CN111593071A - 脑组织特异性pltp过表达模型构建方法及测定方法 - Google Patents
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Abstract
本发明属于PLTP过表达模型构建技术领域,公开了一种脑组织特异性PLTP过表达模型构建方法及测定方法,10月龄3×Tg‑AD雄鼠,随机分为模型组,实验组,以同月龄具有相同遗传背景的雄性C57BL/6J作为对照组;AD模型注射携带PLTP基因增强型绿色荧光蛋白报告基因的腺相关病毒AAV‑PLTP‑EGFP,诱导PLTP的高表达;对照组与模型组均注射不携带PLTP基因只携带增强型绿色荧光蛋白报告基因的腺相关病毒AAV‑EGFP。本发明的PLTP过表达可改善3×Tg‑AD小鼠的学习记忆能力、对3×Tg‑AD小鼠学习记忆能力的改善;通过促进GSK‑3β失活进而减少Aβ产生和Tau蛋白磷酸化有关。
Description
技术领域
本发明属于PLTP过表达模型构建技术领域,尤其涉及一种脑组织特异性PLTP过表达模型构建方法及测定方法。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是中枢神经系统退行性疾病,是痴呆最常见的类型。目前对于AD的研究国内外学者广泛关注,提出了多个致病机制学说,并研制出了有一定治疗效果的针对性药物,但其发病机制仍未完全阐明,尚缺少有效的治疗药物。磷脂转运蛋白(phospholipid transfer protein,PLTP)是一种广泛表达于中枢神经系统的糖蛋白,近年,体内、外研究发现,PLTP与AD密切相关。PLTP缺乏可促进老年小鼠认知功能的下降,那么PLTP高表达是否对AD具有改善作用,国内外研究还未见报道。多种方式可用于研究PLTP过表达对AD的影响,如给与重组的PLTP蛋白,但重组的蛋白价格昂贵,而且动物给药还要考虑药物是否能被吸收进入机体,能否透过血脑屏障进入脑组织发挥作用等,因此利用病毒作为载体,脑定位注射后,诱导PLTP基因高表达,进而引起PLTP蛋白表达上调是较为理想的技术,利用这种技术,再结合APP/PS1/Tau三转基因的AD小鼠,可以整体水平明确PLTP高表达对AD的保护作用。AD虽然是危害人类的重要疾病,但目前还未有完全能模拟人类AD的动物模型,这给AD机制及药物开发的研究带来了困难。而APP/PS1/Tau三转基因的AD小鼠目前是众多模型中相对较好的一种AD的模型,通过此模型对PLTP高表达的AD保护作用研究,能更加确证PLTP的抗AD作用。另外,Aβ与Tau都是AD发病过程中的关键分子,而PLTP高表达对两种分子都具有调控作用,也说明其有望成为临床抗AD的成分或靶点之一。而通过病毒介导的基因治疗在肿瘤治疗中已有应用,也时通过病毒介导基因表达的AD治疗具有一定可能性。
通过上述分析,现有技术存在的问题及缺陷为:PLTP缺乏可促进老年小鼠认知功能的下降,PLTP高表达是否对AD具有改善作用,国内外研究还未见报道。
解决以上问题及缺陷的难度为:侧脑室定位注射,腺相关病毒PLTP高表达基因的构建。
解决以上问题及缺陷的意义为:为临床以PLTP为靶点的AD治疗提供依据。
发明内容
针对现有技术存在的问题,本发明提供了一种脑组织特异性PLTP过表达模型构建方法及测定方法。
本发明是这样实现的,一种脑组织特异性PLTP过表达模型构建方法,所述脑组织特异性PLTP过表达模型构建方法包括:
第一步,以3×Tg-AD小鼠作为AD模型,10月龄3×Tg-AD雄鼠,随机分为模型组,实验组,以同月龄具有相同遗传背景的雄性C57BL/6J作为对照组;
第二步,AD模型注射携带PLTP基因增强型绿色荧光蛋白报告基因的腺相关病毒AAV-PLTP-EGFP,诱导PLTP的高表达;
第三步,对照组与模型组均注射不携带PLTP基因只携带增强型绿色荧光蛋白报告基因的腺相关病毒AAV-EGFP。
进一步,所述脑组织特异性PLTP过表达模型构建方法10月龄3×Tg-AD雄鼠24只,随机分为模型组12只,实验组12只。
进一步,所述脑组织特异性PLTP过表达模型构建方法以同月龄具有相同遗传背景的雄性C57BL/6J作为对照组12只。
本发明的另一目的在于提供一种由所述脑组织特异性PLTP过表达模型构建方法构建的脑组织特异性PLTP过表达模型。
本发明的另一目的在于提供一种所述脑组织特异性PLTP过表达模型的测定方法,所述脑组织特异性PLTP过表达模型的测定方法包括:
第一步,以3×Tg-AD小鼠作为AD模型,构建脑组织特异性PLTP过表达模型;诱导PLTP基因的高表达。
第二步,观察PLTP过表达对3×Tg-AD小鼠学习记忆能力的影响;AD主要的临床表现就是认知功能下降,水迷宫等方法可检测动物的认知功能评价PLTP高表达的抗AD作用。
第三步,病理学观察PLTP过表达对3×Tg-AD小鼠的保护作用;老年斑和神经纤维缠结时AD特征性的病理学改变,因此病理学观察可以确证PLTP高表达的抗AD作用。
第四步,观察PLTP过表达对Aβ及其生成相关蛋白的影响;Aβ与Tau是AD发病的关键分子,通过两个通路蛋白的检测,明确PLTP改善AD的可能通路。
第五步,观察PLTP过表达对总Tau蛋白及其pTau蛋白的影响;Aβ与Tau是AD发病的关键分子,通过两个通路蛋白的检测,明确PLTP改善AD的可能通路。
第六步,观察PLTP过表达对GSK-3β及GSK-3β的影响;采用western blot检测GSK-3β及GSK-3β的蛋白表达水平。GSK3β既可以调控Aβ,也可以调控Tau,有文献报道PLTP可调控GSK3β的活性,通过对GSK3β的检测明确PLTP抗AD作用的机制。
进一步,所述第二步观察PLTP过表达对3×Tg-AD小鼠学习记忆能力的影响;小鼠侧脑室注射AAV-PLTP-EGFP病毒2周后,水迷宫实验与穿梭被动回避实验检测小鼠的学习记忆能力;水迷宫实验分为定位航行实验和空间探索实验两部分,以小鼠寻找平台的潜伏期、进入平台的次数为指标评价认知功能;穿梭被动回避实验在明暗穿梭箱中进行,以小鼠进入暗箱的潜伏期及在暗箱中的停留时间为指标,评价学习记忆能力。
进一步,所述第三步病理学观察PLTP过表达对3×Tg-AD小鼠的保护作用;通过HE染色与尼氏染色检测各组病理学损伤,通过Aβ1-42免疫组化染色观察各组老年斑SP的变化,通过Bielschowsky银染观察各组神经纤维缠结NFT的变化。
进一步,所述第四步观察PLTP过表达对Aβ及其生成相关蛋白的影响;行为学实验后,处死小鼠,分离小鼠大脑皮质及海马,制备其组织匀浆,采用蛋白免疫印迹技术检测与Aβ生成相关蛋白APP、PS1、BACE1的表达水平;ELISA法检测Aβ1-40及Aβ1-42水平。
进一步,所述第五步观察PLTP过表达对总Tau蛋白及其pTau蛋白的影响;采用western blot检测大脑皮质及海马总Tau蛋白及pTau蛋白在pSer199、pSer214、pThr231、pSer404四个磷酸化位点的蛋白的表达。
进一步,所述脑组织特异性PLTP过表达模型的测定方法的数据用SPSS16.0软件进行统计,多组样本间比较采用单因素方差分析ANOVO,P<0.05时具有统计学差异。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:本发明以3×Tg-AD(即APPSwe/PS1dE91/TauP301L三重转基因)小鼠作为AD模型,以腺病毒介导PLTP基因侧脑室内转染构建脑组织特异性PLTP过表达模型,采用水迷宫、穿梭被动回避实验、病理学染色、western blot等技术和方法,通过学习记忆能力、老年斑、Aβ相关蛋白、总Tau蛋白、pTau蛋白、GSK-3β通路相关蛋白的检测,探讨PLTP过表达对AD的防治作用及可能机制,以期为通过PLTP寻找AD治疗的新靶点、新思路提供重要依据。
本发明的PLTP过表达可改善3×Tg-AD小鼠的学习记忆能力;PLTP过表达对3×Tg-AD小鼠学习记忆能力的改善,可能通过促进GSK-3β失活进而减少Aβ产生和Tau蛋白磷酸化有关。本发明说明PLTP高表达能明显改善AD小鼠学习能力,其机制可能与调控GSK3B表达,进而影响Aβ和磷酸化tau的形成,进而产生抗AD作用,有望成为AD治疗的新靶点,而通过病毒介导PLTP基因高表达可能成为一种新的方法。
附图说明
图1是本发明实施例提供的脑组织特异性PLTP过表达模型构建方法流程图。
图2是本发明实施例提供的脑组织特异性PLTP过表达模型的测定方法流程图。
图3是本发明实施例提供的PLTP在各组小鼠大脑皮质及海马的表达(n=4,#P<0.05,##P<0.01vs.WT;*P<0.05,**P<0.01vs.3×Tg-AD)示意图;
图中:A:PLTP蛋白条带在模型和3×Tg-AD小鼠海马和皮质内的表达;,B:PLTP在模型和3×Tg-AD小鼠海马和皮质内的表达。
图4是本发明实施例提供的PLTP过表达能改善3×Tg-AD小鼠学习记忆能力(n=12,#P<0.05,##P<0.01vs.WT;*P<0.05,**P<0.01vs.3×Tg-AD)示意图;
图中:A:水迷宫定位航行实验小鼠逃避潜伏期;B:定位航行实验小鼠运行轨迹;C:水迷宫空间探索实验60秒穿越平台次数及小鼠进入平台所在区域的潜伏期;D:穿梭被动回避实验小鼠进入暗箱的潜伏期及暗箱停留时间。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种脑组织特异性PLTP过表达模型构建方法及测定方法,下面结合附图对本发明作详细的描述。
如图1所示,本发明实施例提供的脑组织特异性PLTP过表达模型构建方法包括以下步骤:
S101:以3×Tg-AD小鼠作为AD模型,10月龄3×Tg-AD雄鼠24只,随机分为模型组(12只),实验组(12只),以同月龄具有相同遗传背景的雄性C57BL/6J(Wild Type,WT)作为对照组(12只)。
S102:AD模型注射携带PLTP基因增强型绿色荧光蛋白报告基因的腺相关病毒(AAV-PLTP-EGFP),诱导PLTP的高表达。
S103:对照组与模型组均注射不携带PLTP基因只携带增强型绿色荧光蛋白报告基因的腺相关病毒(AAV-EGFP)。
如图2所示,本发明实施例提供的脑组织特异性PLTP过表达模型的测定方法包括以下步骤:
S201:以3×Tg-AD小鼠作为AD模型,构建脑组织特异性PLTP过表达模型。
S202:观察PLTP过表达对3×Tg-AD小鼠学习记忆能力的影响。
S203:病理学观察PLTP过表达对3×Tg-AD小鼠的保护作用。
S204:观察PLTP过表达对Aβ及其生成相关蛋白的影响。
S205:观察PLTP过表达对总Tau蛋白及其pTau蛋白的影响。
S206:观察PLTP过表达对GSK-3β及GSK-3β(pSer9)的影响;采用western blot检测GSK-3β及GSK-3β(pSer9)的蛋白表达水平。
下面结合具体实施例对本发明的技术方案作进一步的描述。
一、方法
1、以3×Tg-AD小鼠作为AD模型,构建脑组织特异性PLTP过表达模型;10月龄3×Tg-AD雄鼠24只,随机分为模型组(12只),实验组(12只),以同月龄具有相同遗传背景的雄性C57BL/6J(Wild Type,WT)作为对照组(12只)。侧脑室(自前囟向后0.6mm矢状缝旁开1.2mm处)注射携带PLTP基因增强型绿色荧光蛋白报告基因的腺相关病毒(AAV-PLTP-EGFP),诱导PLTP的高表达。对照组与模型组均注射不携带PLTP基因只携带增强型绿色荧光蛋白报告基因的腺相关病毒(AAV-EGFP)。
2、观察PLTP过表达对3×Tg-AD小鼠学习记忆能力的影响;小鼠侧脑室注射AAV-PLTP-EGFP病毒2周后,水迷宫实验与穿梭被动回避实验检测小鼠的学习记忆能力。水迷宫实验分为定位航行实验和空间探索实验两部分,以小鼠寻找平台的潜伏期、进入平台的次数为指标评价其认知功能。穿梭被动回避实验在明暗穿梭箱中进行,以小鼠进入暗箱的潜伏期及在暗箱中的停留时间为指标,评价其学习记忆能力。
3、病理学观察PLTP过表达对3×Tg-AD小鼠的保护作用;通过HE染色与尼氏染色检测各组病理学损伤,通过Aβ1-42免疫组化染色观察各组老年斑(senile plaques,SP)的变化,通过Bielschowsky银染观察各组神经纤维缠结(neurofibrillary tangles,NFT)的变化。
4、观察PLTP过表达对Aβ及其生成相关蛋白的影响;行为学实验后,处死小鼠,分离小鼠大脑皮质及海马,制备其组织匀浆,采用蛋白免疫印迹技术(western blot)检测与Aβ生成相关蛋白APP、PS1、BACE1的表达水平;ELISA法检测Aβ1-40及Aβ1-42水平。
5、观察PLTP过表达对总Tau蛋白及其pTau蛋白的影响;采用western blot检测大脑皮质及海马总Tau蛋白及pTau蛋白在pSer199、pSer214、pThr231、pSer404四个磷酸化位点的蛋白的表达。
6、观察PLTP过表达对GSK-3β及GSK-3β(pSer9)的影响;采用western blot检测GSK-3β及GSK-3β(pSer9)的蛋白表达水平。
7、实验数据用SPSS16.0软件进行统计,多组样本间比较采用单因素方差分析(ANOVO),P<0.05时具有统计学差异。
二、结果
1、PLTP过表达能改善3×Tg-AD小鼠的学习记忆能力;水迷宫结果显示:在定位航行实验、空间探索实验中,3×Tg-AD小鼠与WT小鼠相比,逃避潜伏期明显延长,探索实验中进入平台所在区域的次数明显减少,进入平台所在区域的潜伏期明显增长;而PLTP高表达的3×Tg-AD小鼠逃避潜伏期明显缩短,探索实验中进入平台所在区域的次数明显增多,进入平台所在区域的潜伏期明显缩短。差异具有统计学意义。穿梭被动回避实验结果显示:与WT小鼠相比,3×Tg-AD小鼠进入暗箱的潜伏期明显缩短,暗箱停留时间明显延长;而PLTP高表达的3×Tg-AD小鼠,进入暗箱的潜伏期明显延长,停留时间明显缩短,,差异具有统计学意义。说明PLTP过表达能明显改善3×Tg-AD小鼠的学习记忆能力。
2、PLTP过表达可改善3×Tg-AD小鼠病理学损伤、老年斑、神经纤维缠结的形成;通过HE染色和尼氏染色显示:3×Tg-AD小鼠皮层神经细胞减少,细胞核浓缩,细胞凋亡,Aβ1-42免疫组化染色可见明显的SP,而银染发现明显的NFT;PLTP过表达后,皮质神经元细胞增多,SP减少,NFT也明显减少。
3、PLTP过表达可减少Aβ形成;ELISA结果显示:3×Tg-AD小鼠海马及皮层区,Aβ1-40及Aβ1-42蛋白表达均升高;PLTP过表达后,Aβ1-40及Aβ1-42蛋白表达均降低。
4、PLTP过表达能减少与Aβ生成相关蛋白的表达;Western blot结果显示:3×Tg-AD小鼠海马与皮层区,Aβ生成相关标记物APP、PS1、BACE1表达均增高;PLTP过表达后,APP、PS1、BACE1表达均降低。
5、PLTP过表达能抑制Tau蛋白及其pTau蛋白的表达;Western blot结果显示:3×Tg-AD小鼠海马与皮层区,总Tau及其pTau蛋白在pSer199、pSer214、pThr231、pSer404四个位点的表达均升高;PLTP过表达后总Tau及其pTau蛋白在pSer199、pSer214、pThr231、pSer404四个位点的表达均降低。
6、PLTP过表达能促进GSK-3β9位丝氨酸磷酸化使GSK-3β失活;Western blot结果显示:3×Tg-AD小鼠的海马及皮层区,GSK-3β表达增高,其在9位苏氨酸残基磷酸化生成的GSK3β(pSer9)表达降低;PLTP过表达后,GSK-3β表达降低,其在9位苏氨酸残基磷酸化生成的GSK3β(pSer9)表达增高。
本发明的PLTP过表达可改善3×Tg-AD小鼠的学习记忆能力;PLTP过表达对3×Tg-AD小鼠学习记忆能力的改善,可能通过促进GSK-3β失活进而减少Aβ产生和Tau蛋白磷酸化有关。
下面结合附图对本发明的技术效果作详细的描述。
图3是本发明实施例提供的PLTP在各组小鼠大脑皮质及海马的表达(n=4,#P<0.05,##P<0.01vs.WT;*P<0.05,**P<0.01vs.3×Tg-AD);图中:A:PLTP蛋白条带在模型和3×Tg-AD小鼠海马和皮质内的表达;,B:PLTP在模型和3×Tg-AD小鼠海马和皮质内的表达。
图4是本发明实施例提供的PLTP过表达能改善3×Tg-AD小鼠学习记忆能力(n=12,#P<0.05,##P<0.01vs.WT;*P<0.05,**P<0.01vs.3×Tg-AD);图中:A:水迷宫定位航行实验小鼠逃避潜伏期;B:定位航行实验小鼠运行轨迹;C:水迷宫空间探索实验60秒穿越平台次数及小鼠进入平台所在区域的潜伏期;D:穿梭被动回避实验小鼠进入暗箱的潜伏期及暗箱停留时间。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种脑组织特异性PLTP过表达模型构建方法,其特征在于,所述脑组织特异性PLTP过表达模型构建方法包括:
第一步,以3×Tg-AD小鼠作为AD模型,10月龄3×Tg-AD雄鼠,随机分为模型组,实验组,以同月龄具有相同遗传背景的雄性C57BL/6J作为对照组;
第二步,AD模型注射携带PLTP基因增强型绿色荧光蛋白报告基因的腺相关病毒AAV-PLTP-EGFP,诱导PLTP的高表达;
第三步,对照组与模型组均注射不携带PLTP基因只携带增强型绿色荧光蛋白报告基因的腺相关病毒AAV-EGFP。
2.如权利要求1所述的脑组织特异性PLTP过表达模型构建方法,其特征在于,所述脑组织特异性PLTP过表达模型构建方法10月龄3×Tg-AD雄鼠24只,随机分为模型组12只,实验组12只。
3.如权利要求1所述的脑组织特异性PLTP过表达模型构建方法,其特征在于,所述脑组织特异性PLTP过表达模型构建方法以同月龄具有相同遗传背景的雄性C57BL/6J作为对照组12只。
4.一种由权利要求1~3任意一项所述脑组织特异性PLTP过表达模型构建方法构建的脑组织特异性PLTP过表达模型。
5.一种如权利要求4所述脑组织特异性PLTP过表达模型在治疗阿尔茨海默病靶点中的用途。
6.一种如权利要求4所述脑组织特异性PLTP过表达模型的测定方法,其特征在于,所述脑组织特异性PLTP过表达模型的测定方法包括:
第一步,以3×Tg-AD小鼠作为AD模型,构建脑组织特异性PLTP过表达模型;
第二步,观察PLTP过表达对3×Tg-AD小鼠学习记忆能力的影响;
第三步,病理学观察PLTP过表达对3×Tg-AD小鼠的保护作用;
第四步,观察PLTP过表达对Aβ及其生成相关蛋白的影响;
第五步,观察PLTP过表达对总Tau蛋白及其pTau蛋白的影响;
第六步,观察PLTP过表达对GSK-3β及GSK-3β的影响;采用western blot检测GSK-3β及GSK-3β的蛋白表达水平。
7.如权利要求6所述的脑组织特异性PLTP过表达模型的测定方法,其特征在于,所述第二步观察PLTP过表达对3×Tg-AD小鼠学习记忆能力的影响;小鼠侧脑室注射AAV-PLTP-EGFP病毒2周后,水迷宫实验与穿梭被动回避实验检测小鼠的学习记忆能力;水迷宫实验分为定位航行实验和空间探索实验两部分,以小鼠寻找平台的潜伏期、进入平台的次数为指标评价认知功能;穿梭被动回避实验在明暗穿梭箱中进行,以小鼠进入暗箱的潜伏期及在暗箱中的停留时间为指标,评价学习记忆能力。
8.如权利要求6所述的脑组织特异性PLTP过表达模型的测定方法,其特征在于,所述第三步病理学观察PLTP过表达对3×Tg-AD小鼠的保护作用;通过HE染色与尼氏染色检测各组病理学损伤,通过Aβ1-42免疫组化染色观察各组老年斑SP的变化,通过Bielschowsky银染观察各组神经纤维缠结NFT的变化。
9.如权利要求6所述的脑组织特异性PLTP过表达模型的测定方法,其特征在于,所述第四步观察PLTP过表达对Aβ及其生成相关蛋白的影响;行为学实验后,处死小鼠,分离小鼠大脑皮质及海马,制备其组织匀浆,采用蛋白免疫印迹技术检测与Aβ生成相关蛋白APP、PS1、BACE1的表达水平;ELISA法检测Aβ1-40及Aβ1-42水平。
10.如权利要求6所述的脑组织特异性PLTP过表达模型的测定方法,其特征在于,所述第五步观察PLTP过表达对总Tau蛋白及其pTau蛋白的影响;采用western blot检测大脑皮质及海马总Tau蛋白及pTau蛋白在pSer199、pSer214、pThr231、pSer404四个磷酸化位点的蛋白的表达;
所述脑组织特异性PLTP过表达模型的测定方法的数据用SPSS16.0软件进行统计,多组样本间比较采用单因素方差分析ANOVO,P<0.05时具有统计学差异。
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