染色移 植区及胼胝体内未见普鲁士蓝染颗粒(图13c)及Bcl2+细胞(图14g)。
实验结果表明,FerritinH-Bcl2-EGFP-NSCs移植入梗死区对侧脑组织后能够 通过胼胝体向梗死区迁徙,并通过NSCs替代作用及分化为星形胶质细胞治疗脑 梗死。
序列表
<110> 中山大学
<120> 一种FerritinH、Bcl2及EGFP基因联合修饰的神经干细胞制备方法及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Homo sapiens
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gaggatcccc gggtaccggt cgccaccatg accaccgcgt ctccct 46
<210> 2
<211> 39
<212> DNA
<213> Homo sapiens
<400> 2
tcaccatggt ggcgaccggc ttgtggccca ggtatgcac 39
<210> 3
<211> 1311
<212> DNA
<213> Homo sapiens
<400> 3
atgaccaccg cgtctccctc gcaagtgcgc cagaactacc accaggactc ggaggctgcc 60
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tattttgacc gggatgatgt ggccctgaag aactttgcca aatactttct ccatcaatct 180
catgaagaga gggagcatgc tgagaaactg atgaagctgc agaaccagcg aggtggacga 240
atcttcctgc aggatataaa gaaacctgac cgtgatgact gggagagcgg gctgaatgca 300
atggagtgtg cactgcactt ggaaaagagt gtgaatcagt cactactgga acttcacaaa 360
ctggctactg acaagaatga tccccactta tgtgacttca ttgagacgca ttacctgaat 420
gagcaggtga aatccattaa agaactgggt gaccacgtga ccaacttacg caagatggga 480
gcccctgaat ctggcatggc agaatatctc tttgacaagc acaccctggg acacggtgat 540
gagagcgagg gcaggggaag tcttctaaca tgcggggacg tggaggaaaa tcccggcccc 600
atggcgcaag ccgggagaac agggtatgat aaccgggaga tcgtgatgaa gtacatccat 660
tataagctgt cacagagggg ctacgagtgg gatactggag atgaagactc cgcgcccctg 720
agggctgccc ccacccctgg catcttctcc ttccagcctg agagcaaccg aacgcccgct 780
gtgcaccgag acacggctgc caggacgtcg cctctacggc cccttgtcgc caacgctggg 840
cctgcgctca gccctgtgcc acctgtggtc cacctgaccc tccgccgggc tggggatgac 900
ttctctcgtc gctaccgtcg cgactttgca gagatgtcca gtcagctgca cctgacgccc 960
ttcaccgcga ggggacgctt tgccacggtg gtggaggaac tcttcaggga tggggtgaac 1020
tgggggagga ttgtggcctt ctttgagttc ggtggggtca tgtgtgtggg gagcgtcaac 1080
agggagatgt cacccctggt ggacaacatc gctctgtgga tgactgagta cctgaaccgg 1140
catctgcaca cctggatcca ggataacgga ggctgggatg cctttgtgga actatatggc 1200
cccagcatgc gacctctgtt tgatttctcc tggctgtctc tgaagacgct gctcagcctg 1260
gccctggtgg gggcctgcat cactctgggt gcatacctgg gccacaagtg a 1311
<210> 4
<211> 436
<212> PRT
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Met Thr Thr Ala Ser Pro Ser Gln Val Arg Gln Asn Tyr His Gln Asp
1 5 10 15
Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser
20 25 30
Tyr Val Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp Val Ala
35 40 45
Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg
50 55 60
Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg
65 70 75 80
Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp Asp Trp Glu Ser
85 90 95
Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Ser Val Asn
100 105 110
Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro
115 120 125
His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys
130 135 140
Ser Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly
145 150 155 160
Ala Pro Glu Ser Gly Met Ala Glu Tyr Leu Phe Asp Lys His Thr Leu
165 170 175
Gly His Gly Asp Glu Ser Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly
180 185 190
Asp Val Glu Glu Asn Pro Gly Pro Met Ala Gln Ala Gly Arg Thr Gly
195 200 205
Tyr Asp Asn Arg Glu Ile Val Met Lys Tyr Ile His Tyr Lys Leu Ser
210 215 220
Gln Arg Gly Tyr Glu Trp Asp Thr Gly Asp Glu Asp Ser Ala Pro Leu
225 230 235 240
Arg Ala Ala Pro Thr Pro Gly Ile Phe Ser Phe Gln Pro Glu Ser Asn
245 250 255
Arg Thr Pro Ala Val His Arg Asp Thr Ala Ala Arg Thr Ser Pro Leu
260 265 270
Arg Pro Leu Val Ala Asn Ala Gly Pro Ala Leu Ser Pro Val Pro Pro
275 280 285
Val Val His Leu Thr Leu Arg Arg Ala Gly Asp Asp Phe Ser Arg Arg
290 295 300
Tyr Arg Arg Asp Phe Ala Glu Met Ser Ser Gln Leu His Leu Thr Pro
305 310 315 320
Phe Thr Ala Arg Gly Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg
325 330 335
Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly
340 345 350
Val Met Cys Val Gly Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp
355 360 365
Asn Ile Ala Leu Trp Met Thr Glu Tyr Leu Asn Arg His Leu His Thr
370 375 380
Trp Ile Gln Asp Asn Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly
385 390 395 400
Pro Ser Met Arg Pro Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr
405 410 415
Leu Leu Ser Leu Ala Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Tyr
420 425 430
Leu Gly His Lys
435
Claims (9)
1.一种基因工程化的可示踪神经干细胞的制备方法,其特征在于,包括以下步骤:
S1. 合成融合基因FerritinH-T2A-Bcl2,其核苷酸序列如SEQ ID NO:3所示;
S2. 将融合基因FerritinH-T2A-Bcl2插入含有EGFP基因的慢病毒表达载体,得到重组质粒;
S3. 将重组质粒转染宿主细胞,制备FerritinH-T2A-Bcl2-EGFP过表达慢病毒;
S4. 将FerritinH-T2A-Bcl2-EGFP过表达慢病毒转染神经干细胞,得到基因工程化的可示踪神经干细胞FerritinH-Bcl2-EGFP-NSCs。
2.根据权利要求1所述制备方法,其特征在于,慢病毒表达载体为GV218载体。
3.根据权利要求1所述制备方法,其特征在于,宿主细胞为293T细胞。
4. 根据权利要求1所述制备方法,其特征在于,步骤S2中,线性化载体DNA与融合基因FerritinH-T2A-Bcl2 DNA的摩尔比为1:(3~9)。
5.根据权利要求1所述制备方法,其特征在于,步骤S4中,MOI指数为10。
6. 根据权利要求1所述制备方法,其特征在于,步骤S4中,转染时间为24 h。
7.根据权利要求1~6任一所述制备方法制备得到的基因工程化的可示踪神经干细胞FerritinH-Bcl2-EGFP-NSCs。
8.根据权利要求7所述基因修饰的可示踪神经干细胞,其特征在于,该神经干细胞同时过表达FerritinH、Bcl2及EGFP基因。
9.权利要求2中所述的融合基因FerritinH-T2A-Bcl2、重组质粒、FerritinH-T2A-Bcl2-EGFP过表达慢病毒和/或基因工程化的可示踪神经干细胞FerritinH-Bcl2-EGFP-NSCs在制备治疗脑梗死的制剂中的应用。
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Application publication date: 20181113 |