CN115819552A - 一种多肽用于预防和治疗补体相关紊乱的用途 - Google Patents
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Abstract
一种多肽用于预防和治疗补体相关紊乱的用途,涉及生物医学领域。所述多肽包含TREM2受体蛋白的胞外段第31‑71位氨基酸,其氨基酸序列如SEQ ID NO:1所示,其核苷酸序列如SEQ ID NO:2所示。所述多肽可在制备预防和治疗补体紊乱相关疾病药物中的应用。所述多肽可以与补体蛋白C1q结合,能够抑制经典补体级联反应,并具有降低神经退行性疾病模型小鼠脑中补体蛋白C3沉积,减少小胶质细胞对神经突触的吞噬,增加突触蛋白密度,改善神经突触功能和学习记忆能力,因而可在制备经典补体通路抑制剂中应用,具有制备预防和治疗补体紊乱相关疾病药物的用途。
Description
技术领域
本发明涉及生物医学领域,特别是涉及一种多肽在制备预防和治疗补体紊乱相关疾病药物中的用途。
背景技术
补体系统是人体先天免疫的重要组成部分,近年来人们发现补体除协助抗体发挥免疫效应外,还参与炎症、神经退行性疾病以及癌症等过程中。补体系统由数量众多的蛋白质组成,除补体级联经典组分C1-C9之外,还包含其他50多种蛋白。这些蛋白组成复杂的调控网络,介导免疫监视作用和组织稳态维持。正常生理状态下,补体蛋白以非活化的形式存在于体液中,在某些激活物的作用下,补体通过级联酶促反应被激活,发挥生物学活性。补体系统可由经典途径(Classical Pathway,CP)、凝集素途径(Lectin pathway,LP)以及替代途径(Alternative Pathway,AP)激活,他们的激活启动物不一样,但是均汇聚于补体第三组份(C3)并导致效应分子的产生。补体级联可通过C3b的调理作用补充抗体和吞噬细胞对入侵微生物的清除能力;通过C3a和C5a促进炎症反应;通过C5b-9攻膜复合物溶解病原体。补体系统与其他免疫和生理系统一起合作,将先天免疫和适应性免疫整合到一起,共同介导免疫复合物、细胞碎片以及凋亡细胞的清除,促进正常组织和器官的发育以及损伤后的组织修复。
尽管补体激活能够有效保护机体免受病原体入侵和感染,但过量或失调的补体激活可能导致一系列疾病的发生。目前,已发现补体紊乱导致的多种急性和慢性疾病,急性病包含抗体介导的免疫排斥、脓毒症、缺血性中风等,慢性病包括自身免疫性疾病、癌症、肾脏疾病、慢性溶血性疾病、牙周病、眼部疾病以及神经退行性疾病等。由于补体系统失调引发多种疾病的发生,因此针对补体系统的药物开发受到广泛的关注。
C1q是构成补体C1的核心组分,是经典补体级联反应的起始分子。C1的异常激活可触发小胶质细胞识别和吞噬被补体标记的神经突触,最终导致神经突触丢失和神经退行性病变。在阿尔茨海默氏病、亨廷顿氏病、额颞叶痴呆、青光眼等疾病的几种动物模型中,敲除C1q基因能够抑制神经突触的丢失,改善小鼠的行为学表现,提示抑制C1q对上述疾病具有保护意义。目前,通过脑立体定位注射C1q特异性的抗体已被证实能够显著改善多种神经退行性疾病模型小鼠的病理,但这些抗体分子量较大,跨越血脑屏障困难,因此临床上尚没有可用的针对补体C1q的神经退行性疾病干预药物。
发明内容
本发明的第一目的在于提供一种多肽TREM2 31-71。
本发明的第二目的在于提供所述多肽在制备预防和治疗补体紊乱相关疾病药物中的用途。
本发明提供一种多肽TREM2 31-71,包含TREM2受体蛋白的胞外段第31-71位氨基酸,其氨基酸序列如SEQ ID NO:1所示,其核苷酸序列如SEQ ID NO:2所示。
本发明提供所述多肽在制备预防和治疗补体紊乱相关疾病药物中的用途。
所述补体为C1,由C1q、C1r、C1s三个糖蛋白亚单位组成。
所述补体紊乱相关疾病包括具有淀粉样蛋白病变或Tau蛋白病变的神经退行性疾病,或由于补体蛋白C1q过度蓄积导致的疾病。
所述多肽具有与补体蛋白C1q结合的用途。
所述多肽具有抑制补体蛋白C1对C2切割的用途。
本发明提供所述多肽在制备经典补体通路抑制剂中应用。所述多肽具有抑制经典补体通路激活的功能。
本发明提供所述多肽在防治脑神经退行性病变、脑损伤、脑功能障碍疾病的药物中的应用。
所述多肽具有抑制脑神经退行性病变脑中补体蛋白C3沉积的功能。
所述多肽具有降低神经退行性病变脑中小胶质细胞吞噬神经突触的功能。
所述多肽具有增加神经退行性病变脑中神经突触蛋白密度的功能。
所述多肽具有增加神经退行性病变脑中神经突触功能。
本发明提供所述多肽在用于防治和/或缓解阿尔茨海默氏病引发的学习记忆功能障碍药物中的应用。所述多肽具有增加神经退行性病变学习记忆功能的功能。
与现有利用补体特异性抗体进行靶向干预的技术相比,本发明为包含41个氨基酸的多肽,比抗体更容易穿透血脑屏障进入脑内发挥抑制补体活性的功能,因而所述多肽在治疗神经退行性疾病中具有良好的应用前景。此外,本发明首次报道TREM2 31-71多肽能够改善补体紊乱相关疾病,具体体现在:TREM2 31-71多肽能够与补体蛋白C1q结合,能够抑制C1对C2的切割,能够抑制经典补体级联反应。通过脑立体定位注射或尾静脉注射TREM231-71多肽至神经退行性疾病模型小鼠,能够显著减少C3蛋白沉积,减少小胶质细胞对神经突触的吞噬,增加神经突触蛋白密度,改善神经突触功能和学习记忆能力。因而,TREM231-71多肽可在制备经典补体通路抑制剂中应用,在制备预防和治疗补体紊乱相关疾病药物中将具有广阔的应用前景。
附图说明
图1是TREM2 31-71多肽与补体C1q结合的结果图;
图2是TREM2 31-71多肽抑制经典补体通路激活的结果图;
图3是TREM2 31-71多肽抑制C1对C2切割的结果图;
图4是TREM2 31-71多肽减少淀粉样病变小鼠脑中C3蛋白水平的结果图;
图5是TREM2 31-71多肽降低淀粉样病变小鼠脑中C3沉积的结果图;
图6是TREM2 31-71多肽降低淀粉样病变小鼠脑中小胶质细胞吞噬神经突触的结果图;
图7是TREM2 31-71多肽增加淀粉样病变病小鼠脑中突触蛋白水平的结果图;
图8是TREM2 31-71多肽改善淀粉样病变病小鼠突触功能的结果图;
图9是TREM2 31-71多肽降低Tau蛋白病变小鼠脑中C3蛋白水平的结果图;
图10是TREM2 31-71多肽降低Tau蛋白病变小鼠脑中C3沉积的结果图;
图11是TREM2 31-71多肽降低Tau蛋白病变小鼠脑中小胶质细胞吞噬神经突触的结果图;
图12是TREM2 31-71多肽增加Tau蛋白病变小鼠脑中突触蛋白水平的结果图;
图13是TREM2 31-71多肽降低Tau蛋白病变小鼠认知功能缺陷的结果图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1 TREM2 31-71多肽的获得
2型髓系细胞触发受体(Triggering receptor expressed on myeloid cells 2,TREM2)属于Ig(Immunoglobulin,Ig)超家族,其编码基因位于染色体6p21上,编码的核苷酸和氨基酸序列在Genbank中的编号为NM_018965.3。本发明利用序列截短实验,筛选获得TREM2蛋白胞外段第31-71位氨基酸,介导TREM2与C1q的结合作用。通过委托科生景肽公司合成,获得的多肽序列如下:
TREM2 31-71的氨基酸序列为:
SLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHNLWL SEQ ID NO:1
TREM2 31-71的核苷酸序列为:
TCCCTGCAGG TGTCTTGCCC CTATGACTCC ATGAAGCACT GGGGGAGGCG CAAGGCCTGGTGCCGCCAGC TGGGAGAGAA GGGCCCATGC CAGCGTGTGG TCAGCACGCA CAACTTGTGG CTG SEQ IDNO:2
实施例2 TREM2 31-71多肽和补体C1q的结合能力检测
参照图1,利用酶联免疫吸附试验(ELISA)检测TREM2 31-71多肽和作为对照的杂乱肽(Scrambled)与C1q的结合能力,TREM2 31-71多肽和Scrambled多肽在科生景肽公司合成。将10μM的TREM2 31-71多肽和Scrambled多肽固定在96孔板中,封上塑料膜,4℃摇床过夜。第二天,弃去板内液体,先利用0.1%PBST(1×PBS+0.1%Tween 20)洗涤三次后,再用4%BSA在37℃烘箱中封闭1h,分别加入不同浓度的C1q蛋白(0、0.78125、1.5625、3.125、6.25、12.5、25、50nM),C1q稀释于1×PBS中,37℃孵育1h;0.1%PBST洗三次后再利用C1q生物素化抗体(Hycult Biotech,HM1096BT,1︰200)在37℃烘箱中孵育1h;一抗孵育结束后,洗板,再加入链霉亲和素Poly-HRP40偶联试剂(Fitzgerald,65R-S104PHRP,1︰3000)37℃孵育30min;0.1%PBST洗三次后利用TMB(Sigma Aldrich,T5569)显色液显色,记录656nm处的吸光值。结果显示:Scrambled多肽不与C1q结合,TREM2 31-71多肽与C1q存在较强的相互作用,其结合的平衡解离常数KD值为1.41nM。
实施例3 TREM2 31-71多肽和补体C1q结合对补体通路激活的影响
利用人IgM特异性激活经典补体途径,再利用ELISA方法检测C3沉积以评估补体活化水平。将溶于75mM碳酸钠(pH 9.6)的人IgM(Calbiochem,4017991MG,2μg/mL)加入ELISA板中4℃孵育过夜,第二天用0.1%PBST清洗孔板3次。利用GVB++(Gelatin Veronal Buffer,Complement Technology,B100)稀释正常人血清(Normal Human Serum,ComplementTechnology,NHS),再加入不同浓度的Scrambled或TREM2 31-71多肽于4℃下孵育2h,然后加入板中于37℃孵育25min。使用0.1%PBST清洗孔板3次,加入C3抗体(Santa Cruz,sc-20137,1︰100),室温下孵育1h。用0.1%PBST清洗孔板3次,加入HRP标记的二抗(ThermoFisher Scientific,31460,1︰2000),室温下孵育40min。最后,加入TMB显色并记录每孔在656nm的吸光值。图2给出TREM2 31-71多肽抑制补体C3沉积的结果图,结果显示:TREM2 31-71多肽显著抑制补体蛋白C3的沉积。
实施例4 TREM2 31-71多肽和补体C1q结合对C1切割C2的影响
将TREM2 31-71多肽(10μM)或C1酯酶抑制剂(C1-INH)(Peprotech,130-20,5μg/mL)与C1蛋白(Complement Technology,A098,0.1μg/mL)在GVB++缓冲液中4℃孵育2h,然后加入C2蛋白(Complement Technology,A112,2μg/mL),37℃孵育1h,C1-INH作为阳性对照。最后,加入等体积不含DTT的2×SDS上样缓冲液终止裂解反应,样品于100℃煮10min。通过蛋白印迹分析C2切割产物的水平,结果如图3所示。与对照组相比,TREM2 31-71多肽使得C1对C2切割效率下降27.10%,表明TREM2 31-71多肽能够显著抑制C1对C2的切割。
实施例5 TREM2 31-71多肽对淀粉样病变模型小鼠脑中补体C3蛋白水平的影响
取6只7月龄的淀粉样病变模型小鼠——5xFAD小鼠,异氟烷麻醉后置于脑立体定位仪上,小心剪开头皮找到前囟位置,以前囟为原点找到位置(海马区域:±2.0mm,-2.0mm),并在此位置打孔。利用微量注射器以0.2μL/min速度、深度2.0mm注射多肽。左右半脑分别注射Scrambled对照多肽和TREM2 31-71多肽(即实施例1所合成的多肽),蛋白浓度为5μg/μL,各注射2μL。注射完成后让针头停留5min使多肽充分扩散,缓慢抽出注射器,缝合伤口并涂上红霉素,将小鼠放回饲养笼饲养。注射7天后,收取脑组织进行匀浆裂解,免疫印迹检测补体C3的变化(Abcam,ab200999,1︰1000)。结果如图4所示,与Scrambled对照多肽相比,TREM2 31-71多肽使得C3和iC3b蛋白水平分别降低37.40%和45.44%。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著降低小鼠海马区的C3和iC3b蛋白水平。
实施例6 TREM2 31-71多肽对淀粉样病变病模型小鼠脑中补体C3沉积的影响
小鼠的使用和注射的方法与实施例4相同,注射7天后,收取小鼠脑袋,先置于4%PFA固定过夜,再用25%和30%的蔗糖脱水,最后用OCT包埋鼠脑后冰冻切片,免疫荧光染色补体蛋白C3(Abcam,ab200999,1︰100),统计补体蛋白C3的沉积水平。结果如图5所示,注射Scrambled对照多肽组C3沉积为0.14/μm2,注射TREM2 31-71组C3沉积为0.10/μm2。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著降低小鼠海马区域的C3蛋白沉积。
实施例7 TREM2 31-71多肽对淀粉样病变病模型小鼠脑中小胶质细胞吞噬神经突触的影响
小鼠的使用和注射的方法与实施例5相同,注射7天后,收取小鼠脑袋,先置于4%PFA固定过夜,再用25%和30%的蔗糖脱水,最后用OCT包埋鼠脑后冰冻切片,免疫荧光染色小胶质细胞Iba1(Wako,019-19741,1:200)和突触蛋白PSD95(EDM Millipore,MAB1596,1:100),通过统计PSD95与Iba1的共定位反映小胶质细胞对神经突触的吞噬水平。结果如图6所示,注射Scrambled对照多肽组小胶质细胞对突触的吞噬百分比为3.29%,注射TREM231-71组小胶质细胞对突触的吞噬百分比为2.23%。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著降低5xFAD小鼠海马区域小胶质细胞对神经突触的吞噬作用。
实施例8 TREM2 31-71多肽对淀粉样病变病模型小鼠脑中突触蛋白水平的影响
小鼠的使用和注射的方法与实施例5相同,注射7天后,收取小鼠脑袋,先置于4%PFA固定过夜,再用25%和30%的蔗糖脱水,最后用OCT包埋鼠脑后冰冻切片,免疫荧光染色突触蛋白PSD95(EDM Millipore,MAB1596,1:100),统计突触蛋白PSD95的密度。结果如图7所示,注射Scrambled对照多肽组PSD95密度为0.38/μm2,注射TREM2 31-71组PSD95密度为0.43/μm2。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著增加小鼠海马区域突触蛋白PSD95的密度。
实施例9 TREM2 31-71多肽对淀粉样病变病模型小鼠突触功能的影响
小鼠的使用和注射的方法与实施例6相同,注射7天后,急性取脑并利用电生理检测5xFAD小鼠海马区微型兴奋性突触后电流(mEPSC)的变化。结果如图8所示,注射Scrambled对照多肽的mEPSC频率和振幅分别为0.46Hz和7.65pA,注射TREM2 31-71多肽组的mEPSC频率和振幅分别为0.78Hz和8.60pA。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够增加5xFAD小鼠海马区兴奋性神经元mEPSC的频率和振幅,表明TREM231-71多肽能够改善5xFAD小鼠的突触功能。
实施例10 TREM2 31-71多肽对Tau蛋白病变模型小鼠脑中补体C3蛋白水平的影响
选择8月龄Tau蛋白病变模型小鼠——PS19进行尾静脉注射实验(每只小鼠每次分别注射50μg Scrambled对照多肽和TREM2 31-71多肽,每3天注射一次),注射30天后,收取脑组织进行匀浆裂解,免疫印迹检测补体C3的变化(Abcam,ab200999,1︰1000)。结果如图9所示,与Scrambled对照多肽相比,TREM2 31-71多肽使C3和iC3b蛋白水平分别降低46.74%和38.44%。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著降低小鼠海马区的C3和iC3b蛋白水平。
实施例11 TREM2 31-71多肽对Tau蛋白病变模型小鼠脑中补体C3沉积的影响
小鼠的使用和注射的方法与实施例10相同,注射30天后,收取小鼠脑袋,先置于4%PFA固定过夜,再用25%和30%的蔗糖脱水,最后用OCT包埋鼠脑后冰冻切片。免疫荧光染色补体蛋白C3(Abcam,ab200999,1︰100),统计补体蛋白C3的沉积水平。结果如图10所示,注射Scrambled对照多肽组C3沉积为0.30/μm2,注射TREM2 31-71组C3沉积为0.27/μm2。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著降低小鼠海马区域的C3蛋白沉积。
实施例12 TREM2 31-71多肽对Tau蛋白病变模型小鼠脑中小胶质细胞吞噬神经突触的影响
小鼠的使用和注射的方法与实施例10相同,注射30天后,收取小鼠脑袋,先置于4%PFA固定过夜,再用25%和30%的蔗糖脱水,最后用OCT包埋鼠脑后冰冻切片,免疫荧光染色小胶质细胞Iba1(Wako,019-19741,1:200)和突触蛋白PSD95(EDM Millipore,MAB1596,1︰100),通过统计PSD95与Iba1的共定位反映小胶质细胞对神经突触的吞噬水平。结果如图11所示,注射Scrambled对照多肽组小胶质细胞对突触的吞噬百分比为2.34%,注射TREM2 31-71组小胶质细胞对突触的吞噬百分比为1.53%。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著降低Tau蛋白病变模型小鼠海马区域小胶质细胞对神经突触的吞噬作用。
实施例13 TREM2 31-71多肽对Tau蛋白病变模型小鼠脑中突触蛋白水平的影响
小鼠的使用和注射的方法与实施例10相同,注射30天后,收取脑组织进行匀浆裂解,免疫印迹检测突触后蛋白PSD95(Cell Signaling Technology,3450S,1︰2000)和突触前蛋白vGluT1(Millipore,MAB5502,1:2000)的水平。结果如图12所示,与注射Scrambled对照多肽组相比,注射TREM2 31-71组PSD95和vGluT1的密度分别增加1.53和1.33倍。以上结果表明,TREM2 31-71多肽注射到海马区域后,能够显著增加小鼠海马区域突触蛋白PSD95和vGluT1的蛋白水平。
实施例14 TREM2 31-71多肽缓解Tau蛋白病变模型小鼠的认知功能缺陷
小鼠的使用和注射的方法与实施例10相同,注射30天后进行新物体识别和水迷宫实验检测小鼠的学习记忆能力。结果如图13所示:注射Scrambled对照多肽的PS19小鼠平均花费53.21s探索新物体,注射TREM2 31-71组的PS19小鼠平均花费93.77s探索新物体。水迷宫实验结果显示:注射Scrambled对照多肽的PS19小鼠60s内平台穿梭次数为1.46,注射TREM2 31-71组的PS19小鼠60s内平台穿梭次数为2.23。以上结果表明,尾静脉注射31-71多肽能够缓解Tau蛋白病变模型小鼠的认知功能缺陷。
综上所述,TREM2 31-71多肽能够改善补体相关紊乱,具体体现在:TREM2 31-71多肽能够与补体蛋白C1q结合,能够抑制C1对C2的切割,能够抑制经典补体级联反应。通过脑立体定位注射或尾静脉注射TREM2 31-71多肽至神经退行性疾病模型小鼠,能够显著减少C3蛋白的沉积,减少小胶质细胞对神经突触的吞噬,增加突触蛋白PSD95的密度,改善神经突触功能和小鼠的学习记忆能力。因而,TREM2 31-71多肽可在制备经典补体通路抑制剂中应用,在制备预防和治疗补体紊乱相关疾病药物中将具有广阔的应用前景。
尽管上面已经示出和描述本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.氨基酸序列如SEQ ID NO:1所示的多肽,包含TREM2受体蛋白的胞外段第31-71位氨基酸,其核苷酸序列如SEQ ID NO:2所示。
2.氨基酸序列如SEQ ID NO:1所示的多肽在制备预防和治疗补体紊乱相关疾病药物/试剂中的用途。
3.如权利要求2所述用途,其特征在于所述补体为C1,由C1q、C1r、C1s三个糖蛋白亚单位组成。
4.如权利要求2所述用途,其特征在于所述补体紊乱相关疾病包括具有淀粉样蛋白病变或Tau蛋白病变的神经退行性疾病,或由于补体蛋白C1q过度蓄积导致的疾病。
5.氨基酸序列如SEQ ID NO:1所示的多肽与补体蛋白C1q结合的用途。
6.氨基酸序列如SEQ ID NO:1所示的多肽抑制补体蛋白C1对C2切割的用途。
7.氨基酸序列如SEQ ID NO:1所示的多肽在在制备经典补体通路抑制剂中应用,所述多肽具有抑制经典补体通路激活的功能。
8.氨基酸序列如SEQ ID NO:1所示的多肽在制备脑神经退行性病变、脑损伤、脑功能障碍防治药物中的应用。
9.如权利要求8所述应用,其特征在于所述多肽具有抑制神经退行性病变脑中补体蛋白C3沉积的功能,所述多肽具有降低神经退行性病变脑中小胶质细胞吞噬神经突触的功能,所述多肽具有增加神经退行性病变脑中神经突触蛋白密度的功能,所述多肽具有增加神经退行性病变脑中神经元电生理活性的功能。
10.如权利要求1所述多肽在用于防治和/或缓解阿尔茨海默氏病引发的学习记忆功能障碍药物中的应用,所述多肽具有增加神经退行性病变学习记忆功能的功能。
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