CN110367202B - 一种肠菌外膜囊泡在制备痴呆动物模型中的应用 - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种肠菌外膜囊泡(OMVs)在制备痴呆动物模型中的应用,从罹患痴呆的阿尔茨海默病(AD)患者粪便中提取的OMVs作为肠道和大脑之间的媒介物,加重GSK‑3β和tau高磷酸化、海马神经炎症,从而导致小鼠出现痴呆行为改变。
Description
技术领域
本发明属于生物技术领域,具体涉及一种肠菌外膜囊泡在制备痴呆动物模型中的应用。
背景技术
阿尔茨海默病(AD)是一种影响全球数千万人的重要疾病,是最常见的痴呆类型,其发病机制尚未完全阐明。AD是一种持续时间长、进展缓慢的疾病,而糖原合成酶激酶3β(GSK-3β)参与病理生理过程的调节可能是一个关键因素。GSK-3β诱导神经炎症、氧化应激、细胞凋亡等,增加乙酰胆碱酯酶、磷酸化tau蛋白活性,形成双螺旋丝。此外,还发现使用GSK-3β抑制剂可以缓解神经炎症的病理过程,减少淀粉样蛋白异常聚集。鉴于GSK-3β是AD发病机制中的一种重要蛋白,它已成为研究的热点,有助于揭开AD的神秘面纱。,
肠道细菌(GM)与中枢神经系统(“微生物-肠道-脑轴”)的相互作用是目前研究的热点,而AD与GM的异常组成有关。大多数情况下,GM的代谢产物,如短链脂肪酸,会以多种方式影响神经系统。然而,作用于大脑的具体物质、机制和信号通路仍需进一步研究。
由细菌分泌的外膜囊泡(OMVs),直径20-250nm,携带细菌的脂多糖(LPS)、蛋白酶、膜受体、DNA、RNA和ETC。有几项研究证实了OMVs在宿主信息通信和调节中起着至关重要的作用,甚至可以推测OMVs与AD存在相关。
发明内容
本发明的目的是提供一种肠菌外膜囊泡在制备痴呆动物模型中的应用,从AD患者粪便中提取的OMVs是否可以作为肠道和大脑之间的媒介物,加重GSK-3β和tau高磷酸化、海马神经炎症,从而可能诱发痴呆行为改变。
为了解决以上技术问题,本发明采用以下技术方案:
一种肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,将阿尔茨海默病患者的肠菌外膜囊泡用于构建痴呆动物模型。
进一步地,所述肠菌外膜囊泡来自于阿尔茨海默病患者的粪便。
进一步地,所述肠菌外膜囊泡的模式直径为100-120nm。
进一步地,所述肠菌外膜囊泡的提取和纯化方法为:将阿尔茨海默病患者的粪便充分搅拌成浆液,匀浆转移至玻璃坛中,滤网过滤2次,过滤后将滤液分装至50ml离心管中,1200g,4℃离心5min;离心后取上清液,3000g,4℃离心20min;离心后取上清液,6000g,4℃离心40min;离心后取上清液,10000g,4℃离心1h;离心后取上清液,15000g,4℃离心1h;离心后取上清液,用0.45μm膜过滤器过滤上清液,取滤液,用0.22μm膜过滤器过滤,取滤液。滤液集中装到耐超高速离心管中,170000g,4℃超高速离心2h,离心后去滤液,沉淀用0.5MPBS重悬。
进一步地,所述痴呆动物模型的动物为小鼠。
进一步地,所述痴呆动物模型的动物血脑屏障损伤,学习记忆功能明显受损,出现痴呆行为改变。
进一步地,所述痴呆动物模型的动物的炎症因子、肿瘤坏死因子α的表达显著增加。
进一步地,所述痴呆动物模型的动物的海马中IBA1和GFAP阳性细胞,IBA1和GFAP蛋白表达增加。
进一步地,所述肠菌外膜囊泡加重痴呆动物模型的动物的GSK-3β和tau高磷酸化、海马神经炎症。
本发明具有以下有益效果:
本发明中AD患者肠菌的OMVs作为肠-脑交流的媒介,破坏血脑屏障(BBB)通过糖原合成酶激酶3β(GSK3β)诱导tau磷酸化增加,激活星形胶质细胞和小胶质细胞,增加炎症细胞因子(NF-κB、TNF-α、IL-1β)分泌,促进学习和记忆的障碍。病理学及行为学上出现痴呆的表现。
本发明通过AD患者肠菌能够方便的构建具有显著痴呆特征的动物模型,方便其他科学研究。
附图说明
图1为动物处理的时间轴。图1A:将小鼠移入实验区,适应新环境1周,同时提取纯化人的OMVs。在第二周始,AD-OMVs组和HL-OMVs组的小鼠每天注射相同剂量(50μm)的OMVs,持续8周。对照组注射生理盐水(0.9%)100ul,频率和周期与其他组相匹配。第八周,小鼠接受Morris水迷宫试验,如文中所述。在第八周末处死小鼠,收集样本。图1B为实验操作流程图。
OMVs:外膜囊泡;AD-OMVs:阿尔茨海默病患者粪便的OMVs;HL-OMVs:健康志愿者粪便的OMVs;MWM:Morris水迷宫。
图2为AD患者和健康对照者OMVs的电子显微镜图像(A,B)和NTA(C,D)中OMVs的特征。
图3为OMVs对体重的影响的统计结果图。A、B为三组小鼠尾静脉注射前及注射8周后的体重,C为三组小鼠体重变化情况。以平均值±SEM表示的值。使用单因素方差分析确定显著性。AD-OMVs组(n=5),对照组(n=5),HL-OMVs组(n=4)。OMVs,外膜囊泡;AD-OMVs,来自阿尔茨海默病患者粪便的OMVs;HL-OMVs,来自健康志愿者粪便的OMVs。
图4为OMVs进入大脑并影响血脑屏障通透性的结果图。A)显微镜检测海马组织中pkh26标记的OMVs(浅色)。B)小鼠脑内EB浓度。C)claudin-5的表达。*P<0.05,*P<0.01。EB,伊文斯蓝。OMVs,外膜囊泡;AD-OMVs,来自阿尔茨海默病患者粪便的OMVs;HL-OMVs,来自健康志愿者粪便的OMVs。
图5为AD-OMVs诱发痴呆行为的水迷宫评估结果图。A)AD-OMVs组小鼠、HL-OMVs组小鼠和对照组小鼠的逃逸潜伏期。B-D)第6天结束时三组在水迷宫中的游泳路径。E)三组跨平台频率;F)三组跨平台象限时间百分比;G)三组平台目标象限路径/总路径(n=8,每组)。以平均值±SEM表示。使用单因素方差分析确定显著性,*P<0.05,*P<0.01。OMVs,外膜囊泡;AD-OMVs,来自阿尔茨海默病患者粪便的OMVs;HL-OMVs,来自健康志愿者粪便的OMVs。
图6为AD-OMVs增加GSK3β和Tau的统计结果图。A)WB分析p-GSK3β和GSK3β。B)p-tau396和tau46。以平均值±SEM表示。使用单因素方差分析确定显著性,*P<0.05,*P<0.01。OMVs,外膜囊泡;AD-OMVs,阿尔茨海默病患者粪便的OMVs;HL-OMVs,健康志愿者粪便的OMVs;GSK3β:糖原合酶激酶3β。
图7为AD-OMVs增加了炎症细胞因子的统计结果图。A)三组IL-1β的Western blot结果。B)三组TNF-αWestern blot结果。C)三组中NF-κBWestern blot结果。以平均值±SEM表示的值。使用单因素方差分析确定显著性,*P<0.05,*P<0.01。OMVs,外膜囊泡;AD-OMVs,阿尔茨海默病患者粪便的OMVs;HL-OMVs,健康志愿者粪便的OMVs;NF-κB,核因子kappab;TNF-α,肿瘤坏死因子-α;IL-1β,白细胞介素-1β。
图8为AD-OMVs激活小胶质细胞和星形胶质细胞的结果图。海马中表达IBA1(B)和GFAP(A)的细胞的免疫荧光成像和Western blot(C、D)结果。OMVs,外膜囊泡;AD-OMVs,来自阿尔茨海默病患者粪便的OMVs;HL-OMVs,来自健康志愿者粪便的OMVs。IBA1,离子化钙结合接头分子1;GFAP,胶质纤维酸性蛋白。
具体实施方式
为便于更好地理解本发明,通过以下实例加以说明,这些实例属于本发明的保护范围,但不限制本发明的保护范围。
1参与者
根据入选标准和排除标准招募年龄和性别匹配的16名AD患者和18名健康对照者。经患者同意,经医院伦理委员会批准,患者和对照组均从广东医科大学附属医院招募。AD粪便提供者的来源需要符合AD的标准(NINCDS-ADRDA)。
2粪便收集
每天收集AD患者和健康志愿者粪便,10%甘油-80℃冰箱冷冻保存,直至所有样本采集完成。从每个样本中取1.5g粪便混合物,用于纯化OMVs。
3粪便中OMVs的提取与纯化
OMVs方法的提取和纯化如下:
将粪便充分搅拌成浆液。匀浆转移至玻璃坛中,滤网过滤2次,过滤后将滤液分装至50ml离心管中,1200g,4℃离心5min;离心后取上清液,3000g,4℃离心20min;离心后取上清液,6000g,4℃离心40min;离心后取上清液,10000g,4℃离心1h;离心后取上清液,15000g,4℃离心1h;离心后取上清液,用0.45μm膜过滤器过滤上清液,取滤液,用0.22μm膜过滤器过滤,取滤液。滤液集中装到耐超高速离心管中,170000g,4℃超高速离心2h,离心后去滤液,沉淀用0.5M PBS重悬。最后,BCA分析OMVs的蛋白质浓度。OMVs实验使用浓度为50μg/ml。
用扫描电镜观察OMVs的形态特征,确定OMVs的形状。此外,单纳米颗粒跟踪分析技术(NTA)检测OMVs颗粒尺寸,密度分布。来自AD患者、健康对照者的OMVs分别被指定为ADOMVs和HOMVs。
4动物
从南京动物实验研究中购买36只正常健康雄性C57BL/6J小鼠,年龄约9个月,体重约26-35g。在广东医科大学动物实验中心(湛江)提供的无特定病原体(SPF)条件饲养小鼠,采用适宜的垫料、标准的啮齿动物饲料、25℃的室温、54%至64%的湿度和12小时的明暗循环下饲养,垫料5天更换一次。这项研究得到了广东省湛江市动物中心伦理委员会的批准。动物处理时间点如图1所示。
5注射OMVs
将9个月龄的正常C57BL小鼠分为三组:AD患者(AD-OMVs组)、健康成人(HL-OMVs组)和生理盐水注射组(对照组)。使用小鼠固定器固定小鼠,暴露尾巴,用胰岛素针将OMVs注射到尾静脉。
AD-OMVs组和HL-OMVs组的小鼠每天注射相同剂量(50μm)的OMVs,持续8周。对照组采用生理盐水(0.9%)一次注射100ul,频率和周期与其他组相匹配。观察和比较注射前后小鼠体重的变化。
6 Morris水迷宫试验
MWM行为测试系统主要由平台、环形水槽、摄像机和数据采集装置组成。水槽直径为1.2m,高度为50cm。标志槽壁上的四个入口分别为东、西、南、北。两个对称点之间的直线平均可将水槽分成四个象限。在其中一个象限(一般为第三象限)内固定一个直径10cm高28cm的圆形平台,平台顶部在实验时被水淹没,距离水面1cm,水中加入二氧化钛使水变白,制成黑鼠白水背景以便观察,水温维持在24℃-26℃。采用定位导航实验和空间探索实验,测量对潜伏时间、游动距离及相关指标。
7在大脑中跟踪OMVs
纯化的OMVs分别用pkh26红色荧光连接试剂盒标记膜,孵育2h,离心,尾静脉注射到C57BL/6J小鼠体内。小鼠被禁止进食或饮水12小时后处死取材。
8 BBB渗透性
随机选择各组小鼠,分离脑前通过快速尾静脉注射2%埃文斯蓝(EB)(3mg/kg,sigma)。一小时后,用生理盐水进行心脏灌注,将半球组织粉碎,并加入两倍量体积的二甲基甲酰胺。将混合物在60℃水浴72h,1500g离心10分钟,除去上清液,在635nm处检测吸光度。通过标准曲线计算EB含量,进一步计算EB浓度(μg/g)=EB含量(μg/ml)×甲酰胺容量(ml)/脑重量(g)。
9蛋白质印迹分析
Western blot分析检测炎症通路的相关指标和GSK3β/tau通路。
使用以下主要抗体:兔抗肿瘤坏死因子-α抗体(1:1000;CST,美国);兔抗claudin5抗体(1:1000;abcam,英国);小鼠抗IL-1β(0.25μg/ml,Bio-Techne,美国);NF-κB(p65)(1:1000,CST,美国);兔抗GSK3β(1:1000,Santa Cruz Biotechnology,加拿大);兔抗GSK3β(1:5000;abcam,英国),兔抗tau(phospho S396)(1:50000,abcam,英国),小鼠抗tau46(1:1000,CST,美国),小鼠抗IBA1(1:1000;Abcam,英国)、小鼠抗GFAP(1:1000;Abcam,英国),小鼠抗GAPDH(1:1000,Santa Cruz Biotechnology,加拿大)。二级抗体为山羊抗兔IgG和山羊抗鼠IgG(1:1000;Jackson,美国)。
10免疫荧光
免疫荧光染色检测星形胶质细胞和小胶质细胞激情况。脑组织冷冻切片用100%冷丙酮固定5分钟。用5%驴血清和0.3%TrITO-X100PBS封闭。采用抗IBA1(1:1000;Abcam,英国)、GFAP(1:1000;Abcam,英国)的一抗孵育12h,然后用驴抗兔IgG H&L(1:400,BioWorldTechnology,美国)标记Alexa Fluor 488,室温孵育60分钟。随后与Hoechst 33342(1:100,中国北京索拉比科技有限公司)孵育10分钟,用激光扫描共聚焦显微镜(LSM 780)观察和拍摄(Carl Zeiss,Jena,德国)。
11统计分析
实验分别重复三次,用SPSS 17.0软件对实验数据进行分析。除特殊说明外,数据表示为:均值、标准差、均值±标准差。采用单因素方差分析(ANOVA)和t检验(Student's T-test)两种方法对测量数据的组间差异进行比较。绘图方法软件为graphpad prism 7.0。另外,*P<0.05为统计学意义。
12结果
(1)OMVs提供者的基线特征
为了研究OMVs在AD中的作用,我们收集了34份粪便样本,包括16名AD患者和18名对照者。在个体因素中,性别、年龄、婚姻状况、高血压、糖尿病、糖尿病、高脂血症、心脏病、吸烟史、文化程度等无显著性差异(P>0.05)。表1显示了OMVs提供者的基线特征。
表1 OMVs提供者的基线特征
备注
OMVs:外膜囊泡;AD:阿尔茨海默病。
(2)OMVs的特征
我们首先比较了AD患者和健康对照组的OMVs大小差异。我们使用透射电镜拍摄了纳米结构,结果表明,两种来源OMVs的平均直径分别为113.3±6.4和109.7±5.6nm(图2A和2B)。NTA测量时,两组的OMVs大小没有差异(图2C和图2D)。AD患者的OMVs模式直径为108.7±2.6nm,平均直径为107.5±2.6nm,而对照组的OMVs模式直径为107.2±3.9nm,平均直径为106.5±4.1nm。
(3)AD-OMVs对体重的影响
图3所示,36只9个月龄正常的C57小鼠随机分为三组:AD-OMVs、对照组和HL-OMVs。对尾静脉注射前后8周的数据进行称重,并对结果进行统计分析。三组的体重变化无差异,但AD-OMVs组体重有减轻倾向。
(4)OMVs穿过血脑屏障和损伤血脑屏障
为确定来自GM的OMVs是否能通过血脑屏障进入大脑。pkh26标记的OMVs尾静脉注射后,可在海马体中检测到(图4A)。采用EB渗透试验观察BBB的渗透性。结果表明,与对照组和HL-OMVs组相比,AD-OMVs组的EB浓度明显升高。而HL-OMVs组与对照组的差异无统计学意义(图4B)。我们还用蛋白质印迹法检测了海马中的claudin-5(图4C),结果表明,与对照组和HL-OMVs组相比,AD-OMVs组明显降低。这与大脑中的EB浓度一致。
(5)AD-OMVs损伤小鼠学习记忆能力
随着训练时间的延长,三组中寻找平台的时间逐渐缩短。在训练试验中,与野生型小鼠相比,AD-OMVs小鼠的逃逸潜伏期更长。而与对照组相比,HLOMVs小鼠没有表现出不同的逃逸潜伏期(图5A)。为了探索不同组的空间记忆能力,在第6天结束时移除平台。水迷宫中的游泳路径如图5B(AD-OMVs组)、C(Control组)、D(HL-OMVs组)所示。AD-OMVs组的跨平台时间、目的象限时间和平台象限时间百分比明显低于其他两组(P<0.05)。(图5E、F和G)。结果提示AD-OMVs小鼠学习记忆功能明显受损,出现痴呆行为改变。
(6)AD-OMVs增加小鼠GSK3β活性和tau高磷酸化
我们分析了不同组海马tau和gsk3β的水平。Western blot结果(图6A,B)显示,AD-OMVs小鼠的p-GSK3β/GSK3β和p-tau396/tau46明显增加。对照组与HL-OMVs组比较差异无统计学意义(P>0.05)。三组小鼠的非磷酸化GSK3β-和tau46无明显差异。
(7)AD-OMVs增加小鼠炎症
图6和图7所示,AD-OMVs小鼠海马内NF-κB、炎症因子IL-1β(白细胞介素-1β)和肿瘤坏死因子α(TNF-α)的表达显著增加。但HL-OMVs小鼠与对照组无明显差异。
(8)AD-OMVs激活小鼠小胶质细胞和星形胶质细胞
如图8A、8B、8C和8D所示,AD-OMVs小鼠海马中IBA1和GFAP阳性细胞和IBA1和GFAP蛋白表达增加,而HL-OMVs组和对照组之间没有显著差异。
以上内容不能认定本发明具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思前提下,还可以做出若干简单推演或替换,都应当视为属于本发明由所提交的权利要求书确定的专利保护范围。
Claims (8)
1.一种肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,将阿尔茨海默病患者的肠菌外膜囊泡用于构建痴呆动物模型,所述痴呆动物模型的动物的海马中IBA1和GFAP阳性细胞、IBA1和GFAP蛋白表达增加。
2.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述肠菌外膜囊泡来自于阿尔茨海默病患者的粪便。
3.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述肠菌外膜囊泡的模式直径为100-120nm。
4.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述肠菌外膜囊泡的提取和纯化方法为:将阿尔茨海默病患者的粪便充分搅拌成浆液,匀浆转移至玻璃坛中,滤网过滤2次,过滤后将滤液分装至50ml离心管中,1200g,4℃离心5min;离心后取上清液,3000g,4℃离心20min;离心后取上清液,6000g,4℃离心40min;离心后取上清液,10000g,4℃离心1h;离心后取上清液,15000g,4℃离心1h;离心后取上清液,用0.45µm膜过滤器过滤上清液,取滤液,用0.22µm膜过滤器过滤,取滤液,滤液集中装到耐超高速离心管中,170000g,4℃超高速离心2h,离心后去滤液,沉淀用0.5M PBS重悬。
5.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述痴呆动物模型的动物为小鼠。
6.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述痴呆动物模型的动物血脑屏障损伤,学习记忆功能明显受损,出现痴呆行为改变。
7.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述痴呆动物模型的动物的炎症因子的表达显著增加。
8.根据权利要求1所述的肠菌外膜囊泡在制备痴呆动物模型中的应用,其特征在于,所述肠菌外膜囊泡加重痴呆动物模型的动物的GSK-3β和tau高磷酸化、海马神经炎症。
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