CN114503954A - 髓鞘形成不足以及髓鞘再生障碍动物模型的构建方法 - Google Patents
髓鞘形成不足以及髓鞘再生障碍动物模型的构建方法 Download PDFInfo
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Abstract
本发明公开了髓鞘形成不足以及髓鞘再生障碍动物模型的构建方法。本发明研究证明c‑Abl敲除可以导致小鼠髓鞘形成不足,还可以导致小鼠髓鞘再生发生延迟出现障碍,基于上述研究成果,可以通过敲除c‑Abl构建髓鞘形成不足以及髓鞘再生障碍动物模型,为研究髓鞘形成机制提供研究基础。
Description
技术领域
本发明属于生物医药领域,涉及髓鞘形成不足及髓鞘再生障碍动物模型的构建方法。
背景技术
轴突髓鞘形成对于神经回路内信息流调节过程中动作电位的快速传播至关重要;此外,髓鞘为轴突提供代谢和营养支持以防止其病变。因此,中枢神经系统(CNS)髓鞘形成不足或髓鞘再生障碍极大影响大脑功能的实现,表现为髓鞘减少、白质营养不良、多发性硬化症(MS)、神经退行性病变等病症。
少突胶质细胞(OL)负责中枢神经系统的髓鞘形成。在发育过程中,少突胶质前体细胞(OPC)—特异性表达神经胶质细胞抗原2(NG2)和血小板衍生生长因子受体α(PDGFRα)—在中枢神经系统中迁移并依次分化为OL。在分化过程中,程序性表达髓鞘少突胶质细胞糖蛋白(MOG)、髓鞘碱性蛋白(MBP)和蛋白脂蛋白。 OL的产生是动态调节过程,其在成年期通过脑室下区OPC的增殖和分化而持续。在年龄增长过程中,髓鞘再生减少及白质损伤的积累,暗示OPC在髓鞘形成、髓鞘维持和再髓鞘化方面的能力减弱。因此,促进OPC增殖、髓鞘形成和再髓鞘化的信号途径和重要分子研究受到越来越多的关注。而髓鞘形成不足和髓鞘再生障碍模型在髓鞘相关疾病的研究显得尤为重要。
多种酪氨酸激酶受体及其配体,如PDGFRα、ErbB1/EGFR、TrkA/B/C、 FGFR1/2和cMET参与了OPC增殖和分化及OL成熟。OL谱系细胞生理调节的潜在机制可为脱髓鞘相关疾病的治疗提供依据。
非受体酪氨酸激酶c-Abl参与氧化应激激活、生长因子信号转导、细胞粘附和DNA损伤等生理过程。C-Abl被发现是一种癌基因,由于9号和22号染色体易位导致c-Abl与B细胞受体(BCR)融合,从而触发慢性粒细胞白血病(CML)的发生。在中枢神经系统中,c-Abl在氧化应激诱导的神经细胞死亡中起关键作用。据报道,c-Abl抑制剂Bosutinib可以适度增加SOD1突变的肌萎缩侧索硬化症患者的存活率。
我们前期分析,c-Abl磷酸化激活哺乳动物MST1-FOXO信号,增加氧化应激诱导的神经元死亡。C-Abl磷酸化并激活p38α,在MPTP诱导的帕金森病发病机制中促进多巴胺能神经元死亡。近期,我们报道了c-Abl激酶通过磷酸化并激活线粒体抗病毒信号(MAVS)蛋白调节小胶质细胞的激活和自噬。然而,c-Abl 在OL谱系细胞中的作用尚不清楚,在髓鞘相关疾病模型的构建中的应用未见报道。
发明内容
本发明提供了一种髓鞘形成不足动物模型的构建方法,所述构建方法包括抑制动物体内的c-Abl基因表达。
抑制c-Abl基因表达的试剂不受限制,只要是可以抑制c-Abl基因表达水平或抑制c-Abl功能活性即可。
有效抑制基因表达的方法主要有:基因敲除、反义核苷酸技术和RNAi技术。
基因敲除方法包括CRISPR/Cas9技术、锌指核酸酶(ZFN)技术、转录激活因子样效应物核酸酶(TALEN)技术。
反义寡核酸技术是应用反义寡核苷酸类药物通过Waston-Crick碱基配对与细胞内核酸(DNA或RNA)特异结合形成杂交分子,从而在转录和翻译水平抑制特定基因表达的基因治疗技术。
将与mRNA序列对应的正义和反义RNA组成的双链RNA导入细胞,可以使RNA降解和基因沉默,这种转录后基因沉默(post-transcriptional gene silencing, PTGS)被称为RNAi。常用的RNAi技术使用的试剂包括shRNA和siRNA。
优选地,抑制动物体内的c-Abl基因表达的方法是敲除动物体内的c-Abl基因。
进一步,所述构建方法包括以下步骤:
1)获得c-Ablf/f;
2)获得内源性Olig2启动子控制Cre重组酶表达的动物Olig2-Cre;
3)将c-Ablf/f和Olig2-Cre杂交,获得c-Ablf/f;Olig2-Cre条件性敲除动物。
更进一步,所述构建方法还包括以下步骤:收获出生14天的c-Ablf/f;Olig2-Cre动物。
本发明提供了一种脱髓鞘再生障碍动物模型的构建方法,其特征在于,所述构建方法包括抑制脱髓鞘动物模型体内的c-Abl基因表达。
抑制c-Abl基因表达的试剂不受限制,只要是可以抑制c-Abl基因表达水平或抑制c-Abl功能活性即可。
有效抑制基因表达的方法主要有:基因敲除、反义核苷酸技术和RNAi技术。
基因敲除方法包括CRISPR/Cas9技术、锌指核酸酶(ZFN)技术、转录激活因子样效应物核酸酶(TALEN)技术。
反义寡核酸技术是应用反义寡核苷酸类药物通过Waston-Crick碱基配对与细胞内核酸(DNA或RNA)特异结合形成杂交分子,从而在转录和翻译水平抑制特定基因表达的基因治疗技术。
将与mRNA序列对应的正义和反义RNA组成的双链RNA导入细胞,可以使RNA降解和基因沉默,这种转录后基因沉默(post-transcriptional gene silencing, PTGS)被称为RNAi。常用的RNAi技术使用的试剂包括shRNA和siRNA。
优选地,抑制脱髓鞘动物模型体内的c-Abl基因表达的方法是敲除脱髓鞘动物模型体内的c-Abl基因。
进一步,所述构建方法包括以下步骤:
1)根据前面所述的构建方法获得c-Ablf/f;Olig2-Cre条件性敲除动物;
2)使用导致动物发生脱髓鞘病变的药物诱导步骤1)获得的动物发生脱髓鞘病变;所述药物是Cuprizone。
更进一步,所述构建方法包括以下步骤:
1)根据前面所述的构建方法获得c-Ablf/f;Olig2-Cre条件性敲除动物,培养至成年;
2)使用导致动物发生脱髓鞘病变的药物诱导步骤1)获得的动物发生脱髓鞘病变。
更进一步,所述构建方法包括以下步骤:
1)根据前面所述的构建方法获得c-Ablf/f;Olig2-Cre条件性敲除动物,培养至成年;
2)使用导致动物发生脱髓鞘病变的药物诱导步骤1)获得的动物发生脱髓鞘病变,诱导时间为6周。
优选地,成年动物是小鼠,成年小鼠为出生8周。
本发明提供了c-Abl基因的应用,所述应用包括以下任一项:
1)c-Abl在构建髓鞘形成不足动物模型中的应用;
2)c-Abl在构建髓鞘再生障碍动物模型中的应用;
3)c-Abl在制备促进髓鞘形成的药物中的应用;
4)c-Abl在制备促进髓鞘再生的药物中的应用;
5)c-Abl在制备治疗脱髓鞘疾病的药物中的应用。
进一步,所述脱髓鞘疾病包括多发性硬化症、视神经脊髓炎、肌萎缩性侧索硬化症、炎性脱髓鞘疾病、中枢神经系统神经病变、中央脑桥髓鞘溶解症、脊髓病变、脊髓痨、梅毒性脊髓病变、脑白质病变、脑白质营养不良、阿尔茨海默病、格林-巴利综合征、慢性炎性脱髓鞘多发性神经病、抗MAG外周神经病变、夏柯-馬利-杜斯氏病、易于压迫性麻痹的遗传性神经病变、外周神经病变、视神经病变、进行性炎性神经病变、巴洛同心圆硬化、谢尔德病、慢性炎性脱髓鞘性多发性神经病、进行性炎性神经病、急性播散性脑脊髓炎、肾上腺脊髓神经病、进行性多灶性白质脑病、帕金森氏病、中风、糖尿病神经并发症、脑小血管病变。
本发明的术语“脱髓鞘疾病”,为发生在神经系统中的髓鞘被损害的病理过程。髓鞘是由称为少突胶质细胞的神经胶质细胞在CNS中以及雪旺细胞在PNS 中形成的脂肪物质。对髓鞘的破坏损害了在受影响的神经中的信号传导。因此,传导能力的降低导致感觉、运动、认知和/或取决于所涉及的神经的其他功能的不足。脱髓鞘过程可能是由于遗传、传染原、自身免疫反应以及未知因素引起的。根据神经系统中脱髓鞘的主要部位,这些疾病分为涉及CNS的中枢脱髓鞘和影响PNS的外周脱髓鞘。脱髓鞘疾病还可以根据炎症的存在或缺乏分为炎性和非炎性疾病,并且可以根据脱髓鞘的根本原因进一步分为髓鞘碎裂性疾病(myelino-clastic disease),其中正常和健康的髓鞘被有毒、化学或自身免疫性物质破坏;和脱髓鞘性白细胞营养不良疾病,其中该髓鞘异常并退化。
本发明的术语“治疗”涉及以脱髓鞘为特征或与脱髓鞘相关的疾病、障碍或病症,是指如上所述的治疗有效量的药物组合的施用,其有效缓解与所述疾病、障碍或病症相关的不良症状;在此类症状出现之前预防它们出现;减慢所述疾病、障碍或病症的进展;减缓症状的恶化;增强缓解期的开始;减缓在所述疾病、障碍或病症的进展性慢性阶段引起的不可逆的损害;延迟所述进展阶段的开始;减轻所述疾病、障碍或病症的严重性或治愈所述疾病、障碍或病症;提高存活率或更快恢复;和/或预防所述疾病、障碍或病症的发生。
文中c-Ablf/f是指c-Abl两端带有flox位点的纯合动物。
附图说明
图1显示c-Abl基因敲除效率检测结果图,其中,A:WB图;B:WB统计结果; C:qRT-PCR统计结果;
图2显示Black-gold II髓鞘染色结果图;
图3显示TEM分析结果图,其中,A:电镜图;B:统计结果;
图4显示利用Black-gold II染色检测WT小鼠脱髓鞘再生结果图;
图5显示8周龄WT小鼠和c-Abl CKO小鼠胝体区中的MBP蛋白水平结果图,其中,A:WB图;B:WB统计结果;
图6显示8周龄WT小鼠和c-Abl CKO小鼠Olig2和CC1+Olig2+细胞免疫染色结果图,其中,A:荧光图;B:Olig2+统计结果;C:CC1+Olig2+统计结果;
图7显示TEM分析8周龄WT小鼠和c-Abl CKO小鼠髓鞘形成结果图,其中, A:电镜图;B:统计结果;
图8显示开放旷场结果图,其中,A:总距离;B:平均速度;
图9显示高架迷宫试验结果图;
图10显示利用Black-gold II染色检测c-Abl CKO小鼠脱髓鞘再生结果图;
图11显示c-Abl CKO小鼠胼胝体区MBP表达的免疫荧光图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均为常规生化试剂,可从商业途径得到。
本发明使用的统计分析方法
使用GraphPad Prism 8(GraphPad 16Software Inc,USA)进行统计分析。使用多组的单向或双向方差分析和两组学生t检验分析变量。数据表示为平均值的平均值±标准误差(SEM)。以下P值表示显着性阈值:*P<0.05、**P<0.01和 ***P<0.001。
实施例1c-Abl基因敲除对髓鞘发育的影响
一、方法
1、实验动物
C57BL/6雄性小鼠(8-10周龄)购自北京维通利华实验动物科技有限公司 (中国北京)。通过将c-Ablf/f小鼠(浙江大学生命科学研究院的仓勇教授赠予) 与Olig2-Cre小鼠(南方科技大学生物学系肖波教授赠予)杂交,获得了OL谱系特异性敲除c-Abl小鼠。雄性后代c-Ablf/f;Olig2-Cre(c-Abl条件性敲除 [c-Abl CKO])和c-Ablf/f(野生型[WT])用于实验。将小鼠维持在12:12h的明暗循环中,并提供充足食物和水。所有实验均使用雄性小鼠以避免行为测试中性别的影响,实验方案经北京基础医学研究所实验动物伦理委员会批准。本发明所有附图中的f/f;Olig2-Cre指代c-Ablf/f;Olig2-Cre条件性敲除小鼠;f/f指代c-Ablf/f小鼠。
2、Black-Gold II髓鞘染色
Black-Gold II髓鞘染色试剂盒购自Millipore(AG105),对中枢神经系统内髓鞘进行染色。将脑片水化两次,每次3分钟,在Black-Gold II溶液(预热)中以60℃孵育10-15分钟。此后,每隔3-4分钟进行监测以确认标记范围。当有髓纤维呈暗红色时,染色停止。随后,将切片在水中冲洗三次,每次2分钟,并在 60℃下浸入硫代硫酸钠溶液(1%,预热)中3分钟,用双蒸馏水冲洗3次后置于载玻片上脱水封片。
3、透射电子显微镜(TEM)
用戊巴比妥钠麻醉小鼠后灌注生理盐水。取大脑胼胝体,用3%戊二醛在4 ℃预固定2h,用0.19M蔗糖溶液冲洗3~5次后,浸泡在1%渗透酸固定剂中脱水,包埋于EPON 812中。取60~70nm厚的超薄切片,收集于铜网上经醋酸铀酰 (10min)、柠檬酸铅(10min)染色。用透射电子显微镜(H-7650,日立,日本)拍照。
4、qRT-PCR
使用TRIzol(15596018,Invitrogen,USA)和氯仿-异丙醇提取细胞或脑组织的总RNA。用随机引物和cDNA合成试剂盒(AT341,TransGen Biotech,China) 进行第一链cDNA合成。qPCR使用QuantStudio 3(Agilent Technologies,USA) 设备和2×RealStar GreenFast Mixture(with ROX)(A303-01,Genestar,China) 操作。以β-actin为内参用2-ΔΔCt)统计。用于qRT-PCR的引物序列如表1所示。
表1用于qRT-PCR的引物序列
5、免疫荧光
脑片用磷酸盐缓冲液(PBS)冲洗2次,然后用磷酸盐缓冲液(含0.4%Triton X-100、1%BSA和2%马血清)中封闭1.5h,分别用anti-c-Abl(1:1000,#2862, CellSignaling,USA),anti-Olig2(1:500,AB9610,Millipore,USA)和anti-BrdU (1:1000,ab6326,Abcam,UK)抗体在4℃过夜,加入荧光标记的二抗(Jackson ImmunoResearch,USA),室温下放置2h。激光共聚焦拍照分析。
二、结果
用c-Abl f/f和Olig2-Cre构建c-Abl f/f;Olig2-Cre小鼠(c-Abl CKO小鼠)。出生后第14天从脑组织中分离胼胝体,通过WB和qRT-PCR分别检测少突胶质细胞相关基因的蛋白和mRNA水平。大脑少突胶质细胞特异性c-Abl基因敲除后降低了c-Abl、MBP和Olig2的表达(图1)。出生后第14天,Black-gold II髓鞘染色显示c-Abl CKO小鼠在胼胝体区和外囊处显示髓鞘化不足(图2)。此外,TEM 分析显示c-Abl CKO小鼠脑区有髓轴突的比例减少(图3)。
实施例2c-Abl敲除对体内再髓鞘化的影响
一、方法
1、Cuprizone(CPZ)诱导的小鼠脱髓鞘模型
取8周龄雄性C57BL/6小鼠,用0.2%(w/w)的CPZ(中国,美仑生物)碾磨成粉掺入动物饲料中喂养6周,诱导脱髓鞘。停止CPZ饮食,髓鞘得以再生。实验动物(CPZ饮食后0、2、4周)麻醉后经心内灌注生理盐水。取动物脑组织,4℃多聚甲醛固定24h后蔗糖梯度脱水,切成40μm厚脑片进一步分析。
2、开放旷场实验
开放旷场仪是方形盒子(50cm×50cm×20cm),用于分析小鼠自发活动。将小鼠置于旷场中(用75%乙醇溶液擦拭干净)并允许自由探索盒子5分钟。使用ANY-maze软件(Global Biotech,USA)分析速度、总距离和在中心场(25 cm×25cm)中的距离。
3、高架十字迷宫测试
迷宫由两个开放臂(35cm×5cm)和两个闭合臂(35cm×5cm×15cm)、中央平台(5cm×5cm)组成,放置距离地面50厘米高度,在中央平台上方灯光照亮。用75%乙醇溶液清洗迷宫后放入小鼠,在5min内测量小鼠在开放臂和闭合臂的时间和距离。
二、结果
脱髓鞘后髓鞘再生:8周龄的小鼠喂养CPZ 6周后发生脱髓鞘,在正常喂养 4周后再髓鞘化。Black-gold II染色显示,CPZ喂养6周后,小鼠胼胝体区、纹状体和大脑皮质脱髓鞘,正常喂养4周后髓鞘再生(图4)。为探讨c-Abl基因在脱髓鞘后髓鞘再生模型中的作用,对8周大的WT和c-Abl CKO小鼠进行测试,发现8周龄WT小鼠和c-Abl CKO小鼠胼胝体区中的MBP蛋白水平没有差异(图 5)。8周龄WT小鼠和c-Abl CKO小鼠Olig2+和CC1+Olig2+细胞免疫染色结果无明显差异(图6)。TEM发现WT和CKO小鼠髓鞘形成无障碍(图7)。8周龄的 WT和c-Abl CKO小鼠在开放旷场和高架迷宫试验中没有显著差异(图8和图9)。这些数据表明,c-Abl缺乏导致髓鞘形成延迟,而不是永久性的髓鞘不足。
此后,对8周大的WT、c-Abl CKO小鼠按图4处理,CPZ喂养6周后, Black-gold II染色显示WT和c-Abl CKO小鼠在胼胝体区脱髓鞘;而c-Abl CKO 小鼠在恢复正常饮食后2周和4周时表现出髓鞘再生障碍(图10)。此外,免疫染色显示CKO小鼠胼胝体区在恢复期MBP显著降低,这与black-gold II染色结果一致(图11)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 髓鞘形成不足以及髓鞘再生障碍动物模型的构建方法
<141> 2022-01-29
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Claims (10)
1.一种髓鞘形成不足动物模型的构建方法,其特征在于,所述构建方法包括抑制动物体内的c-Abl基因表达;优选地,抑制动物体内的c-Abl基因表达的方法是敲除动物体内的c-Abl基因。
2.根据权利要求1所述的构建方法,其特征在于,所述构建方法包括以下步骤:
1)获得c-Ablf/f;
2)获得内源性Olig2启动子控制Cre重组酶表达的动物Olig2-Cre;
3)将c-Ablf/f和Olig2-Cre杂交,获得c-Ablf/f;Olig2-Cre条件性敲除动物。
3.根据权利要求2所述的构建方法,其特征在于,所述构建方法还包括以下步骤:收获出生14天的c-Ablf/f;Olig2-Cre条件性敲除动物。
4.一种髓鞘再生障碍动物模型的构建方法,其特征在于,所述构建方法包括抑制脱髓鞘动物模型体内的c-Abl基因表达;优选地,抑制脱髓鞘动物模型体内的c-Abl基因表达的方法是敲除脱髓鞘动物模型体内的c-Abl基因。
5.根据权利要求4所述的构建方法,其特征在于,所述构建方法包括以下步骤:
1)根据权利要求2所述的构建方法获得c-Ablf/f;Olig2-Cre条件性敲除动物;
2)使用导致动物发生脱髓鞘病变的药物诱导步骤1)获得的动物发生脱髓鞘病变。
6.根据权利要求5所述的构建方法,其特征在于,所述构建方法包括以下步骤:
1)根据权利要求2所述的构建方法获得c-Ablf/f;Olig2-Cre条件性敲除动物,培养至成年;
2)使用导致动物发生脱髓鞘病变的药物诱导步骤1)获得的动物发生脱髓鞘病变。
7.根据权利要求6所述的构建方法,其特征在于,所述构建方法包括以下步骤:
1)根据权利要求2所述的构建方法获得c-Ablf/f;Olig2-Cre条件性敲除动物,培养至成年;
2)使用导致动物发生脱髓鞘病变的药物诱导步骤1)获得的动物发生脱髓鞘病变,诱导时间为6周。
8.根据权利要求5-7中任一项所述的构建方法,其特征在于,导致动物发生脱髓鞘病变的药物是Cuprizone。
9.根据权利要求6-8中任一项所述的构建方法,其特征在于,成年动物是小鼠,成年小鼠为出生8周。
10.c-Abl基因的应用,所述应用包括以下任一项:
1)c-Abl在构建髓鞘形成不足动物模型中的应用;
2)c-Abl在构建髓鞘再生障碍动物模型中的应用;
3)c-Abl在制备促进髓鞘形成的药物中的应用;
4)c-Abl在制备促进髓鞘再生的药物中的应用;
5)c-Abl在制备治疗脱髓鞘疾病的药物中的应用。
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WO2017172976A1 (en) * | 2016-03-29 | 2017-10-05 | The Regents Of The University Of California | Methods for promoting oligodendrocyte regeneration and remyelination |
CN110101704A (zh) * | 2019-05-28 | 2019-08-09 | 中国人民解放军军事科学院军事医学研究院 | c-Abl激酶抑制剂在FoxM1高表达肿瘤治疗中的应用 |
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WO2017172976A1 (en) * | 2016-03-29 | 2017-10-05 | The Regents Of The University Of California | Methods for promoting oligodendrocyte regeneration and remyelination |
CN110101704A (zh) * | 2019-05-28 | 2019-08-09 | 中国人民解放军军事科学院军事医学研究院 | c-Abl激酶抑制剂在FoxM1高表达肿瘤治疗中的应用 |
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