CN109942714B - 一种功能多肽及应用 - Google Patents
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Abstract
本发明提供一种功能多肽,包括多肽片段Ⅰ:识别及阻断淀粉样蛋白Aβ聚集的多肽片段和多肽片段Ⅱ:apoE受体结合区域的模拟肽,所述多肽片段Ⅰ与多肽片段Ⅱ通过连接肽或直接连接。本发明所述多肽可以抑制淀粉样蛋白Aβ聚集、抑制Aβ的细胞毒性、加速小胶质细胞对Aβ的清除,可用于制备治疗阿尔茨海默病的药物。
Description
技术领域
本发明涉及医药领域,尤其涉及一种功能多肽,可用于制备治疗阿尔茨海默病的药物。
背景技术
阿尔茨海默病(AD)是最常见的神经退行性疾病,是老年人群中痴呆的主要原因。根据2018年世界老年痴呆症报告,到2018年全世界有5000万人患有痴呆症,到2050年,这一数字将增加三倍,达到1.52亿。估计2018年全球痴呆症总费用为1万亿美元,到 2030年这个数字将增加到2万亿美元。AD发病机制复杂,涉及淀粉样蛋白-β(Aβ)代谢异常,tau蛋白过度磷酸化,氧化应激,反应性胶质细胞和小胶质细胞改变等病理事件。其中,脑内Aβ的异常积聚和聚集,称为“淀粉样蛋白假说”,被认为是发病机制中的一个重要事件。并且Aβ斑块的沉积被用作AD的病理标志之一。Aβ衍生自淀粉样蛋白前体蛋白(APP)通过两种酶活性的蛋白水解加工:β-和γ-分泌酶。APP在脑细胞和外周器官和组织中广泛表达,并且细胞外支配的APP由BACE1基因编码的β-分泌酶切割。然后,膜内的剩余部分APP被γ-分泌酶切割,形成Aβ。众所周知,可溶性Aβ在有毒低聚物,纤维和淀粉样蛋白斑中的积累引发了该疾病的所有其他标志,包括tau病理学,炎症,突触功能障碍,神经元丢失和痴呆。它暗示了降低Aβ产生,抑制Aβ积聚或增加其清除率足以治愈该疾病。基于此,在过去的十年中,通过抑制γ-分泌酶和β-分泌酶,加速Aβ的去除或抑制Aβ的聚集和沉积,已经进行了许多研究来减少脑内Aβ的产生。100多项临床试验在1998年至2017年宣布失败,导致研发失败的原因很多,著名神经学医生Yan-Jiang Wang也在其发表在Nature Reviews Neurology上的文章中提出AD病理发生涉及多种机制,对AD发病机制复杂性的忽视,采用单分子或单通路的靶向治疗是大多数靶向Aβ临床试验失败的原因。多策略、多效应联用,从而减少Aβ低聚物和纤维的形成以及清除可能是一种新的治疗AD的策略。
研究表明与Aβ的过度产生相比,Aβ的清除受损是迟发性AD的标志。通过蛋白酶水解降解,转运至外周,通过小胶质细胞,星形胶质细胞和神经元通过受体介导的转运或受体介导的细胞清除是Aβ的清除的主要途径。载脂蛋白E(apoE)是胆固醇的主要转运蛋白,在中枢神经系统中起着多种作用。它与Aβ形成的复合物通过与外周细胞以及小胶质细胞表面LDL家族受体(包括LDL受体相关蛋白1(LPR1),LDL受体(LDLR)和VLDL 受体(VLDLR))来影响Aβ的清除。受体与单独的Aβ或与apoE复合的Aβ结合,递送至溶酶体或通过转胞吞作用透BBB进入血浆。然而LOAD中的神经病理学检查表明, APOEε4等位基因剂量与脑内Aβ,Aβ寡聚体和斑块积聚增加有关。实验表明,游离的ApoE 能够促进Aβ寡聚体的形成以及斑块的沉积。由此可见,ApoE与生物体中的Aβ息息相关。
发明内容
本发明模拟ApoE设计了一种多肽,通过将Aβ同源肽与ApoE同源模拟肽连接,使其具备ApoE促进Aβ被小胶质细胞清除的优点同时又避免其促进Aβ寡聚体的形成并抑制Aβ聚集的特点,可有效降低Aβ寡聚体的含量。
本发明具体技术方案如下:
本发明提供一种功能多肽,包括多肽片段Ⅰ:识别及阻断淀粉样蛋白Aβ聚集的多肽片段和多肽片段Ⅱ:apoE受体结合区域的模拟肽,所述多肽Ⅰ与多肽Ⅱ通过连接肽或直接连接。
优选地,所述多肽片段Ⅱ为apoE的氨基酸序列的第130位到150位的受体结合区域的模拟肽。
优选地,所述多肽片段Ⅰ和/或多肽片段Ⅱ中的一个或几个氨基酸残基的可以被取代和 /或缺失和/或添加,具有相同或相似的生物学功能。
优选地,多肽片段Ⅰ的氨基酸序列如SEQ ID NO:1~5所示,多肽片段Ⅱ的氨基酸序列如SEQ ID NO:6-10所示。具体信息如下:
SEQ ID NO:1:Lys-Leu-Val-Phe-Phe(KLVFF)(Lowe,T.L.;Strzelec,A.;Kiessling,L.L.; Murphy,R.M.Structure-Function Relationships for Inhibitors ofβ-Amyloid Toxicity Containing the Recognition Sequence KLVFF.Biochemistry2001,40,7882-7889.)。
SEQ ID NO:2:Lys-Leu-Val-Phe-Phe-Ala(KLVFFA)(Chalifour,R.J.;McLaughlin,R.W.; Lavoie,L.;Morissette,C.;Tremblay,N.;Boulé,M.;Sarazin,P.;Stéa,D.;Lacombe,D.; Tremblay,P.;Gervais,F.Stereoselective Interactions ofPeptide Inhibitors with theβ-Amyloid Peptide. J.Biol.Chem.2003,278,34874-34881.)
SEQ ID NO:3:Lys-Leu-Val-Phe-Phe-Lys(KLVFFK) (Pallitto,M.M.;Ghanta,J.;Heinzelman,P.;Kiessling,L.L.;Murphy,R.M.Recognition Sequence Design forPeptidyl Modulators ofβ-Amyloid Aggregation and Toxicity.Biochemistry 1999,38, 3570-3578.)。
SEQ ID NO:4:Arg-Gly-Lys-Leu-Val-Phe-Phe-Gly-Arg(RGKLVFFGR)(Austen,B.M.; Paleologou,K.E.;Ali,S.A.E.;Qureshi,M.M.;Allsop,D.;El-Agnaf,O.M.A.Designing Peptide Inhibitors for Oligomerization and Toxicity ofAlzheimer’sβ-Amyloid Peptide. Biochemistry 2008,47,1984-1992)
SEQ ID NO:5:Lys-Leu-Val-Phe-Ala(KLVFA)(Tjernberg LO,Naslund J,Lindqvist F,et al.Arrest of-Amyloid Fibril Formation by a Pentapeptide Ligand[J].Journal of Biological Chemistry,1996,271(15):8545-8548.)
SEQ ID NO:6:Ala-Ser-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu(ASHLRKLRKRLL) (Laskowitz D T,Fillit H,Yeung N,et al.Apolipoprotein E-derivedpeptides reduce CNS inflammation:implications for therapy of neurologicaldisease[J].Acta Neurologica Scandinavica,2010,114(s185):15-20.)
SEQ ID NO:7: Leu-Arg-Val-Arg--Leu-Ala-Ser-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu (LRVRLASHLRKLRKRLL)(Laskowitz D T,Fillit H,Yeung N,etal.Apolipoprotein E-derived peptides reduce CNS inflammation:implications fortherapy of neurological disease[J]. Acta Neurologica Scandinavica,2010,114(s185):15-20.)
SEQ ID NO:8:Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys-Arg-Leu-Leu (AS-Aib-LRKL-Aib-KRLL)Aib具体为氨基异丁酸。(Laskowitz D T,Fillit H,Yeung N,etal.Apolipoprotein E-derived peptides reduce CNS inflammation:implications fortherapy of neurological disease[J].Acta Neurologica Scandinavica,2010,114(s185):15-20.)
SEQ ID NO:9:Leu-Arg-Val-Arg-Ala-Ala-Ser-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu (LRVRAASHLRKLRKRLL)(Laskowitz D T,Fillit H,Yeung N,etal.Apolipoprotein E-derived peptides reduce CNS inflammation:implications fortherapy of neurological disease[J]. Acta Neurologica Scandinavica,2010,114(s185):15-20.)
SEQ ID NO:10: Leu-Arg-Val-Arg-Leu-Ala-Ala-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu (LRVRLAAHLRKLRKRLL)(Laskowitz D T,Fillit H,Yeung N,etal.Apolipoprotein E-derived peptides reduce CNS inflammation:implications fortherapy of neurological disease[J]. Acta Neurologica Scandinavica,2010,114(s185):15-20.)
本发明一个优选的方案为多肽片段Ⅰ的氨基酸序列如SEQ ID NO:2所示;多肽片段Ⅱ的氨基酸序列选自如SEQ ID NO:8。
优选地,本发明所述连接肽可以为本领域常规使用的连接肽,如一个或多个甘氨酸或赖氨酸,优选SEQ ID NO:11-15所示的连接肽。具体为
SEQ ID NO:11:Gly。
SEQ ID NO:12:Gly-Gly-Gly。
SEQ ID NO:13:Lys。
SEQ ID NO:14:Lys-Lys。
SEQ ID NO:15:Lys-Lys-Lys。
本发明一个优选的方案,所述多肽的多肽片段的C端和多肽片段Ⅱ的N端直接偶联。
更进优选的,所述多肽优选如下结构:C端-KLVFFAAS-Aib-LRKL-Aib-KRLL-N端(赖氨酸-亮氨酸-颉氨酸-苯丙氨酸-苯丙氨酸-丙氨酸-丙氨酸-丝氨酸-氨基异丁酸-亮氨酸-精氨酸-赖氨酸-亮氨酸-氨基异丁酸-赖氨酸-精氨酸-亮氨酸-亮氨酸)(MOP),作为以上所述的多功能多肽。氨基酸序列如SEQ ID NO:16所示。
本发明另一目的在于提供本发明所述多肽在制备治疗阿尔茨海默病的药物中的应用。
本发明所述抑制淀粉样蛋白Aβ聚集、抑制Aβ的细胞毒性、加速小胶质细胞对Aβ的清除。
更优选地,Aβ具体为Aβ40的单体及寡聚体;小胶质细胞具体为BV-2细胞;
本发明另一目的在于提供一种药物组合物,含有所述本发明所述多肽或其盐作为活性成分。
进一步的,还可以含有药学上可接受的载体。
本发明所述药物组合物为抑制Aβ细胞毒性的制剂,所述细胞具体为SH-SY5Y细胞。
本发明的实验证明,本发明的多肽可与Aβ40结合,从而抑制Aβ40的聚集,进一步抑制Aβ40的细胞毒性,保护SH-SY5Y神经母细胞瘤细胞免受Aβ40毒性的影响。本发明的多肽与Aβ40可形成复合物,并通过LDL家族受体介导的胞吞促进BV-2小胶质细胞对Aβ40 的清除。
本发明优点:本发明所述多功能多肽可以具有同时抑制淀粉样蛋白Aβ聚集并且加速小胶质细胞对Aβ的清除的功能,从而达到多通道减少脑区Aβ寡聚体的含量的目的,为阿尔茨海默病的临床治疗提供了新的思路。
附图说明
图1为实施例1制备的MOP的结构式。
图2为实施例1制备的MOP的高效液相色谱图。
图3为实施例1制备的MOP的质谱图。
图4为实施例2中不同浓度MOP抑制Aβ40单体聚集的ThT荧光图。
图5a为实施例2中Aβ40单体聚集的透射电镜图。图5b为MOP抑制Aβ40单体聚集的透射电镜图。
图6为实施例2中MOP抑制Aβ40寡聚体聚集的ThT荧光图。
图7为实施例2中MOP抑制Aβ40寡聚体聚集的斑点印记图。
图8为实施例3中MOP抑制Aβ40的细胞毒性。
图9为实施例4中MOP与Aβ40结合的荧光能量共振转移图。
图10为实施例4中MOP与Aβ40结合的荧光滴定图。
图11为实施例5中浓度与MOP促进BV-2细胞对Aβ40单体清除能力关系图。
图12为实施例5中时间与MOP促进BV-2细胞对Aβ40单体清除能力关系图。
图13为实施例5中MOP促进BV-2细胞对Aβ40寡聚体清除图。
具体实施方式
为了获得具备同时抑制Aβ聚集和加速Aβ清除的多肽分子,本发明设计合成了多种不同类型和结构的融合多肽,包括,不使用连接子,通过16Aβ20的衍生肽序列SEQ ID NO:1-5与130Apo E150的模拟肽序列SEQ ID NO:6-10的直接连接的融合多肽SEQ ID NO:16-19;使用SEQ ID NO:11-15作为连接子构建的融合多肽SEQ ID NO:20-22。进一步,对上述多肽进行结构表征及功能性评价,结果发现,通过KLVFFA的C端与AS-Aib-LRKL-Aib-KRLL 的N端直接连接的融合多肽SEQ ID NO:16(MOP)具有极好的抑制Aβ聚集、抑制Aβ的细胞毒性以及加速Aβ清除的能力。
为了更好的理解本发明的技术方案,下面结合实施例对本发明作进一步的说明。
实施例1:本发明所述功能多肽的制备及纯化
本实施例采用固相合成方法进行多功能多肽MOP的合成。具体合成步骤如下:
用9-芴甲氧羰基固相肽来合成所述的SEQ ID NO:16:Lys-Leu-Val-Phe-Phe-Ala-Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys-Arg-Leu-Leu
(KLVFFA-AS-Aib-LRKL-Aib-KRLL)。这些程序使用FocusXC自动合成仪(AAPPTEC)完成的。从KLVFFAAS-Aib-LRKL-Aib-KRLL序列基 (Rink-KLVFFA-AS-Aib-LRKL-Aib-KRLL-Fmoc)除去MTT保护基团,脱保护用3:5:92 的TFA/TIS/DCM的混合液振摇5分钟脱去MTT,并且过程重复两次。将亮氨酸的一端氨基转化成羧酸形式,在DMF中加入琥珀酸干和DIEA与多肽进行反应,室温下振摇过夜。反应均通过游离胺的茚三酮试验监测。用比例95:2.5:2.5的TFA/TIS/H20的混合液振摇3 小时将KLVFFA-AS-Aib-LRKL-Aib-KRLL-Fmoc-COOH肽从树枝上切断。通过旋转蒸发以及加冷乙醚沉淀得到粗肽出去过量的TFA。将沉淀的粗肽和乙醚用离心法(6000rpm, 1min),将乙醚溶液弃去得到分离的多肽。过程重复3次。所有离心管用封口膜密封,以尽量减少乙醚在高速离心期间的蒸发。
分析提纯和质谱检测:将上述得到的多功能多肽用制备液相纯化得到产物,其结构式如图1所示。并利用ESI-MS和HPLC鉴别分析。
将实施例1制备的KLVFFA-AS-Aib-LRKL-Aib-KRLL利用HPLC检测,参见图2,由图2中的谱峰面积可计算其纯度已达到90%以上。将实施例1制备的 KLVFFA-AS-Aib-LRKL-Aib-KRLL采用ESI-MS检测,参见图3,说明其分子量为2073是正确的。
参照上述方案,分别制备得到SEQ ID NO:17-22所示的融合蛋白:
SEQ ID NO:17: Lys-Leu-Val-Phe-Phe-Ala-Ser-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu, (KLVFF-ASHLRKLRKRLL)。
SEQ ID NO:18: Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys-Arg-Leu-Leu-Lys-Leu-Val-Phe-Phe-Ala, (AS-Aib-LRKL-Aib-KRLL-KLVFFA)。
SEQ ID NO:19:Lys-Leu-Val-Phe-Ala-Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys-Arg-Leu-Leu,(KLVFA-AS-Aib-LRKL-Aib-KRLL)。
SEQ ID NO:20:Lys-Leu-Val-Phe-Phe-Ala-Gly-Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys- Arg-Leu-Leu,(KLVFFA-G-AS-Aib-LRKL-Aib-KRLL)。
SEQ ID NO:21: Lys-Leu-Val-Phe-Phe-Ala-Gly-Gly-Gly-Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys-Arg-Leu-Leu, (KLVFFA-GGG-AS-Aib-LRKL-Aib-KRLL)。
SEQ ID NO:22: Lys-Leu-Val-Phe-Phe-Ala-Lys-Lys-Lys-Ala-Ser-Aib-Leu-Arg-Lys-Leu-Aib-Lys-Arg-Leu-Leu, (KLVFFA-KKK-AS-Aib-LRKL-Aib-KRLL)。
实施例2:本发明所述功能多肽抑制Aβ的聚集的活性
1.抑制Aβ单体聚集的ThT实验
用磷酸盐缓冲盐水(PBS;50mM,pH7.4,含有150mM KCl)稀释1mM储备浓度的Aβ40和本发明所述功能多肽(SEQ ID NO:16-22所示的融合蛋白,分别配制),分别得到100μM 单体Aβ40(mAβ40)和本发明所述功能多肽溶液。将Aβ40(100μM,10μL)的新鲜单体化溶液与本发明所述功能多肽溶液(100μM,0,1,5,10μL)混合,并将混合物用PBS 稀释至Aβ40的最终浓度为20μM。在黑色96孔平底板中测定本发明所述功能多肽对mAβ聚集的抑制活性。随后,将板在37℃下恒温摇动(750rpm)孵育48小时。通过ThT染料 (100μM,150μL/孔)(λex=445nm和λem=485nm)监测聚集过程。空白组包含除Aβ40 和M本发明所述功能多肽之外的所有物质。
结果表明,SEQ ID NO:16-22所示的功能多肽在以与Aβ40(20μM)等摩尔比存在情况下,与Aβ40组相比,均具有抑制Aβ40的聚集的活性,其中SEQ ID NO:17-22所示的功能多肽ThT荧光水平虽有所降低,但仍呈现一定的荧光值,表明Aβ40的聚集并不能被完全抑制。详细地,融合蛋白SEQ ID NO:16-22抑制Aβ单体聚集的能力分别为SEQ ID NO: 16(ThT相对荧光强度:0)>SEQ ID NO:18~SEQ ID NO:21(ThT相对荧光强度:0.2)>SEQ ID NO:17~SEQID NO:20(ThT相对荧光强度:0.4)>SEQ ID NO:22(ThT相对荧光强度: 0.5)>SEQ ID NO:19(ThT相对荧光强度:0.7)。SEQ ID NO:16所示的MOP的ThT结果如图4所示,其中,20μMAβ40的动力学曲线显示出特征性的S形响应,其中滞后期约4小时之后是4至10小时的延伸期和10小时后的稳态。其中,20μMAβ40的聚集产生t50约为6.5h。在MOP存在的情况下,聚集动力学导致较长的滞后期并且在稳态下其ThT荧光水平降低。随着MOP比例的增加,20μMAβ40聚集的t50延长。稳态中的ThT强度以MOP 剂量依赖性方式降低,其中亚化学计量比在1:0.1至1:1之间(Aβ40:MOP)。特别是,在MOP以等摩尔比存在的情况下,Aβ40的聚集被完全抑制,在稳定状态下t50被延长 (>30h)和ThT荧光水平几乎为0。
2.本发明所述功能多肽抑制Aβ单体聚集的透射电镜显微镜检查
(1)用磷酸盐缓冲盐水(PBS;50mM,pH7.4,含有150mM KCl)稀释1mM储备浓度的 Aβ40和MOP,分别得到100μM单体Aβ40(mAβ40)和MOP溶液。将Aβ40(100μM, 10μL)的新鲜单体化溶液与MOP溶液(100μM,10μL)混合,并将混合物用PBS稀释至 Aβ40的最终浓度为20μM。在37℃下恒温摇动(750rpm)孵育48小时。
(2)将10μL样品加载到碳涂覆的铜网格上3分钟。然后除去过量的溶液并用水洗涤网格。最后用5%乙酸铀酰染色网格2分钟。除去过量的乙酸双氧铀,并在室温下干燥网格。过夜。通过透射电子显微镜(HITACHIH-7000FA)在100kV的加速电压下获得图像。
实验结果如图5所示,左图表明20μMAβ40孵育48小时后,形成长且分枝的原纤维。而右边的TEM图像显示在MOP存在下以等摩尔比率在Aβ40的聚集过程中未检测到纤维。
3.MOP抑制Aβ寡聚体聚集的ThT实验
用磷酸盐缓冲盐水(PBS;50mM,pH7.4,含有150mMKCl)稀释1mM Aβ40储备溶液 (20μM)来触发Aβ寡聚化过程,然后将板在37℃下恒温摇动(750rpm)孵育一段时间后以等摩尔比将MOP加入到该溶液中。通过ThT染料(100μM,150μL/孔)(λex=445nm和λem=485nm)监测聚集过程。空白组包含除Aβ40和MOP之外的所有物质。对照组为仅包含Aβ40的溶液。
结果如图6所示,当在4小时和6小时加入时,MOP不能有效的消除预先形成的oAβ,但可有效抑制oAβ进一步纤维化。
参照上述方案,分别进行融合蛋白SEQ ID NO:18和21抑制Aβ寡聚体聚集的ThT实验。结果显示,融合蛋白SEQ ID NO:18和21并不能有效的消除预先形成的oAβ,也不能有效抑制oAβ进一步纤维化。
4.MOP抑制Aβ40寡聚体聚集的斑点印记实验
通过在不存在和存在MOP的等摩尔比下以不同的时间间隔孵育Aβ40来制备样品。然后将样品施加到硝酸纤维素膜上并在室温下干燥。在4℃下1小时或过夜。然后将膜用5%脱脂奶在缓冲溶液(20mM Tris,pH7.4)中在室温下封闭1小时。然后用补充有 0.01%Tween-20(TBST)的20mM Tris,pH7.4洗涤(×3)硝酸纤维素膜,并与多克隆A11 抗体(TBST中5%脱脂牛奶中1/1000稀释)在4℃过夜。然后用TBST缓冲液洗涤样品(×3),并与辣根过氧化物酶(HRP)偶联的抗兔IgG(在TBST中的5%脱脂牛奶中1/500稀释) 在室温下孵育。1小时然后用TBST缓冲液(×3)洗涤斑点印迹,使用ECL试剂盒(Amersham, Piscataway,NJ,USA)显影,并使用Typhoon FLA 9000仪器(GE Healthcare Life Sciences, Pittsburgh,PA,USA)成像。
图像如图7所示,在没有MOP的情况下,Aβ40样品的信号强度从0到24小时逐渐增加。在整个时间内逐渐增加的点强度表明了oAβ的形成。然而,在MOP以等摩尔比存在下,相同条件,在淀粉样蛋白反应的整个时间过程中仅观察到弱强度点。
实施例3:MOP抑制Aβ聚集体的诱导的细胞毒性:
将生长良好的SH-SY5Y的单细胞悬液以5×103细胞/孔的密度接种在96孔板中,每孔体积200μL。细胞于37℃,CO2浓度为5%的条件下培养24小时后,弃去原培养基,每孔加入如下样品:
共孵育2小时的Aβ40(200μM)与MOP的混合物(Aβ:MOP=1:1,1:0.5;1:0.25),Aβ40的终止浓度是2μM;孵育2小时的的Aβ40(200μM),Aβ40的终止浓度是2μM;Aβ40阴性对照组包含除Aβ和MOP(100%细胞活力)之外的所有物质。阳性对照组组接受2%SDS 溶液(0%细胞活力)。
将以上6组细胞继续培养48小时后,每孔加MTT溶液(5mg/mL)10μL,37℃孵育3 小时,终止培养,每孔加入100μLDMSO溶解结晶物,在多功能酶标仪上以570nm波长检测吸光度值。
实验重复三次,结果如图8所示,Aβ诱导的毒性降低了对60%对照和MOP的活力,剂量依赖性地降低了对SH-SY5Y诱导细胞活力的毒性从60%增加到90%。在等摩尔比例的Aβ和MOP下,观察到细胞活力的最大增加。
实施例4:MOP与Aβ40的结合能力
1.MOP与Aβ40的荧光能量共振转移实验
精密配制200nM的FAM-Aβ40,200nM的TAMRA-MOP以及FAM-Aβ40与 TAMRA-MOP的混合溶液(FAM-Aβ40:TAMRA-MOP=1:0.1,1:0.2,1:0.5,1:1,1:2;FAM-Aβ40的浓度为200nM),用配备有1.0-cm石英细胞并具有1.5-nm狭缝宽度的分光荧光光度计(Thermo ScientificLumina,USA),在494nm处激发,记录样品溶液在505nm 至610nm的荧光光谱。
荧光光谱图如图9所示,结果显示,FRET的大小与MOP的剂量相对应。随着MOP 浓度从Aβ的0倍增加到2倍,平衡向复合物方向移动。
2.MOP与Aβ40的荧光滴定实验
在96孔板中用增量的MOP溶液(2mM-200nM)滴定新鲜的FAM-Aβ40(20nM,100μL) 的单体化溶液。然后用稀释液(20mM NaPi,1%TFE,pH7.4)稀释混合物至FAM-Aβ40 的终浓度10nM,用酶标仪(Thermo Electron Corporation,USA),记录522nm的荧光强度。将荧光强度(λmax=522nm)的变化作为MOP浓度的函数作图。该图使用S形结构来提取明显的结合效率。
图像如图10所示,在达到平台之前,随着MOP的滴定,FAM-Aβ的荧光强度降低。基于作为log[MOP]的函数的荧光强度的变化形成S形曲线。通过该图计算出的表观结合亲和力为0.378±0.023μM(app.Kd),其反映了MOP和Aβ之间的强结合相互作用。
实施例5:MOP促进BV-2细胞对Aβ40的清除
1.MOP促进BV-2细胞对Aβ40单体的清除
浓度的影响:BV-2细胞在含10%FBS(Gibco Company,USA)的高糖DMEM(KeyGENBioTECH,中国)中,在5%CO2中在37℃恒温培养箱中培养,将生长良好的细胞以5×104个细胞/孔的密度接种在24孔板中,每孔400μL,培养24小时以允许细胞附着。除去培养基,细胞在不存在或存在MOP(5,2,1,0.5,0.2,0.1μM)的情况下与FAM-Aβ(1μM) 在37℃培养3小时。之后,用PBS洗涤细胞三次,并用流式细胞术(Becton Dickinson and Company,USA)对FAM-Aβ的细胞摄取进行定量分析。
实验图像如图11所示,在MOP存在下,相对荧光强度随着MOP浓度的增加而持续增加。特别是,当MOP以1:0.5的Aβ:MOP浓度比存在时,Aβ的细胞摄取达到对照组的 1.5倍,这表明MOP对小胶质细胞摄取Aβ具有稍强的促进作用。而随着MOP浓度的进一步增加,荧光强度降低,表明高浓度的MOP可抑制Aβ的细胞摄取。我们注意到在存在 MOP(0.5eq.)下Aβ水平在相对于不存在MOP时强烈增加。同时,Aβ图像中在高浓度 MOP(5eq.)的影响下的荧光信号明显减少。时间的影响:BV-2细胞在含10%FBS(Gibco Company,USA)的高糖DMEM(KeyGENBioTECH,中国)中,在5%CO2中在37℃恒温培养箱中培养,将生长良好的细胞以5×104个细胞/孔的密度接种在24孔板中,每孔 400μL,培养24小时以允许细胞附着。除去培养基,细胞在37℃下在不存在或存在MOP (0.5μM)的情况下将细胞与FAM-Aβ(1μM)在无血清DMEM中孵育0.25,0.5,1,3, 5小时。类似地,通过流式细胞术获得定量结果。
实验图像如图12所示,随着时间增加,Aβ的细胞摄取增加。在MOP的作用下在短时间(15min,1h)后没有显著的变化,但共孵育后3和5h后,MOP显著的增加了Aβ的细胞摄取。
2.MOP促进BV-2细胞对Aβ40寡聚体的清除
BV-2细胞含10%FBS(Gibco Company,USA)的高葡萄糖DMEM(KeyGEN BioTECH,中国)中,在5%CO2中在37℃恒温培养箱中培养,将生长良好的细胞以5×104个细胞/孔的密度接种在24孔板中,每孔400μL,培养24小时以允许细胞附着。除去培养基,细胞在37℃下在不存在或存在MOP(1,0.5,0.2,0.1,0.05,0.02,0.01μM)的情况下,与 oAβ40(1μM)孵育3小时。用ELISA试剂盒定量细胞外残留的Aβ40水平。各组中DMSO 的含量相等且低于0.5%。并且实验重复三次。
实验结果如图13所示,在存在MOP的情况下,随着MOP浓度的增加,oAβ的细胞摄取持续增加。特别是当MOP以0.2eq.存在时,Aβ的细胞摄取达到对照组的1.8倍,这表明MOP对小胶质细胞摄取oAβ具有很强的促进作用。类似地,随着MOP浓度的进一步增加,oAβ的细胞摄取减少,表明高浓度的MOP可以抑制oAβ的细胞摄取。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国药科大学
<120> 一种用于治疗阿尔茨海默病的多肽和应用
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Claims (7)
1.一种功能多肽,其特征在于包括多肽片段Ⅰ:识别及阻断淀粉样蛋白 Aβ聚集的多肽片段和多肽片段Ⅱ:apoE 受体结合区域的模拟肽,所述多肽片段Ⅰ的氨基酸序列如 SEQ IDNO:2所示,所述多肽片段Ⅱ的氨基酸序列如 SEQ ID NO:8所示,所述多肽Ⅰ与多肽Ⅱ通过连接肽或直接连接。
2.根据权利要求 1 所述的功能多肽,其特征在于所述连接肽的氨基酸序列如SEQ IDNO:11-15 所示。
3.根据权利要求 1 所述的功能多肽,其特征在于所述多肽的氨基酸序列如SEQ IDNO:16 所示。
4.根据权利要求 1-3任一项所述的功能多肽在制备治疗阿尔茨海默病的药物中的应用。
5.根据权利要求4 所述的应用,其特征在于所述的功能多肽抑制淀粉样蛋白Aβ聚集、抑制Aβ的细胞毒性、加速小胶质细胞对Aβ的清除。
6.一种药物组合物,其特征在于含有权利要求1-5任一项所述的功能多肽或其盐作为活性成分。
7.根据权利要求6所述的药物组合物,其特征在于还含有药学上可接受的载体。
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