CN110787284A - 一种治疗缺血性脑损伤的混合小肽tat-shc及其应用 - Google Patents
一种治疗缺血性脑损伤的混合小肽tat-shc及其应用 Download PDFInfo
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Abstract
本发明公开了一种治疗缺血性脑损伤的混合小肽TAT‑SHC(由TAT‑sinker、TAT‑hook、TAT‑C227三种小肽按分子量等比例混合)及其应用,所述混合小肽TAT‑SHC的全序列为:TAT‑sinker:H‑YGRKKRRQRRR‑DISDTIYPR‑OH;TAT‑hook:H‑YGRKKRRQRRR‑DYLLRTGQV‑OH;TAT‑C227:H‑YGRKKRRQRRR‑PNCFF‑OH。使用本发明混合小肽TAT‑SHC后,可以降低脑缺血及复灌后XIAP与proaspase‑3(Casp3)的结合,减轻proaspase‑3与XIAP转亚硝基化以及proaspase‑3的活化,最终减轻脑缺血诱导的细胞凋亡。另外TAT能够直接将融合的小肽带入细胞,解决了药物进入细胞的途径问题,方便给药。最后,小肽没有免疫源性,不会使机体产生免疫反应,减少了副反应,保证了二次以上给药的有效性。
Description
技术领域
本发明涉及医学技术领域,具体是一种治疗缺血性脑损伤的混合小肽TAT-SHC及其应用。
背景技术
脑卒中是世界范围内导致人口死亡的三大原因之一。其中,缺血性脑卒中是脑卒中的主要类型,具有高致残率、高死亡率的特点。随着人口老龄化和各种危险因素的增加,缺血性卒中所占比例还在增加。缺血性脑卒中(脑中风)包括脑血栓形成、腔隙性梗死和脑栓塞等,约占全部脑中风的70%。脑中风过程中神经元大量死亡,造成神经元的不可逆损伤,而脑中风后所遗留的神经***后遗症会严重影响患者生活质量。然而现在临床上很多神经保护药物并不能有效的治疗脑中风所致的神经元损伤。因此,积极探索缺血性卒中的发生机制以及预防和临床治疗方法,寻找一种有效治疗缺血性脑中风导致神经元损伤和死亡的药物,有着重要的医学价值和社会意义。
一氧化氮(NO)是调节正常信号转导的一种重要气态自由基,但过量会导致神经元损伤、死亡。NO除了通过cGMP信号通路激活下游蛋白激酶外,还可以使蛋白亚硝基化来调控其功能,如Parkin蛋白、二硫键异构酶(protein disulphide isomerase,PDI)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)和基质金属蛋白酶-9(matrix metallopeptidase 9,MMP-9)的亚硝基化会促进神经细胞的损伤与坏死,而含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,Caspase)与N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)的亚硝基化则有神经保护作用。蛋白质的亚硝基化修饰是一个可逆过程,在生理状态下,蛋白质的亚硝基化与去亚硝基化保持动态平衡。目前已经证实介导去亚硝基化的酶***有亚硝基谷胱甘肽还原酶(GSNOR)***和硫氧还蛋白(Trx)***。研究表明,在静息的人淋巴细胞中,Trx1介导了胞浆caspase-3的去亚硝基化,使其维持在一个稳定的低亚硝基化水平;Trx2介导了Fas诱导的线粒体相关caspase-3的去亚硝基化,促进了凋亡的发生;静息状态下,抑制Trx后发现caspase-9、蛋白酪氨酸磷酸酶1B(PTP1B)和GAPDH的亚硝基化水平显著升高,说明它们也是Trx介导去亚硝基化的底物。Trx本身也可以被亚硝基化,其中Trx1的第73位半胱氨酸被亚硝基化后可以介导转亚硝基化,使caspase-3的活性位点上第163位半胱氨酸亚硝基化并抑制其活性。Trx1介导去亚硝基化或转亚硝基化与其本身的氧化还原状态有关。
XIAP由Duekett等人于1996年首次鉴定,基因定位于Xq25,分子量55kDa,cDNA全长2540bp,mRNA全长9kb,编码区位于129-1622bp之间。XIAP蛋白主要由3个BIR区以及一个C末端的约含70个左右氨基酸残基的RING结构域组成。其中BIR1区与TAB1、TAK1结合,BIR2区与caspase-3、-7结合并抑制他们的活性。
半胱天冬酶(cysteinyl aspartate-specific proteinases,caspase)是一类富含半胱氨酸的蛋白酶家族,是导致细胞凋亡解体的蛋白酶***,目前已经发现的家族成员达14种。正常情况下,caspases以无活性的酶原(proenzymes)形式存在,必须经过线粒体途径、死亡受体途径、内质网途径等激活以后才可以发挥作用。当受到凋亡信号刺激时,上游的caspases能次序的激活下游的Caspases,形成级联反应,将凋亡信号逐级传到凋亡底物[7]。Caspase-3是Caspase家族中最重要凋亡执行者之一,广泛分布于各种类型的细胞中。其前体procaspase-3含有277个氨基酸残基,分子量约32kD,在活化过程中从Asp28~Ser29和Asp175~Ser176两处被剪切,形成P17(29~175)和P10(182~277)两个片段,两种亚基再组成活性形式的caspase-3。
静息状态下,procaspase-3是以亚硝基化的形式存在的,在受到凋亡刺激后,通过TrxR***发生去亚硝基化而活化,然后介导细胞凋亡,而XIAP的抗凋亡活性则因被亚硝基化而受到抑制,并且XIAP与caspase-3能相互作用。既然caspase-3在静息状态下以亚硝基化的状态存在,那么在受到凋亡信号刺激时,它是如何活化的呢?Moran Benhar等人研究发现[8],TrxR***能够介导caspase-3的去亚硝基化使其活化而发挥凋亡执行者的作用。那么procaspase-3的去亚硝基化与XIAP的亚硝基化之间是否存在着转亚硝基化的关系呢?Tomohiro等人就此做了研究,在试管中将提纯得到的caspase-3和XIAP用外源性NO供体GSNO刺激使他们发生亚硝基化,然后在分别将产生的SNO-caspase-3与XIAP,SNO-XIAP与caspase-3混合,通过生物素转化法检测,发现SNO-caspase-3能够将NO转给XIAP而SNO-XIAP不能转亚硝基给caspase。
如图1所示,缺血性脑中风中,产生大量自由基NO,使XIAP发生亚硝基化(图1中①所示);同时,与XIAP相结合的procaspase-3(Casp3)转亚硝基到XIAP(图1中②所示)。XIAP亚硝基化后对procaspase-3的抑制作用降低,同时,procaspase-3去亚硝基化后被活化,剪切为有活性的caspase-3,引起细胞凋亡。
发明内容
本发明的目的在于提供一种治疗缺血性脑损伤的小肽TAT-SHC及其应用,利用一种人工合成的小肽,抑制procaspase-9与XIAP在缺血性脑卒中的结合及转亚硝基化增加,从而抑制procaspase-9活化所介导的凋亡信号通路,减轻脑缺血所致的神经元损伤。
目前发现的转亚硝基化都是蛋白之间直接相互作用发生的,procaspase-3与XIAP也是这样?本研究前期用免疫沉淀证实了这一点:缺血复灌促进了二者的结合且复灌3h结合程度最高,这与procaspase-3、XIAP的亚硝基化变化趋势一致,由此得出结论:在脑中风发生后,导致procaspase-3与XIAP结合增加,由此提高了procaspase-3与XIAP之间转亚硝基化,紧接着去亚硝基化的procaspase-3激活,作用于下游信号通路,最终引起神经细胞凋亡,加重脑损伤。
由于脑中风引起procaspase-3与XIAP结合增加,因而触发了一系列反应,最终加重了脑损伤。因此,本发明根据procaspase-3与XIAP结合位点的氨基酸序列,设计了小肽TAT-SHC(XIAP上与procaspase-3上结合的3个位点基序,负责与procaspase-3结合;TAT来源于慢病毒上的一段11个氨基酸的序列,能够携带融合的小肽进入细胞内),能够与XIAP竞争结合procaspase-3,抑制缺血再灌注诱导的procaspase-3与XIAP的结合,减少XIAP的亚硝基化及procaspase-3的去亚硝基化,即减少procaspase-3与XIAP之间转亚硝基化,降低由此引起的procaspase-3的去亚硝基化和水解活化,从而抑制脑缺血诱导的Procaspase-3的活化激活的下游凋亡信号通道,对缺血性脑损伤起保护作用。
为实现上述目的,本发明提供如下技术方案:
一种治疗缺血性脑损伤的混合小肽TAT-SHC,基于Procaspase-3与XIAP之间结合及转亚硝基化设计而成,其中TAT为慢病毒上的一段11个氨基酸的序列YGRKKRRQRRR,所述的混合小肽TAT-SHC的全序列为:TAT-sinker:H-YGRKKRRQRRR-DISDTIYPR-OH;TAT-hook:H-YGRKKRRQRRR-DYLLRTGQV-OH;TAT-C227:H-YGRKKRRQRRR-PNCFF-OH。
所述的混合小肽TAT-SHC在缺血性脑损伤保护药物中的应用。
与现有技术相比,本发明的有益效果是:
使用本发明小肽TAT-SHC后,可以降低XIAP与procaspase-3的结合,减轻procaspase-3与XIAP转亚硝基化以及procaspase-3的活化,最终减轻脑缺血诱导的细胞凋亡。另外TAT能够直接将融合的小肽带入细胞,解决了药物进入细胞的途径问题,方便给药。最后,混合小肽TAT-SHC没有免疫源性,不会使机体产生免疫反应,减少了副反应,保证了二次以上给药的有效性。
附图说明
图1缺血性脑中风中,TAT-SHC拮抗Procaspase-3与XIAP结合从而抑制转亚硝基化、激活procaspase-3导致细胞凋亡的过程示意图:缺血性脑中风中,产生大量自由基NO,使XIAP发生亚硝基化(图中①所示);同时,与XIAP相结合的procaspase-3(Casp3)转亚硝基到XIAP(图中②所示)。XIAP亚硝基化后对procaspase-3的抑制作用降低,同时,procaspase-3去亚硝基化后被活化,引起细胞凋亡。使用小肽TAT-SHC后,降低XIAP与procaspase-3的结合,减轻procaspase-3与XIAP转亚硝基化以及procaspase-3的活化,最终减轻脑缺血诱导的细胞凋亡。
图2脑缺血再灌注后XIAP与procaspase-3的结合增加。
图3脑缺血再灌注诱导XIAP的亚硝基化及procaspase-3的去亚硝基化(即二者的转亚硝基化)和procaspase-3水解活化
图4混合小肽TAT-SHC抑制XIAP与procaspase-3的结合;混合小肽TAT-SHC抑制procaspase-3与XIAP转亚硝基化及procaspase-3的活化;
图5混合小肽TAT-SHC抑制大鼠在缺血再灌注诱导后的脑损伤效果图。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
本发明实施例中,一种治疗缺血性脑损伤的混合小肽TAT-SHC,基于procaspase-3与XIAP之间结合及转亚硝基化设计而成,其序列为:TAT-sinker:H-YGRKKRRQRRR-DISDTIYPR-OH;TAT-hook:H-YGRKKRRQRRR-DYLLRTGQV-OH;TAT-C227:H-YGRKKRRQRRR-PNCFF-OH。
脑缺血后内源性procaspase-3发生了去亚硝基化并水解活化,进而导致细胞凋亡。而caspases的活性又受到凋亡抑制蛋白家族(Inhibitors of apoptosis,IAPs)的调控,其中X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)通过其BIR结构域直接与procaspase-3结合并抑制其活性。脑缺血后,XIAP发生了亚硝基化,从而抑制了自身的抗凋亡活性。
根据XIAP与procaspase-3的3个结合位点基序氨基酸序列,设计了3个小肽TAT-sinker、TAT-hook、TAT-C227(XIAP上DISDTIYPR、DYLLRTGQV、PNCFF三个基序,负责与procaspase-3结合;TAT来源于慢病毒上的一段11个氨基酸的序列YGRKKRRQRRR,能够携带融合的小肽进入细胞内)。由此,干扰肽全序列分别为:TAT-sinker:H-YGRKKRRQRRR-DISDTIYPR-OH;TAT-hook:H-YGRKKRRQRRR-DYLLRTGQV-OH;TAT-C227:H-YGRKKRRQRRR-PNCFF-OH。为了验证是否其它肽也有类似作用,同时合成了与结合位点氨基酸序列不同的对照肽:H-YGRKKRRQRRR-MMLL-OH。小肽交由专门合成肽类和蛋白质的生物公司合成。
采用SD大鼠全脑缺血模型为研究对象,缺血15min再灌注3h,procaspase-3与XIAP结合增强在最高点(参阅图2),并且二者之间的转亚硝基化和procaspase-3的水解活化相一致(参阅图3)。实验组用竞争性抑制procaspase-3与XIAP结合的干扰肽TAT-SHC于缺血前20min侧脑室注射,对照组给予对照肽TAT-MMLL,然后于复灌3h取海马为样品,用免疫沉淀和免疫印迹检测procaspase-3与XIAP的结合、转亚硝基化及procaspase-3的水解活化。结果显示:抑制实验组小肽TAT-SHC能抑制procaspase-3与XIAP结合(参阅图4)及XIAP的亚硝基化、procaspase-3的去亚硝基化和水解活化(参阅图4),表明procaspase-9与XIAP转亚硝基化是与结合有关的,并且实验肽能够抑制这种作用。
用焦油紫染色技术来观察TAT-SHC对缺血再灌注导致的海马CA1区神经元损伤的影响。结果显示:在假手术组,细胞形态为圆形、细胞核淡染。而在单纯缺血组,细胞为皱缩状的形态且数量明显减少。TAT-SHC组的细胞数量明显多于单纯缺血组,并且形态多为圆形、细胞核淡染。这表明TAT-SHC抑制procaspase-3与XIAP的结合、转亚硝基化能减轻缺血再灌注导致的海马CA1区神经元损(参阅图5)伤。
动物模型制备:选择健康成年雄性Sprague Dawley(SD)大鼠,体重250±10g。动物以20%水合氯醛(300~350mg/kg)腹腔注射麻醉后,分离双侧颈总动脉,电凝椎动脉。手术第2天动物于清醒状态下结扎双侧颈总动脉,全脑缺血15min,再灌注特定时间点。缺血时保持其直肠温度在36.5~37.5℃。鉴定大鼠脑缺血模型标准如下:缺血前大鼠处于完全清醒状态,其生命体征正常。缺血后30-60秒内大鼠即失去知觉和活动能力,翻正反射消失,呈现角弓反张,同时痛觉消失,双侧瞳孔散大,对强光刺激闭睑反射消失,眼球颜色灰白(正常为鲜红色)。再灌后眼球颜色立即恢复红色,瞳孔回缩。缺血15min需再灌约1h后才能恢复自主活动。假手术组处理同手术组,但不结扎双侧颈总动脉。
小肽给药:TAT融合肽100μg溶于10μl生理盐水,于缺血前20min侧脑室注射给药。对照组注射相同体积的对照肽(以生理盐水表示)。
样品制备:缺血不同时间后,断头快速取脑,分离双侧海马,沿海马裂分离出CA1区,置液氮中冻存备用。以下操作均在冰水浴中进行:从液氮中取出海马加匀浆缓冲液,用Glas-col匀浆器高速匀浆(10s×6次),冰浴上超声破碎(10s×3次),10000g 4℃离心15min,小心移取上清液。
蛋白含量测定:采用BCA微量法,以牛血清白蛋白(BSA)5mg/ml为标准蛋白。
蛋白S-亚硝基化测定:采用Jaffrey和Snyder报道的‘生物素转化法’(Biotin-Swich method),在实验过程中要避光和使用毛面试管。检测procaspase-9与XIAP的转亚硝基化。
免疫沉淀与免疫印迹:以下操作均在4℃条件下进行:含相同蛋白量(400μg)的样品中,加入5倍体积IP缓冲液,以25μl蛋白A/G-琼脂糖预吸附1h,12000g离心2min,取上清,加入1-2μg抗体,旋转混匀器上反应4h或过夜,再加入25μl蛋白A/G-琼脂糖,旋转混匀器上反应2h,12000g离心2min,沉淀用IP缓冲液洗3遍。加入1/3体积4×sample buffer,混匀后置沸水浴中5min以洗脱琼脂糖上结合的蛋白,10000g离心2min,吸取上清用于免疫印迹。参考Sambrook等方法,含相同蛋白量的样品中加入1/3体积4×sample buffer,置沸水浴中5min变性处理。取等量的变性蛋白质样品(100μg)或IP处理后样品,经SDS-聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis;PAGE)分离后,以半干转法电转移至NC膜上。将NC膜置封闭液中室温孵育6h后,加入一抗工作液,4℃孵育4h或过夜,TBST洗膜(5min×3)后,加入碱性磷酸酶标记的二抗的工作液,室温孵育2h,TBST洗膜(5min×3)。使用NBT/BCIP试剂盒,在新鲜配制的AP显色液中显色,流水洗涤终止反应。扫描膜上显色条带并以Quantity One软件分析处理。
组织学方法:大鼠经腹腔注射水合氯醛麻醉后,开左胸暴露心脏,将针头自心尖穿过左心室***主动脉,先用38℃左右生理盐水约100ml快速灌洗,然后复灌入冰冷的4%的多聚甲醛250-300ml,30分钟内灌完,灌注后的大鼠断头取脑将其直接用4%的多聚甲醛后固定12小时左右。流水冲洗,冲洗时间同固定时间。然后70%(4h)、80%(4h)、90%(4-12h)、95%(2h×2次)、100%(2h×2次)梯度酒精脱水,二甲苯透明(1.5h)、60℃浸蜡(1h×3次)。取出包埋,4℃保存。石蜡切片机切片,厚5μm。45℃温水中贴片于明胶处理过的载玻片上,40℃烘烤过夜。切片经三次二甲苯脱蜡,100%、90%、70%梯度酒精浸水,0.1%焦油紫(Cresyl violet)染色10-20分钟,按常规方法70%、80%、95%、100%梯度酒精分色,光镜下观察神经元紫色而背景基本无色为止。梯度酒精脱水,二甲苯透明,中性树脂封片后,光镜观察。在高倍镜下观察海马CA1区神经元,有清楚的细胞核,胞体着色良好的计为成活神经元,成活神经元密度表示为1mm2面积内成活大锥体神经元的数量。
数据处理:所有数据以均数±标准差(mean±SD)表示,统计学方法采用ANOVA,多个实验组与一个组的比较采用Dunnett检验,多个实验组之间的比较采用q检验(Newman-keuls法),P<0.05为差异有统计学意义。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (2)
1.一种治疗缺血性脑损伤的混合小肽TAT-SHC由三种小肽TAT-sinker、TAT-hook、TAT-C227按分子量等比例组成。TAT-SHC根据procaspase-3与XIAP的3个结合位点而设计,能阻断procaspase-3与XIAP之间在脑缺血及复灌后结合、转亚硝基化增加。其中TAT为慢病毒上的一段11个氨基酸的序列YGRKKRRQRRR,其特征在于可以携带融合的小肽跨膜进入细胞,所述的混合小肽TAT-SHC全序列为TAT-sinker:H-YGRKKRRQRRR-DISDTIYPR-OH;TAT-hook:H-YGRKKRRQRRR-DYLLRTGQV-OH;TAT-C227:H-YGRKKRRQRRR-PNCFF-OH。
2.如权利要求1所述的混合小肽TAT-SHC(TAT-sinker、TAT-hook、TAT-C227)在缺血性脑损伤保护中作为药物的应用。
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