CN114814232A - Rassf6作为阿尔茨海默病标志物的应用 - Google Patents
Rassf6作为阿尔茨海默病标志物的应用 Download PDFInfo
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Abstract
本发明涉及一种Rassf6作为阿尔茨海默病标志物的应用,属于疾病靶点研究技术领域。本发明公开了Rassf6作为疾病标志物在制备用于诊断和/或治疗阿尔茨海默病试剂和/或药物中的用途。如可通过检测Rassf6作为AD辅助诊断的依据,也可通过药物作用于Rassf6靶点,抑制Rassf6‑Hippo信号通路,从而改善AD的行为学及病理特征,起到治疗AD的作用。将为发掘治疗AD的潜在靶点提供重要的临床意义,并具有广阔的应用前景。
Description
技术领域
本发明涉及疾病靶点研究技术领域,特别是涉及一种Rassf6作为阿尔茨海默病标志物的应用。
背景技术
阿尔茨海默病(AD)是一种神经退行性病变,以进行性神经元丧失和大脑萎缩为病理表现,其临床特征为健忘症、进行性认知障碍、定向障碍、行为障碍和日常功能丧失,是痴呆的首要病因。其社区发病率为15.8‰,据Alzheimer Disease International(ADI)估算,到2050年AD病例可能达到1.31亿。
然而,除多奈哌齐作为胆碱脂酶抑制剂在AD症状控制方面有一定的受益外,别的治疗方案尚无满意的效果。因此,寻找安全有效的抗AD治疗方法已成为医学上迫切解决的问题。
发明内容
基于此,有必要针对上述问题,提供一种Rassf6作为疾病标志物在制备用于诊断和/或治疗阿尔茨海默病试剂和/或药物中的用途。
本发明公开了Rassf6作为疾病标志物在制备用于诊断和/或治疗阿尔茨海默病试剂和/或药物中的用途。
本发明人在研究中通过调研和实验发现,Hippo通路是进化上保守的信号级联通路,其控制多种重要的生物学过程,包括细胞生长、命运决定、器官大小和细胞再生。该通路核心包括一个激酶级联,丝/苏氨酸蛋白激酶MST1/2,及下游效应因子原癌蛋白YAP/TAZ。作为通路上游抑制因子,Cdh1的表达结合于粘附连接刺激蛋白激酶MST1/2,抑制Hippo通路的激活。Rassff6的高表达可直接与MST1/2结合,通过抑制MST1/2活性拮抗通路的激活,从而抑制Hippo-YAP通路激活,细胞蛋白质发生降解。此外,Hippo通路与Wnt通路存在交叉调节监视组织平衡,如wnt6可激活Fzd5-YAP信号轴促进细胞增殖,对AD进展起负性调节作用。
常规技术中,Hippo信号通路的失控与肿瘤形成,免疫损伤和心肌细胞增殖低下有关,并未提示与阿尔茨海默病相关。然而,本发明人通过研究发现,Hippo信号通路在AD发病中具有一定的作用,特别是Rassff6靶点与AD的发病具有较大关联。
进而,本发明人发现Rassf6可通过Rassf6-Hippo通路对阿尔茨海默病的发病产生影响,从而可以Rassf6作为老年痴呆的一个新的疾病标志物,作为诊断标志物以及药物治疗靶点应用。如可通过检测Rassf6作为AD辅助诊断的依据,也可通过药物作用于Rassf6靶点,抑制Rassf6-Hippo信号通路,从而改善AD的行为学及病理特征,起到治疗AD的作用。
在其中一个实施例中,所述阿尔茨海默病具有携带CLU位点突变的特点。
在其中一个实施例中,所述药物通过抑制Rassf6-Hippo信号通路改善阿尔茨海默病。
本发明还公开了一种用于检测阿尔茨海默病的试剂盒,包括用于检测生物样本中Rassf6含量的试剂。
本发明还公开了Rassf6抑制剂在制备用于改善于阿尔茨海默病药物中的用途。
在其中一个实施例中,所述Rassf6抑制剂为γ-氨基丁酸能神经前体细胞。
研究表明,γ-氨基丁酸(GABA)能神经元在细胞增殖、突触连接和神经网络的可塑性方面起重要的作用。长时程GABA能投射神经元耦合脑区控制神经干细胞的动态和海马神经发生。再者,GABA能神经的传递驱动神经突起的发育和突触的成熟。而本发明研究发现,γ-氨基丁酸能神经前体细胞可作为Rassf6抑制剂,通过抑制Rassf6-Hippo信号通路改善AD行为学及病理特征。
本发明还公开了一种药物组合物,包括γ-氨基丁酸能神经前体细胞,以及药学上可接受的辅料。
在其中一个实施例中,所述γ-氨基丁酸能神经前体细胞通过以下方法制备得到:
取健康人外周血样本,分离得到单核细胞;并将上述单核细胞扩增培养,加入OCT3/4、SOX2、KLF4、c-MYC和仙台病毒进行转染,并以神经前体细胞培养基培养,得到人源性诱导型神经前体细胞,再给予γ-氨基丁酸能神经前体细胞培养基进行培养,使细胞向MGE分化,即得所述γ-氨基丁酸能神经前体细胞。
本研究运用体细胞重编程技术,把人源的外周血单核细胞(PBMNC)直接重编程为诱导型神经前体细胞(hiNPC),将其分化为GABA能中间神经元前体细胞,克服了采用胚胎干细胞制备MGE所存在的伦理学风险及培养成本高的缺陷。
本发明还公开了上述的药物组合物的制备方法,包括以下γ-氨基丁酸能神经前体细胞的制备方法:
取健康人外周血样本,分离得到单核细胞;并将上述单核细胞扩增培养,加入OCT3/4、SOX2、KLF4、c-MYC和仙台病毒进行转染,并以神经前体细胞培养基培养,得到人源性诱导型神经前体细胞,再给予γ-氨基丁酸能神经前体细胞培养基进行培养,使细胞向MGE分化,即得所述γ-氨基丁酸能神经前体细胞。
在其中一个实施例中,所述γ-氨基丁酸能神经前体细胞的制备方法为:
取健康人外周血样本,分离得到单核细胞;并将上述单核细胞以0.5-1×106密度接种至单核细胞扩增培养基,细胞扩增至14±1天时,加入浓度为(2±0.2)×105CIU/mL的OCT3/4、浓度为(2±0.2)×105CIU/mL的SOX2、浓度为(2±0.2)×105CIU/mL的KLF4、浓度为(2±0.2)×105CIU/mL的c-MYC和浓度为(2±0.2)×105CIU/mL的仙台病毒进行转染,转染第3±0.5天时,换为神经前体细胞培养基培养,培养约21±1天时得到人源性诱导型神经前体细胞,更换为γ-氨基丁酸能神经前体细胞培养基DMEM/F12进行培养,并加入NEAA、N2、SHH激动剂,使细胞向MGE分化,分化培养至第6±1天时,更换为DMEM/F12培养基,并加入1×1NEAA、1×N2、1×B27、Pur,继续分化培养9±1天,即得所述γ-氨基丁酸能神经前体细胞。
与现有技术相比,本发明具有以下有益效果:
本发明对AD发病机制进行研究,该研究方法新颖,视角独特,重复性好,发现了基于Rassf6-Hippo通路的AD发病机制,从而发现老年痴呆的一个新的疾病标志物Rassf6,为AD诊断和治疗提供一个新的靶向通路。也即可将Rassf6作为疾病标志物,在制备用于诊断和/或治疗阿尔茨海默病试剂和/或药物中使用。
因此,本发明将为发掘治疗AD的潜在靶点提供重要的临床意义,并具有广阔的应用前景。
附图说明
图1为将细胞重编程为人源性诱导型神经前体细胞(hiNPC)的流程图;
图2为将hiNPC向γ-氨基丁酸能神经元前体细胞(MGE)分化的流程图;
图3为激光扫描共聚焦显微镜对hiNPC的标记鉴定示意图;
图4为对hiNPC标记进行定量分析结果图;
图5为以RT-PCR对hiNPsC标记和细胞增殖标志的测定结果;
图6为核型分析结果图;
图7为γ-氨基丁酸能神经元前体细胞(MGE)标记NKX2.1和FOX1免疫荧光图片;
图8为γ-氨基丁酸能神经元前体细胞(MGE)标记NKX2.1和FOX1统计结果;
图9为γ-氨基丁酸能神经元标记NKX2.1和GABA免疫荧光图片;
图10为γ-氨基丁酸能神经元标记NKX2.1和GABA统计结果图;
图11为分化成熟的γ-氨基丁酸能神经元表达突触标记Synapsin-1免疫荧光图片;
图12为分化成熟的γ-氨基丁酸能神经元表达突触标记Synapsin-1统计结果图;
图13为细胞分化第20天GABA含量测定代表性HPLC图;
图14为细胞分化第40天GABA含量测定代表性HPLC图;
图15为GABA递质分泌结果图;
图16为Morris水迷宫实验轨迹图;
图17为小鼠到达平台的时间;
图18为小鼠到达原平台的时间、穿越原平台次数及在原平台所在象限停留的时间;
图19为Y迷宫实验中各组动物进入臂的总次数;
图20为Y迷宫实验中各组动物的自发交替反应率;
图21为组成记忆环路的重要脑区脑内示意图;
图22为海马区亚区和分层示意图;
图23为Aβ斑块在记忆环路区的代表性图片;
图24为p-Tau阳性细胞在记忆环路区的代表性图片;
图25为Aβ斑块数量在各脑区的统计结果;
图26为Aβ斑块面积在各脑区的统计结果;
图27为p-Tau阳性细胞在各脑区表达量统计结果;
图28为代表性NeuN阳性和Nissl阳性细胞染色分布于HIP区示意图;
图29为代表性NeuN阳性和Nissl阳性细胞染色分布于Pir区示意图;
图30为代表性NeuN阳性和Nissl阳性细胞染色分布于PFC区示意图;
图31为NeuN阳性和Nissl阳性细胞在HIP区、Pir区和PFC区的表达统计;
图32为GABA信号通路(靶向通路)基因表达热图;
图33为KEGG富集前20位靶向基因气泡图;
图34为目的基因GO富集示意图;
图35为GSEA显示的AD相关基因示意图;
图36为STRING分析目的基因交互网示意图;
图37为对部分目的基因验证的mRNA相对表达量;
图38为HIP、Pir、CC、MS、PFC区Fzd5、IL-1α、Fadd、Wnt 6、Cdh1的代表性图片;
图39为HIP、Pir、CC、MS、PFC区Fzd5、IL-1α、Fadd、Wnt 6、Cdh1表达量统计结果;
图40为临床血清样本中Rassf6因子含量;
图41为hiNPC细胞培养基上清中的Rassf6因子含量。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
定义:浓度单位符号“×”表示稀释倍数,如市售产品标识为浓缩液(100×),即用时稀释100倍至1×。
以下实施例所用试剂,如非特别说明,均为市售可得;以下实施例所用方法,如非特别说明,均为常规方法可实现。
实施例1
γ-氨基丁酸能神经前体细胞的制备,包括以下步骤,其中步骤1-6的重编程流程如图1所示,步骤7的分化流程如图2所示:
(1)筛选健康青壮年,签订医学生物样本库样本收集同意书,无菌条件下在志愿者的右前臂肘静脉采血,约10mL,EDTA-K2抗凝。
(2)外周血和PBS以1:2比例混合,加入人淋巴细胞分离液,750g离心30min,取中间云雾层,提取单个核细胞(PBMNC)。
(3)细胞按0.5-1×106密度接种于6孔板,以单个核细胞扩增培养基培养,隔两天换液一次,进行扩增获得PBMNCs。
(4)单个核细胞细胞扩增到第14天时,于培养基中加入OCT3/4、SOX2、KLF4、c-MYC和仙台病毒进行转染。
转染体系中,转录因子OCT3/4(POU class 5homeobox 1,来源:Invitrogen)的浓度为2×105CIU/mL、转录因子SOX2(来源:Invitrogen)的浓度为2×105CIU/mL、转录因子KLF4(来源:Invitrogen)的浓度为2×105CIU/mL、转录因子c-MYC(来源:Invitrogen)的浓度为2×105CIU/mL、仙台病毒(来源:Invitrogen)的浓度为2×105CIU/mL。
(5)2天后(即转染开始第3天)将单个核细胞扩增培养基换成神经前体细胞培养基。
(6)大约在仙台病毒转染后第10天左右,长出hiNPC克隆,转染后3周左右,可挑人源性诱导型神经前体细胞(hiNPC)克隆至铺有浓度为0.1mg/mL的PDL(多聚赖氨酸)和浓度为0.01mg/mL的Laminin(层粘连蛋白)的48孔板上扩增。
(7)将hiNPC以1×105密度接种于铺有浓度为0.1mg/mL的PDL(多聚赖氨酸)和浓度为0.01mg/mL的Laminin(层粘连蛋白)的6孔板,给予γ-氨基丁酸能神经前体细胞(MGE)培养基使细胞向MGE分化。
该过程约15天,分两个步骤:首先,前6天,用γ-氨基丁酸能神经前体细胞培养基DMEM/F12进行培养,并加入1×1NEAA(Gibco)、1×N2(Stem Cell)、1.5μM SHH激动剂(Pur,Stemgent),使细胞向MGE分化,分化培养至第6天时,更换为DMEM/F12(Gibco)培养基,并加入1×1NEAA(Gibco)、1×N2(Stem Cell)、1×B27(Thermofisher)、1.5μM Pur(Stemgent),继续分化培养9天,即细胞分化至第15天,得到γ-氨基丁酸能神经前体细胞。
(8)得到γ-氨基丁酸能神经前体细胞,也即MGE后,以NEAA、N2、cAMP(来源:Sigma)、IGF1(来源:Peprotech)、GDNF(来源:Peprotech)、BDNF(来源:Peprotech)培养。经继续分化培养后,确认终末分化得到的即是GABA中间神经元。
实施例2
MGE对AD的治疗效果的模型评估。
1、方法。
按照上述实施例1的方法制备得到的γ-氨基丁酸能神经前体细胞(MGE),并按5×106个细胞每只动物的量,通过脑部定位定位仪右侧海马注射给予AD双转基因模型鼠APP/PS1小鼠(购自常州卡文斯实验动物中心),同时以野生型WT小鼠为空白对照Control组。具体如下:
1.1标记γ-氨基丁酸能神经前体细胞制备
利用慢病毒载体pLV-CMV-MCS-EGFP-3FLAG-IRES-puro(来源:广州基旦生物科技有限公司)转染培养得到的hiNPC,使之带上绿色荧光标签,并参照上述实施例1的方法,将带有GFP标记的hiNPC向MGE方向分化,得到具有绿色荧光标签的γ-氨基丁酸能神经前体细胞。
1.2手术
取APP/PS1双转基因小鼠,4月龄,雄性,购自常州卡文斯实验动物中心,该小鼠表达嵌合的小鼠/人淀粉样蛋白前体蛋白(Mo/HuAPP695swe)和突变的人早老蛋白1(PS1-dE9),能够模拟AD患者脑内的beta淀粉状蛋白沉淀。适应性饲养1周后,开始实验。
术前12h禁食,6h禁水。动物经3%戊巴比妥钠生理盐水溶液(1.5ml/kg)腹腔注射麻醉,俯卧位固定于脑部立体定位仪,备皮,碘伏消毒,头部正中切口,暴露颅骨,沿正中线切开大鼠颅顶皮肤,剥离骨膜,暴露前囟。以前囟为准,参照Paxinos和Watson鼠脑立体定位图谱,确定小鼠右侧海马DG区的坐标:前囟后2.0mm,矢状缝右侧0.75mm,颅骨下2.0mm。以牙科钻开颅,用10uL微量注射器经孔缓慢地垂直进针到相应坐标点的深度,注射带有GFP标签的人源的hiNPC来源的MGE或人源的hiNPC,浓度为1×106个细胞/uL,共注射5uL。手术完成后,缝合切口皮肤,待动物清醒后放回笼中饲养。
从术前一天开始,动物每天皮下注射环孢素A(7mg/kg)抑制体内排斥反应。
2、结果。
2.1γ-氨基丁酸能神经前体细胞制备
hiNPC的验证结果如图3-6所示,各图中SOX2是性别决定区Y框蛋白2,为神经干细胞标记物、NESTIN是神经巢蛋白,为神经干细胞标记物、PAX6为是配对盒基6,为神经干细胞标记物、Ki67为细胞增殖标记物,与有丝***密切相关、OCT4是八聚体结合转录因子4,为细胞多能性标记物,Merge为多个通道的免疫荧光合成图。其中,图3和图4分别为hiNPCs标记鉴定及定量分析结果图;图5为以RT-PCR对hiNPsC标记和细胞增殖标志的测定结果,从左至右依次为SOX2、PAX6、NANOG、OCT4和NESTIN组;图6为核型分析结果图。
结果显示,hiNPC细胞系表达经典的神经干细胞标记SOX2、NESTIN和PAX6,并具有很好的增殖活性和正常的核型。
γ-氨基丁酸能神经前体细胞制备及验证结果如图7-15所示,各图中NKX2.1为MGE转录因子、FOXG1为端脑转录因子、GABA为γ-氨基丁酸、Tuj1为成熟神经元标记物,Synapsin-1是神经突触素1,为突触标记物,图7和图8分别为γ-氨基丁酸能神经元前体细胞(MGE)标记NKX2.1和FOX1免疫荧光图片和统计结果图;图9和图10分别为γ-氨基丁酸能神经元标记NKX2.1和GABA免疫荧光图片和统计结果图;图11和图12分别为分化成熟的γ-氨基丁酸能神经元表达突触标记Synapsin-1免疫荧光图片和统计结果图;图13和图14分别为细胞分化第20天和第40天GABA含量测定代表性HPLC图;图15为GABA递质分泌结果图。
结果显示,hiNPC方向分化第15天,80%以上细胞表达前脑腹侧祖细胞标记;随后分化的20天,细胞逐渐生长突出,约90%细胞合并表达NKX2.1和GABA递质标记;在分化第37天,可见95%成熟神经元表达几乎同等量的突触蛋白。HPLC提示GABA递质在分化第20天至37天呈时间依赖式分泌。
2.2 AD小鼠评估
(1)行为学评估
将小鼠饲养至6个月时,以Morris水迷宫实验进行行为学指标检测,评估其学习记忆能力,结果如图16-19所示,各图中,WT为野生型Control组小鼠,APP/PS1为APP/PS1转基因AD模型小鼠,hiNPCs-APP/PS1为APP/PS1转基因AD模型小鼠海马移植hiNPCs,hiNPCs-derived MEGs-APP/PS1为APP/PS1转基因AD模型小鼠海马移植hiNPCs分化的MEGs。
其中,图16a和图16b分别为代表性定位巡航和探索实验轨迹图;图17为各组小鼠到达平台的时间示意图,图18为各组小鼠到达原平台的时间、穿越原平台次数及在原平台所在象限停留的时间,图19为Y迷宫实验中各组动物进入臂的总次数;图20为Y迷宫实验中各组动物的自发交替反应率。
结果显示,细胞移植6个月时,Morris水迷宫实验前5天的定位巡航可见注射hiNPC来源的MGE组小鼠到达平台时间比注射hiNPC组小鼠的时间更短(图16a,图17);第6天的探索实验,与APP/PS1组小鼠比较,注射hiNPC来源的MGE组(hiNPCs-derived MEGs-APP/PS1组)小鼠能明显减少到达原平台的时间,增加原平台的穿越次数及在原平台所在象限的停留时间,而hiNPC组(hiNPCs-APP/PS1)小鼠以上三个指标的变化趋势与hiNPC来源的MGE组一样,但没有统计学意义(图16b,图18)。Y迷宫实验也发现,hiNPC来源的MGE组可明显增加APP/PS1鼠进入臂的总次数和自发交替反应率,而hiNPC组小鼠无统计学差异(图19-20)。
(2)记忆环路AD病理标志物的检测。
组成记忆环路的重要脑区在脑内分布(图21)和海马区分亚区(图22a)和分层(图22b)所示,运用免疫组化法进行检测,结果如图23-27所示,各图中,APP/PS1为APP/PS1转基因AD模型小鼠,hiNPCs-APP/PS1为APP/PS1转基因AD模型小鼠海马移植hiNPCs,hiNPCs-derived MEGs-APP/PS1为APP/PS1转基因AD模型小鼠海马移植hiNPCs分化的MEGs。
其中,图23和图24分别为Aβ斑块和p-Tau阳性细胞在记忆环路区的代表性图片。图25为Aβ斑块数量在各脑区的统计结果;图26为Aβ斑块面积在各脑区的统计结果;图27为p-Tau阳性细胞在各脑区表达量统计结果,各组中由左至右依次为APP/PS1组、hiNPC组和hiNPC-derived MGEs组。
结果显示,如与APP/PS1组比较,hiNPC组和hiNPC来源的MGE组均能明显降低记忆环路脑区海马区(HIP)、梨状皮质(Pir)和额叶皮质(PFC)Aβ和P-Tau蛋白的表达,然而,hiNPC来源的MGE组比hiNPC组更明显。
(3)移植的细胞助于重建神经环路。
以免疫组化方法评估移植的细胞对重建神经环路的影响,结果如图28-31所示,各图中,APP/PS1为APP/PS1转基因AD模型小鼠,hiNPCs为APP/PS1转基因AD模型小鼠海马移植hiNPCs,hiNPCs-derived MEGs为APP/PS1转基因AD模型小鼠海马移植hiNPCs分化的MEGs。
其中,图28-30分别为代表性NeuN阳性和Nissl阳性细胞染色分布于HIP区(图28)、Pir区(图29)和PFC区(图30)示意图。图31为NeuN阳性和Nissl阳性细胞在HIP区(A和B)、Pir区(C和D)和PFC区(E和F)的表达统计。
结果显示,APP/PS1组比较,hiNPC组和hiNPC来源的MGE组均能明显增加记忆环路神经元NeuN阳性和尼氏阳性细胞的比例。
(4)RNA测序及目的基因分析评估MGE细胞对AD小鼠的影响。
为进一步阐明其作用机制,对移植正常人来源的MGE细胞的APP/PS1鼠(hiNPC-derived MGEs)脑进行RNA测序,而APP/PS1鼠作为对照。结果如图32-37所示。
其中,图32为目的基因热图;图33为目的基因KEGG富集示意图;图34为目的基因GO富集示意图;图35为GSEA显示的AD相关基因;图36为STRING分析目的基因交互网示意图;图37为对部分目的基因验证的mRNA相对表达量,每组中从左至右依次为Fzd5、Klf4、IL-1α、Cdh1、Wnt6、Fadd、Rassf6。
结果显示,MGE治疗组目的基因Fzd5、Klf4、IL1a下调,而Cdh1,Wnt6、Fadd、Rassf6上调(图32)。KEGG富集于Alzheimer’s disease、Wnt、Hippo等通路(图33)。GO数据集分析提示目的基因富集于细胞进程、生物进程及信号(图34)。GSEA分析与AD进展正相关和负相关的基因(图35)。基因交互分析示共有16个基因与关注的通路有关(图36)。mRNA验证示目的基因在治疗组表达量的变化与测序的一致(图37)。
(5)关键目的基因的蛋白质的表达验证
对上述分析得到的关键目的基因进行蛋白质表达验证,结果如图38-39所示,各图中,APP/PS1为APP/PS1转基因AD模型小鼠,hiNPCs-derived MEGs为APP/PS1转基因AD模型小鼠海马移植hiNPCs分化的MEGs。
其中,图38为HIP、Pir、CC、MS、PFC区Fzd5、IL-1α、Fadd、Wnt 6、Cdh1的代表性图片;图39为HIP、Pir、CC、MS、PFC区Fzd5(A)、IL-1α(B)、Fadd(C)、Wnt 6(D)、Cdh1(E)表达量的统计。
如图38所示,Fzd5、Fadd、IL-1α免疫阳性细胞主要表达于HIP、Pir、PFC、胼胝体(CC)、内侧隔核(MS),Wnt 6表达于除CC外其余4个脑区,而Cdh1表达于细胞间隙。MGE治疗可降低APP/PS1鼠Fzd5、IL-1α表达及升高Fadd、Wnt 6和Cdh1(主要在HIP、MS、PFC区)的表达(图39)。
上述结果显示,hiNPCs-derived MEGs移植可调节神经环路目的基因表达。
实施例3
Rassf6因子作为疾病靶点在AD发病机制中的作用验证。
1、方法。
1.1临床样本血清含量
经广西壮族自治区人民医院伦理委员会同意后收集AD患者血液标本和正常人血液标本,其中AD标本分为带有CLU位点突变组和非CLU位点突变组。取血清经Elisa法测定Rassf6含量。
1.2细胞培养上清含量
取带有CLU位点突变的AD患者和正常人的PBMNC,按照实施例1的方法重编程为hiNPC。对比AD-hiNPC细胞和Cont-hiNPC细胞(正常人来源)培养基上清Rassf6含量。
2、结果。
临床血清样本中Rassf6因子含量如图40所示,其中,Cont,正常人组;Non-CLUmutation AD,非CLU位点突变的AD患者组;CLU mutation AD,携带CLU位点突变的AD患者组。hiNPC细胞培养基上清中的Rassf6因子含量如图41所示。
结果显示,一方面,带有CLU位点突变的AD患者血清Rassf6含量比正常人明显升高(图40)。另一方面,按照实施例1方法,将带有CLU位点突变AD患者的PBMNCs重编程为NPCs,Elisa法测细胞上清Rassf6水平,结果发现,AD-hiNPC CLU细胞培养基上清的Rassf6含量比正常人来源的hiNPC(Cont-hiNPC)明显升高(图41)。
综上所述,本发明提供的用于治疗AD的正常人来源hiNPC,其分化的MGE,可改善AD的行为学障碍,减少AD的病理改变并重建记忆环路,其作用机制与调节Rassf6-Hippo通路有关。该通路的调节因子也在AD临床样本上得到验证。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.Rassf6作为疾病标志物在制备用于诊断和/或治疗阿尔茨海默病试剂和/或药物中的用途。
2.根据权利要求1所述的用途,其特征在于,所述阿尔茨海默病具有携带CLU位点突变的特点。
3.根据权利要求1所述的用途,其特征在于,所述药物通过抑制Rassf6-Hippo信号通路改善阿尔茨海默病。
4.一种用于检测阿尔茨海默病的试剂盒,其特征在于,包括用于检测生物样本中Rassf6含量的试剂。
5.Rassf6抑制剂在制备用于改善于阿尔茨海默病药物中的用途。
6.根据权利要求5所述的用途,其特征在于,所述Rassf6抑制剂为γ-氨基丁酸能神经前体细胞。
7.一种药物组合物,其特征在于,包括γ-氨基丁酸能神经前体细胞,以及药学上可接受的辅料。
8.根据权利要求7所述的药物组合物,其特征在于,所述γ-氨基丁酸能神经前体细胞通过以下方法制备得到:
取健康人外周血样本,分离得到单核细胞;并将上述单核细胞扩增培养,加入OCT3/4、SOX2、KLF4、c-MYC和仙台病毒进行转染,并以神经前体细胞培养基培养,得到人源性诱导型神经前体细胞,再给予γ-氨基丁酸能神经前体细胞培养基进行培养,使细胞向MGE分化,即得所述γ-氨基丁酸能神经前体细胞。
9.权利要求7或8所述的药物组合物的制备方法,其特征在于,包括以下γ-氨基丁酸能神经前体细胞的制备方法:
取健康人外周血样本,分离得到单核细胞;并将上述单核细胞扩增培养,加入OCT3/4、SOX2、KLF4、c-MYC和仙台病毒进行转染,并以神经前体细胞培养基培养,得到人源性诱导型神经前体细胞,再给予γ-氨基丁酸能神经前体细胞培养基进行培养,使细胞向MGE分化,即得所述γ-氨基丁酸能神经前体细胞。
10.根据权利要求9所述的制备方法,其特征在于,所述γ-氨基丁酸能神经前体细胞的制备方法为:
取健康人外周血样本,分离得到单核细胞;并将上述单核细胞以0.5-1×106密度接种至单核细胞扩增培养基,细胞扩增至14±1天时,加入浓度为(2±0.2)×105CIU/mL的OCT3/4、浓度为(2±0.2)×105CIU/mL的SOX2、浓度为(2±0.2)×105CIU/mL的KLF4、浓度为(2±0.2)×105CIU/mL的c-MYC和浓度为(2±0.2)×105CIU/mL的仙台病毒进行转染,转染第3±0.5天时,换为神经前体细胞培养基培养,培养约21±1天时得到人源性诱导型神经前体细胞,更换为γ-氨基丁酸能神经前体细胞培养基DMEM/F12进行培养,并加入NEAA、N2、SHH激动剂,使细胞向MGE分化,分化培养至第6±1天时,更换为DMEM/F12培养基,并加入1×1NEAA、1×N2、1×B27、Pur,继续分化培养9±1天,即得所述γ-氨基丁酸能神经前体细胞。
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