CN117736270A - 靶向蛋白siglec-10抑制炎症的多肽及其在脓毒症中的应用 - Google Patents
靶向蛋白siglec-10抑制炎症的多肽及其在脓毒症中的应用 Download PDFInfo
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Abstract
本发明公开了靶向蛋白siglec‑10抑制炎症的多肽及其在脓毒症中的应用。靶向siglec‑10的多肽,序列如SEQ ID No.2或SEQ ID No.3所示。所述的靶向siglec‑10的多肽在制备免疫调控药物中的应用。结果表明新多肽2和3可以结合siglec10蛋白。在细胞培养过程中,用多肽处理,可以显著抑制巨噬细胞炎症,还可以在动物水平抑制脓毒症中的炎症反应,改善脓毒症相关器官损伤。因此,该多肽具有免疫调控作用,可以在制备脓毒症相关药物中应用。
Description
技术领域
本发明属于生物药物领域,涉及靶向蛋白siglec-10抑制炎症的多肽及其在脓毒症中的应用。
背景技术
脓毒症是由感染引起的全身炎症反应综合征,严重者可导致器官功能障碍和(或)循环障碍等,也是感染、烧/创伤、休克等急危重症患者的严重并发症。脓毒症病理生理机制复杂,是一种异质性高的综合征,其重要特点是非特异性免疫功能障碍和免疫失衡。正常生理情况下,宿主免疫细胞在识别病原体后,激活免疫反应进行防御,最终一定会达到平衡,而脓毒症被认为是一种不平衡的免疫反应。因为一些已知和未知的原因,病原体逃避宿主免疫系统的防御,保持不断繁殖,持续损伤宿主细胞,也刺激免疫反应,导致免疫稳态不能恢复。在这种不平衡的状态下,许多最初被激活以提供保护的免疫反应已经变得有害,并与过度炎症和免疫抑制有关。
尽管脓毒症的根本发病机制复杂,涉及到多个方面,不可否认,脓毒症相关的过度炎症是其非常重要的特征和机制之一。在脓毒症中,病原体相关分子模式(pathogen-associated molecular patterns, PAMPs)和损伤相关分子模式(damage -associatedmolecular patterns, DAMPs)激活免疫细胞的模式识别受体(pattern recognitionreceptors, PRRs)后引发免疫系统持续激活、器官损伤和功能障碍的恶性循环。感染后炎症的启动对消除感染很重要,而过度炎症可导致组织损伤、多器官功能障碍、败血症死亡。精准调控脓毒症患者机体的过度炎症反应是治疗脓毒症的关键。至今为止,针对脓毒症的临床治疗手段仍十分有限,主要依赖于抗生素的使用和临床重症监护,但临床效果并不显著。动物模型实验证明,消除促炎细胞因子(如TNF、IL-6、IL-1β等)可防止器官损伤,有利于控制脓毒症进展。然而,对促炎因子的靶向中和治疗效果,仍未能有效缓解炎症的进展。因此,开发有效控制疾病早期炎症的药物依然是脓毒症的重要研究方向之一。
唾液酸结合性免疫球蛋白样凝集素(sialic acid-binding immunoglobulin-like lectin,Siglec)是一类细胞表面受体,目前发现 15 种人源和 9 种鼠源的Siglec分子。Siglec受体主要以细胞类型特异性方式在血细胞表面表达,可以通过识别含有唾液酸的糖链结构(如唾液酸化蛋白质、脂质和RNA)介导细胞与细胞或细胞与病原体间的相互作用,因此在固有免疫和适应性免疫中发挥重要的调控作用。大多数Siglec受体作为跨膜受体发挥抑制性的作用,被认为是预防和治疗多种疾病(如感染、自身免疫和癌症)的重要潜在靶标。Siglec-10是近些年发现的Siglec受体中的一种,主要表达在树突状细胞 (DC)、单核细胞、B 细胞、NK 细胞和 T 细胞中,其与 CD24 相互作用可通过结合酪氨酸磷酸酶SHP-1(NF-κB的负调节分子)来抑制DAMP 所产生的免疫应答。在脓毒症中,受损细胞释放的DAMPs可以被模式识别受体TLR4识别,触发炎症反应;而Siglec-10可以通过ITIM结构域激活SHP1,抑制Src激活,调节脓毒症期间DAMPs-TLR4介导的信号传导,进而参与调控脓毒症相关炎症反应。可见,Siglec-10在炎症反应调节中具有重要作用,可能是治疗脓毒症的潜在靶点。
相比化学小分子药物、大分子蛋白药物,多肽类药物,生物活性更强、用药剂量更小、毒副作用更低且疗效显著,生产更简单,成本更低,备受新药研发者的瞩目与青睐。噬菌体展示技术在新型多肽类药物的开发中,具有重要作用。
综上所述,研发获得可靶向Sigec-10,控制炎症反应的多肽分子,可能对于治疗脓毒症具有重要意义。
发明内容
本发明的目的是针对脓毒症中过度炎症反应,提供2条可以抑制炎症的多肽。
本发明的另一目的是提供2条多肽的应用。
本发明的目的可以通过以下技术方案实现:
靶向siglec-10的多肽,选自以下任意一种多肽:
多肽2:HDVKTQKRWAWR(SEQ ID No.2),
多肽3:HDVKTQDRWAWR(SEQ ID No.3)。
以上两条多肽均是基于多肽1(SEQ ID No.1:HFVKTPARWAWG)进行改造得到。
具有医药用途的本发明所述的靶向siglec-10的多肽。
本发明所述的靶向siglec-10的多肽在制备免疫调控药物中的应用。
本发明所述的靶向siglec-10的多肽在制备治疗免疫细胞过度活化和/或炎症反应过度相关疾病药物中的应用。
本发明所述靶向siglec-10的多肽在制备脓毒症药物中的应用。
本发明所述靶向siglec-10的多肽在制备抑制炎症的药物中的应用。
本发明所述靶向siglec-10的多肽在制备减轻器官损伤药物中的应用;优选在制备减轻脓毒症所致器官损伤的药物中的应用;所述的脏器优选肝组织、肺组织、脾组织和/或肾组织。
本发明所述靶向siglec-10的多肽在制备调控siglec-10功能药物中的应用。
本发明所述靶向siglec-10的多肽在制备siglec-10功能异常相关疾病的药物中的应用。
本发明所述多肽的获得可按照氨基酸序列通过固相合成方法获得;或者通过宿主微生物或细胞内克隆并表达携带编码所述多肽之一的核苷酸序列的DNA片段,通过现有的重组DNA技术制备获得。所使用的表达载体和宿主细胞均为重组技术为公众所知的。表达载体例如pET载体、pGEX载体;宿主细胞例如大肠杆菌(E .coli),放线菌(Actinomycetes),芽孢杆菌(Bacillus),链霉菌(Streptomyces)。
有益效果
本发明公开的这2条新多肽,是针对Siglec10特异性序列筛选并改造获得,均含有13个氨基酸,目前尚无这2条多肽的功能报道。序列为:HDVKTQKRWAWR和HDVKTQDRWAWR。合成这2条多肽,进行表面等离子共振实验,结果表明新多肽2和3可以结合siglec10蛋白。在细胞培养过程中,用多肽处理,可以显著抑制巨噬细胞炎症,还可以在动物水平抑制脓毒症中的炎症反应,改善脓毒症相关器官损伤。因此,该多肽具有免疫调控作用,可以在制备脓毒症相关药物中应用。
附图说明:
图1 多肽1促进人巨噬细胞炎性细胞因子产生
图2 多肽1促进人T细胞炎性细胞因子产生
图3 多肽1与siglec10蛋白分子间相互作用力的预测
图4 多肽1结构与siglec10蛋白结合预测
图5 多1和多肽2、多肽3序列的区别,红色字母代表的氨基酸残基为差异位点
图6 SPR实验测定新多肽2和3与靶标siglec10之间的结合亲和力
图7 多肽2和3抑制LPS诱导的巨噬细胞IL-6、TNF-α、IL-1βmRNA的表达
图8 多肽2和3抑制LPS诱导的巨噬细胞IL-6、TNF-α、IL-1β的分泌
图9 多肽2和3抑制炎症信号通路蛋白磷酸化
图10 多肽2和3降低脓毒症小鼠的炎症
图11 多肽2和3缓解脓毒症小鼠肝损伤,保护肝脏功能
图12 多肽2和3缓解脓毒症小鼠肾脏、肝脏、脾脏、肺组织损伤
具体实施方式
下面结合具体实施例对本发明进一步说明。
用 Ficoll/Hypaque 离心法分离得到人外周血单个核细胞(Peripheral BloodMononuclear Cell,PBMC)。用不含FBS的RPMI1640培养基,在37°C、5%的CO2条件下培养3小时,弃掉未粘附的细胞。将所得细胞,更换为含10% FBS 和50 ng/ml 人巨噬细胞集落刺激因子(M-CSF)的新鲜RPMI 1640培养基继续培养,每3天更换一次培养液,在第7天,即为巨噬细胞。诱导完成后,再用多肽1处理,48小时后,收集细胞,提取总RNA,并通过实时定量聚合酶链反应(Real-Time Quantitative polymerase chain reaction,RT-qPCR)方法检测TNFα和IL-6 mRNA的表达情况。结果如图1所示,由图可见与多肽未处理组相比,多肽处理后,可以显著促进TNFα和IL-6的表达水平。
实施例2多肽1促进人T细胞炎性细胞因子产生
为了确定多肽1是否调控T细胞的功能,利用 Ficoll/Hypaque 离心法,从人外周血中分离外周血单个核细胞PBMC,将PBMC与抗CD3 抗体(OKT3)和多肽1共培养。四天后,分别收集培养上清。通过ELISA方法检测TNF-α和IFN-γ的分泌情况。结果如图2所示,与anti-CD3处理的对照相比,多肽1处理可显著促进活化的PBMC 产生IFN-γ和TNF-α。因此,针对Siglec10特异性序列筛选获得的多肽1可以体外促进PBMC 的活化(抗CD3抗体诱导条件下)。
实施例3多肽1与蛋白siglec10结合能力预测和改造
我们使用Autodock对多肽1和siglec10蛋白进行分子对接分析。通过分析发现,siglec10蛋白的核心区域主要为5个β-Sheet片段组成的Domain,多肽1在蛋白上的结合位点主要位于Domain2和Domain3中间形成的空腔中,多肽与蛋白间仅形成了3组氢键作用,参与形成氢键的氨基酸为Ala10和Lys4,两者之间的结合能(Binding energy)为-5.591kcal/mol(图3和4)。我们选择性的将多肽链中Phe和Pro等带有空间位阻较大的环状氨基酸替换为Asp、Gln和Lys等有利于形成氢键的氨基酸,并且将Gly替换为Arg,最终改造获得新多肽2和3:HDVKTQKRWAWR和HDVKTQDRWAWR(图5)。
实施例4 新多肽2和3与蛋白siglec10结合亲和力检测
表面等离子共振( Surface Plasmon Resonance,简称SPR) 是一项分析生物分子之间相互作用的技术,它可以定性的判断两分子之间是否有相互作用,也可以实时定量的测定分子间相互作用的亲和力参数(平衡常数)和动力学参数(速率常数)。进一步,利用该技术测定计算发现,新多肽与蛋白分子的解离常数分别为Kd=6.70E-08M,Kd=4.48E-08M,表明新多肽2和3与siglec10蛋白之间有较强的结合亲和力(图6)。
将单核细胞THP-1按照标准条件(1640培养基,37℃,5%CO2)培养。第一天,将THP-1细胞接种在六孔板中,加入PMA(100ng/ml)24h,使其诱导转化为M0型巨噬细胞后,加入LPS(100ng/ml)刺激24h,再加入1μm浓度的多肽2和3孵育24h后,分别收集各组细胞RNA和细胞培养上清,进行qPCR分析和ELISA分析。其中多肽处理组为实验组,LPS刺激无多肽处理组为对照组。qRT-PCR结果显示,LPS刺激的巨噬细胞中炎性细胞因子IL-6、TNF-α、IL-1β显著升高,而加入多肽2和3处理后,其表达水平显著降低(图7)。ELISA检测也进一步确认,多肽2和3可以抑制巨噬细胞炎症因子IL-6、TNF-α、IL-1β的产生(图8)。
将单核细胞THP-1按照标准条件(1640培养基,37℃,5%CO2)培养。第一天,将THP-1细胞接种在六孔板中,加入PMA(100ng/ml)24h,使其诱导转化为M0型巨噬细胞后,加入LPS(100ng/ml)刺激24h,再加入1μm浓度的多肽2和3孵育24h后,收集各组细胞蛋白,进行WB分析。多肽处理组为实验组,LPS刺激无多肽处理组为对照组。蛋白质印迹分析显示,多肽2和3显著降低了P65、STAT1和IRF-3 蛋白的磷酸(图9)。
我们首先构建了小鼠脓毒症模型:实验采用Siglec-10人源化小鼠,实验分为正常NC组、LPS对照组、新多肽2组、新多肽3组,共四组,每组各5只,多肽浓度为5mg/kg。其中正常组不予任何试剂,其余三组先腹腔注射2mg/kg剂量的LPS,4h后,LPS对照组仅注射多肽溶剂,多肽组分别尾静脉注射5mg/kg剂量的新多肽2和3。24h后,采集各组小鼠眼球血,肝脏、脾脏、肾脏和肺组织进行后续测定。采集的眼球血收集于1.5 ml的EP管中,室温静置1h后,3000rpm,20℃离心10min,吸收的血清置于无菌EP管内送去检测:炎症细胞因子IL-6、TNF-α、IL-1β、IFN-γ和血清谷丙转氨酶(ALT)、转氨酶(AST)、碱性磷酸酶(ALP)。新鲜的肝组织、肺组织、脾组织和肾组织固定在4%PFA(pH=7.4)中,逐渐脱水,包埋在石蜡中,切成3μm的切片,并用HE染色进行光学显微镜检查。结果显示,与LPS对照组相比,多肽2和3处理组小鼠IL-6、TNF-α、IL-1β和IFN-γ的水平显著降低(图10)。此外,与LPS对照组相比,多肽2和3显著地降低了血清肝损伤标志物如天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)和碱性磷酸酶(ALP)的水平(图11)。H&E染色的组织学评估显示,LPS诱导的脓毒症小鼠肝、肺、脾和肾出现了水肿、炎症细胞浸润和严重出血;肝脏出现大量液泡性脂肪变性、肝细胞肿胀和炎症细胞浸润;肺间隔增厚,一些肺泡组织结构被破坏,炎性细胞浸润;还可见肾皮质和间质水肿伴有大量炎性细胞浸润,炎性细胞种类繁多;脾生发中心增加。多肽处理的两组,可明显改善上述病理症状(图12)。
Claims (10)
1.靶向siglec-10的多肽,序列如SEQ ID No.2或SEQ ID No.3所示。
2.具有医药用途的权利要求1所述的靶向siglec-10的多肽。
3.权利要求1所述的靶向siglec-10的多肽在制备免疫调控药物中的应用。
4.权利要求1所述的靶向siglec-10的多肽在制备治疗免疫细胞过度活化和/或炎症反应过度相关疾病药物中的应用。
5.权利要求1所述靶向siglec-10的多肽在制备脓毒症药物中的应用。
6.权利要求1所述靶向siglec-10的多肽在制备抑制炎症的药物中的应用。
7.权利要求1所述靶向siglec-10的多肽在制备减轻器官损伤药物中的应用。
8.根据权利要求7所述的应用,其特征在于权利要求1所述靶向siglec-10的多肽在制备减轻脓毒症所致器官损伤的药物中的应用;所述的脏器优选肝组织、肺组织、脾组织和/或肾组织。
9.权利要求1所述靶向siglec-10的多肽在制备调控siglec-10功能药物中的应用。
10.权利要求1所述靶向siglec-10的多肽在制备siglec-10功能异常相关疾病的药物中的应用。
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WO2016038064A1 (en) * | 2014-09-10 | 2016-03-17 | Innate Pharma | Cross reactive siglec antibodies |
CN114656523A (zh) * | 2022-03-14 | 2022-06-24 | 南京市第一医院 | 靶向siglec-10蛋白的多肽及免疫调控应用 |
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WO2016038064A1 (en) * | 2014-09-10 | 2016-03-17 | Innate Pharma | Cross reactive siglec antibodies |
CN114656523A (zh) * | 2022-03-14 | 2022-06-24 | 南京市第一医院 | 靶向siglec-10蛋白的多肽及免疫调控应用 |
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