CN113788878A - 一类自组装短肽,其在广谱疫苗及生物医学中的用途 - Google Patents
一类自组装短肽,其在广谱疫苗及生物医学中的用途 Download PDFInfo
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- CN113788878A CN113788878A CN202111123602.2A CN202111123602A CN113788878A CN 113788878 A CN113788878 A CN 113788878A CN 202111123602 A CN202111123602 A CN 202111123602A CN 113788878 A CN113788878 A CN 113788878A
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
本发明公开了一类自组装短肽,其在广谱疫苗及生物医学中的用途,涉及自组装短肽领域,如何提高DCs疫苗和其它类别的肿瘤疫苗的有效性,如何降低抗药性,及如何获得稳定、安全、效果更好的疫苗或载体,此外还解决了玻尿酸及同类产品不能原位激活胶原新生的问题,包括两个短肽:Arg Leu Asp Ile Lys Val Glu Phe Cys Cys,Arg Gln Asp Thr Lys Thr Glu Tyr Cys Cys;2种新的自组装短肽,表现出良好的抗肿瘤功能,不仅应用于肿瘤疫苗,也可作为病毒疫苗、药物方面的载体;还可用于细胞、类器官的三维培养,组织的创伤修复,止血材料等;制备的新型自组装短肽类“超级玻尿酸”系统不需要频繁皮肤注射。
Description
技术领域
本发明涉及自组装短肽技术领域,更具体的是涉及自组装短肽在生物学、纳米医学、化妆品及保健品中的应用。
背景技术
短肽在自然界中无处不在。它们被发现为激素、信息素、抗菌素、抗真菌剂在先天免疫系统、毒素和杀虫剂。但没有人认真认为肽可以作为支架水凝胶材料有用。自1990年在酵母蛋白中发现一个非常有趣的重复片段后,已经发生了显著的变化。现在人们认识到,由20种天然氨基酸制成的自组装肽具有真正的材料特性。目前,许多不同的应用已经发展从这些简单的和设计的自组装肽支架水凝胶,并在商业上提供。例子包括:(1)真正的3D组织细胞培养不同的组织细胞和各种干细胞,(2)修复和再生医学以及组织工程,(3)3D组织打印,(4)持续释放小分子,生长因子和单克隆抗体,(5)加速伤口愈合皮肤和糖尿病溃疡以及即时止血应用程序。
分子自组装指的是分子在不受外力介入的情况下,能够进行自我组织,自我聚集形成一种规则的结构,即能够从一个杂乱无序的状态转变成一个有序的状态。近几年来,手性自组装短肽,已经发展成为了一类新兴的纳米生物材料。它是一种仿ECM的生物支架纳米材料,能够模仿ECM的部分功能,从而影响细胞迁移、增殖、分化等生物学行为,可以作为细胞三维培养的基质材料,同时它在创伤修复、组织损伤修复等方面均有一定的作用。
骨组织损伤的修复,特别是软骨的修复,是一个世界性的医学难题,在基础医学和临床医学中有很多重要问题需要突破。在骨组织中,关节软骨是人体最重要的承重组织,具有低摩擦,抗压和拉伸性能。它是一种特殊的结缔组织,由软骨细胞及细胞外基质构成,以蛋白多糖和二型胶原为主。而关节软骨损伤是临床骨科常见的疾病,由于关节软骨缺乏血管,细胞结构单一,一旦损伤,其自身愈合能力很低。有研究表明,直径大于4mm的全层软骨损伤,即缺损超过钙化层,则不能自行修复。因此,一旦发生关节软骨损伤,最终可能导致骨关节炎的发生,严重影响患者的生活质量。
皮肤创伤不仅影响正常生活与美观,严重时将危及生命。皮肤创伤愈合的本质是隔离感染源、修复组织。皮肤创伤修复是一个复杂的过程,主要表现为伤口通过纤维化和瘢痕形成,愈合创面的非特异性愈合形式。其恢复时间分为多个阶段,各阶段之间相互作用、联系,共同完成创伤修复。此外,皮肤创伤修复过程中还有特殊的细胞功能级联机制的参与,如细胞动态参与、生长因子、细胞因子以及细胞外基质等。多种细胞因子和信号转导途径参与、调节着这些过程。急性创伤后几个小时完成修复的第一阶段,在伤口愈合的凝血期可激发炎症反应;在细胞增殖阶段,创伤愈合过程含创面缩小、肉芽组织形成、血管网生成和修复等;在伤口成熟的过程中,细胞外基质成分发生变化;皮肤创伤修复过程的完成时一般有瘢痕形成,其形成机制复杂,与修复过程中的炎症程度、细胞外基质的平衡等直接相关。
人体内DC分为淋巴系DC(lymphoid DC)和髓系DC(myeloid DC),后者DC即为大多数DC的来源。淋巴系DC由胸腺中的前体细胞分化发育而来.活化后主要释放大量Ⅰ型干扰素(interferon 1,IFN1),参与病毒免疫应答;髓系DC主要参与免疫应答的诱导和启动。人体正常条件下,绝大多数DC处于未成熟状态,位于非淋巴组织的上皮和多种实质器官,具有很强的抗原摄取、加工和处理能力,但其表达共刺激分子和主要组织相容性复合体Ⅱ类分子(maior histocompatib ility complex classⅡmolecules,MHCⅡ)类分子、细胞间黏附分子(intercellular cell adhesion molecules,ICAM)的水平低。因此,其抗原提呈能力较弱,在摄取抗原或一些促炎信号的刺激后,由未成熟DC分化发育为成熟DC,此过程中其抗原摄取和加工能力下降,但共刺激分子CD80、CD86、MHC(I、Ⅱ类)、ICAM1、ICAM2、淋巴细胞功能相关抗原1(lymphocyte function associated antigen 1,LFA1)、LFA-3的表达显著提高,激发免疫应答能力强。DC在成熟的分化过程中同时发生迁移,由抗原获取部位通过淋巴管、血循环进入外周免疫器官,提呈抗原后,启动T细胞免疫应答反应。DC抗肿瘤的主要作用机制为∶①DC摄取肿瘤相关抗原并递呈;②为T细胞活化提供信号,诱导T细胞分化产生大量的CD8*T细胞。致敏的DC表面表达大量的MHCⅠ类和Ⅱ类分子、B7-1和B7-2共刺激分子、黏附分子,能显著刺激CD8*T细胞的增殖;③引导效应性T细胞至肿瘤部位,杀灭肿瘤细胞。DC与T细胞结合后可分泌大量的白细胞介素12(interleukin 12,IL-12),可强力诱导T细胞、淋巴因子激活的杀伤(ymphokine activated killer,LAK)细胞、自然杀伤(n atural iller,NK)细胞分泌大量的穿孔素、y干扰素(interferon y,IFN-y)和颗粒酶,增强CD8*的细胞毒性T淋巴细胞(cytotoxic Tlymphocyte,CTL)、NK细胞对肿瘤细胞的杀伤作用;④促进2型辅助T(T helper type 2,Th2)细胞介导的免疫应答继而促进B淋巴细胞的激活,产生抗体;⑥淋巴系DC中的类浆细胞分泌IFN 1,能诱导初始和记忆B细胞分化,产生大量的IgM。研究表明,在肿瘤患者体内,肿瘤的恶性程度与肿瘤组织中浸润的DC数量呈反比关系。这可能是DC数量下降,导致其抗原加工、摄取、提呈能力下降,不能有效的提呈肿瘤细胞抗原,进而不能有效刺激T细胞分化增殖,产生免疫耐受。在DC抗肿瘤过程中,肿瘤组织或细胞会诱导全身性DC分化、发育和功能异常,引起免疫耐受,并使DC不能有效浸润、迁移到肿瘤组织提呈和加工抗原、诱导DC凋亡。
免疫治疗作为一种新兴的具有临床转化价值的治疗模式,已成为改善肿瘤患者预后的治疗手段。肿瘤免疫疗法是一种利用免疫系统来根除肿瘤细胞的疗法。其目的是通过抗体、细胞因子和转化的免疫细胞来刺激或动员身体的免疫系统,并增强肿瘤微环境的抗肿瘤免疫能力,以控制和杀死肿瘤。树突细胞(DCs)是能激活幼稚T细胞的最有效的抗原呈递细胞,它是连接免疫系统固有免疫反应和适应性免疫反应的桥梁,也是激活T细胞细胞介导的免疫反应的起始细胞,以及抗肿瘤疫苗是否能发挥作用的关键。然而,DCs疫苗的效果远未达到预期。
近年来随着化妆品的普及,化妆品深受广大年轻人的喜爱和关注,美白、抗衰老、抗皱等发展日益迅速它一直受到相关学科领域、特别是医学美容、化妆品科学、护肤保健及皮肤抗衰老等领域的高度重视和普遍关注。
众所周知,透明质酸是人类和其他哺乳动物结缔组织的基本成分之一。它是人体表皮、上皮和神经组织中广泛存在的一种物质。透明质酸赋予皮肤独特的抵抗力和保形性。缺乏透明质酸会导致皮肤衰弱,促进皱纹和瑕疵的形成。随着年龄的增长,人体组织中透明质酸的浓度趋于降低,从而削弱了其组织修复功能。随着逐渐衰老并反复暴露于紫外线下,表皮细胞减少了透明质酸的产生,并且衰老速度增加。同时,皮肤失去胶原蛋白,胶原蛋白是维持皮肤年轻、富有弹性的形式所必需的另一种天然物质。随着时间的流逝,表皮组织中透明质酸和胶原蛋白的丢失会导致褶皱和皱纹的形成。从化学角度来看,透明质酸(HA)是高分子化合物,可定义为具有未分支的多糖链的糖胺聚糖,是由数千个二糖单元缩合产生的,而二糖单元又由葡萄糖醛酸和M-乙酰氨基葡萄糖的残基形成。由于其在水中的高度溶解性,与透明质酸相对应的盐分子,即透明质酸盐,能够与许多水分子络合,从而达到高度水合的程度。此外,通过使用特定的交联剂和合适的操作工艺条件(温度、p H等),透明质酸分子的极长长度及其高度水合度使分子能够自我组织以形成网格状的结构(交联透明质酸),以创建分子骨架以维持表皮组织的形状和硬度。近年来,已开发出多种基于透明质酸的制剂用于美容应用,以填充皱纹、褶皱和疤痕,总体上改善脸部外观或填充嘴唇。因此,从工业研究的观点来看,这些制剂正在持续发展,从商业的观点来看是非常有吸引力的。人体天然含有的透明质酸是一种被表皮组织良好耐受的填充剂。由于这个原因,基于HA的制剂今天仍然被认为是市场上最好的表皮填充剂,因为它们没有皮肤过敏反应的风险。最初,基于透明质酸的第一种制剂以悬浮在凝胶中的颗粒或微球形式制备。然而,这些基于胶凝微球的填充剂具有随时间推移稳定性差的缺点,在注入皮肤后数月趋于化学降解。因此,随着时间的推移,需要频繁地重新注入填充剂,以维持修复和表皮生长的恒定。近来已经发现通过使用特定的交联剂使透明质酸经历合适的交联步骤的优点,因此基于交联透明质酸的填充剂被用于面部的美容治疗中。但是还是需要频繁注射,效果,稳定性和安全性一般。
如何提高DCs疫苗和其它类别的肿瘤疫苗的有效性,如何降低抗药性,及如何获得稳定、安全、效果更好的疫苗或载体,此外还解决了玻尿酸及同类产品不能原位激活胶原新生的问题。
发明内容
本发明的目的在于:为了解决上述技术问题,本发明提供一类自组装短肽,其在广谱疫苗及生物医学中的用途。
本发明为了实现上述目的具体采用以下技术方案:一类自组装短肽,包括两个短肽,其氨基酸序列分别:
RCC5:Arg Leu Asp Ile Lys Val Glu Phe Cys Cys,
RCC6:Arg Gln Asp Thr Lys Thr Glu Tyr Cys Cys。
本申请的技术方案中,提供了2种新的自组装短肽,它们都可以与PBS混合后自组装为可注入的水凝胶,即RCC5和RCC6,作为DCs疫苗加载细胞和抗原的输送载体,对改进疫苗的表征研究表明,添加生物材料增强了相关细胞的靶向能力和免疫力,表现出良好的抗肿瘤功能;不仅如此,本申请的自组装短肽水凝胶不仅在DC疫苗中表现出色,还具有在其它类别的肿瘤疫苗中有广阔的应用前景,比如:减毒灭活疫苗、灭活疫苗、类毒素疫苗、重组蛋白疫苗、核酸疫苗、细胞疫苗(如DC疫苗)、全肿瘤疫苗、肿瘤抗原肽疫苗、肿瘤个体化新疫苗、重组亚单位疫苗、合成肽疫苗、抗独特性疫苗、癌症干细胞疫苗等等,作为一种新型的生物材料,本申请的自组装短肽不仅能应用于肿瘤疫苗,也可作为病毒疫苗、药物等方面的载体;本发明以2种新的自组装短肽分别作为化妆品的制备、佐剂的一种新思路,从源头改变化妆品的含量,其降解产物为氨基酸,不仅能作为保湿剂、缓释剂、润滑剂、抗氧化剂、成膜剂、润肤剂,还可以对皮肤的修复具有辅助的作用,相当于皮肤的养料,其降解产物为氨基酸,具有良好的生物相容性;本申请的2种新的自组装短肽,结合其物理性能和微观下的纳米结构,分别与透明质酸联合使用,制备成为比单独使用透明质酸效果更好、更稳定、安全性更好、对降解抵抗力更强的新型“超级玻尿酸”系统,能原位激活胶原新生,从而不需要频繁的皮肤注射,这种在自组装短肽体系下架构的“超级玻尿酸”可作为一种新型的填充剂。
进一步的,每个短肽的中的氨基酸为L型,D型或DL型中的一种或多种。
进一步的,每个短肽的中的氨基酸均为L型。
进一步的,每个短肽的碳端酰胺化,
RCC5:Arg Leu Asp Ile Lys Val Glu Phe Cys Cys-CONH2,
RCC6:Arg Gln Asp Thr Lys Thr Glu Tyr Cys Cys-CONH2。
进一步的,每个短肽形成二级结构,二级结构包括α螺旋、β折叠、β蜷曲和无规则卷曲中的一种或多种。
一类自组装短肽在制备细胞或类器官三维培养纳米支架材料中的应用。
一类自组装短肽在制备软骨、韧带、子宫、心肌、血管、神经或皮肤的再生和损伤修复材料中的应用。皮肤损伤可以是紫外线灼伤、烧伤、烫伤、细菌感染糖尿病溃疡等由物理、化学、生物因素造成的闭合性和开放性创伤。
一类自组装短肽作为主要成分用于制备医美产品,化妆品或保健品。
一类自组装短肽作为疫苗佐剂的应用。
更进一步的,所述疫苗包括DC短肽疫苗。
更进一步的,作为疫苗佐剂的短肽的水混合物制备成短肽水凝胶,然后与DC混合,静置后形成的水凝胶用作疫苗。
以所述应用中短肽作为疫苗佐剂的短肽疫苗。
更进一步的,所述作为疫苗佐剂的短肽与DC物理混合得到疫苗。
一类自组装短肽的水凝胶。
所述的水凝胶的制备方法,包括如下步骤:
步骤1、将两个短肽之一加入水中配制成母液;
步骤2、向母液中加入离子或PBS制备成自组装短肽水凝胶。
进一步的,离子包括Na+、Mg2+、K+、Al3+、Ca2+、Zn2+、Fe3+、Fe2+、H+、NH4 +、Cl-、SO4 2-、NO3 -、CN-、CO3 2-、CH3COO-、HCO3 -、OH-、PO4 3-、HPO4 2-、H2PO4 -、HSO4 -中的一种或多种。
进一步的,制备成的自组装短肽水凝胶的浓度为1ppM及以上。
更进一步的,制备成的自组装短肽水凝胶的浓度为1-10mg/ml。
具体的:将10mg自组装短肽加入1ml水中配置母液,再加入3ml PBS制备为自组装短肽水凝胶,浓度为2.5mg/ml,根据以上方法制备水凝胶,浓度可调整为1-10mg/ml。
含一类自组装短肽的水凝胶在制备抗肿瘤靶向药物中的应用。
含一类自组装短肽的水凝胶作为敷料的应用。
含一类自组装短肽的水凝胶作为止血材料的应用。
进一步的,一种生物医学材料,它包含所述的含一类自组装短肽的水凝胶。
本申请的技术方案中,在盐离子环境下触发短肽自组装,其中盐离子包括且不限于:Na+、Mg2+、K+、Al3+、Ca2+、Zn2+、Fe3+、Fe2+、H+、NH4 +、Cl-、SO4 2-、NO3 -、CN-、CO3 2-、CH3COO-、HCO3 -、OH-、PO4 3-、HPO4 2-、H2PO4 -、HSO4 -等。
本申请的自组装短肽上调或下调相关组织的促修复相关细胞的相关生长因子,对创伤组织修复有直接、正向修复的作用。本申请的自组装短肽上调了皮肤组织增殖基因VEGF、EGF和TGF-β1的表达,激活FAK信号通路;自组装短肽上调了软骨细胞增殖基因R-Col、PDGF-BB、BMP-2的表达。激活SOX-9信号通路;自组装短肽上调了子宫细胞修复相关基因MMP-9、VEGF的表达。
提供一类新型的自组装短肽,具有提高机体免疫力,抗肿瘤、抗感染的作用,作为药物、疫苗的佐剂。
提供一类新型的自组装短肽,负载肿瘤疫苗、药物、细胞、抗体、蛋白质,在体内持续稳定释放,特别是负载肿瘤疫苗(包括且不限于减毒灭活疫苗、灭活疫苗、类毒素疫苗、重组蛋白疫苗、核酸疫苗、细胞疫苗(如DC疫苗)、全肿瘤疫苗、肿瘤抗原肽疫苗、肿瘤个体化新疫苗、重组亚单位疫苗、合成肽疫苗、抗独特性疫苗、癌症干细胞疫苗)对抗肿瘤的应用。
提供一类新型的自组装短肽,可作在化妆品、保健品中的成分之一,其中包含且不限于持久保湿剂、皮肤填充剂、缓释剂、抗菌剂、润滑剂、流变调节剂、抗氧化剂、防晒剂、成膜剂、润肤剂、稳定剂、缓冲剂、胶原蛋白促成剂、美白剂、透明质酸促进剂联合使用、皱纹改善剂、增稠剂、油性基剂、表面活性剂、醇类、、高分子-增粘-凝胶化剂、抗氧化剂、防腐剂、杀菌剂、螯合剂、pH调整剂-酸-碱、紫外线吸收剂、溶剂、止痒剂、消炎剂、止汗剂、清凉剂、抗组胺剂、收敛剂、刺激剂、生发用药剂、血循环促进剂、还原剂-氧化剂、高分子粉体、羟基酸、维生素类及其衍生物类、糖类及其衍生物类、有机酸类、酶类、核酸类、激素类、无机粉体类、香料、和色素等的用途。其中剂型可为水剂、乳剂、膏剂、胶冻状剂、锭状剂型或汽溶胶型剂型,乳液、精华液、面霜、面膜、冻干粉或洗面奶等。
本发明所述短肤在金属盐离子、细胞培养基等环境下,能进行自组装形成纳米纤维。
原子力显微镜、冷冻扫描电镜下所形成的纳米纤维网状结构可应用于化妆品领域,作为其中的缓释剂、抗菌剂、持久保湿剂、润滑剂、流变调节剂、抗氧化剂、成膜剂、润肤剂、稳定剂、缓冲剂等。
本发明所述自组装短肤形成的纳米纤维,细胞能在其上进行三维劲附、生长,所以此自组装短肤可应用到人、动、植物细胞三维培养中。
本发明所述自组装短肽所构建的三维培养的细胞,部分蛋白的表达在二维和三维中有显著不同。人皮肤成纤维细胞(HFF-1)三维培养中的重要蛋白逐渐增加,表明本发明短肽启动了皮肤细胞中的某些信号通路中的重要蛋白的上调或者下调。
本发明所述自组装短肽形成的纳米纤维支架,支持软骨细胞能在其上进行黏附、生长;所以,自组装短肽能使用于骨组织中细胞方面的三维培养,并能有效修复软骨组织。
本发明所述自组装短形成的纳米三维支架,可支持子宫细胞有序生长,有修复子宫创伤的功能。
本发明所述自组装短肽形成的纳米纤维支架调控韧带细胞的生长因子、胶原蛋白,能有效修复韧带损伤。
本发明所述自组装短肽形成的纤维网状结构负载了模式抗原OVA并持续释放。
本发明所述自组装短肽形成的纤维网状结构负载DC疫苗,具有显著的抗肿瘤效果。
本发明所述的自组装短肽具有治疗紫外线灼伤的效果,有效去除疤痕。
本发明的有益效果如下:
1、提供了一类新型自组装短肤,增加自组装短肤类型。
2、提供了一类新型自组装短肽这些材料能够形成稳定的纳米纤维,该纳米纤维能够应用于细胞、类器官三维培养,模拟细胞内的生存环境,提供细胞体外三维微环境。
3、提供一类新型生物功能的自组装材料,该类材料能够目的性的修复损伤组织,减少炎症的发生、增加新生肉芽组织的生成、诱导修复干细胞的迁移分化和组织重建。
4、提供一类新型具有载药功能的自组装材料,该材料能够负载肿瘤疫苗,具有显著的抗肿瘤作用,为疫苗佐剂开辟了新路径。
5、提供一类新型疫苗佐剂功能的自组装材料,能够增强体内免疫能力,可用于保健品、药品的制备。
6、提供一类新型应用于化妆品领域的自组装材料,该材料可作为化妆品中的缓释剂、抗菌剂、持久保湿剂、润滑剂、流变调节剂、抗氧化剂、成膜剂、润肤剂、稳定剂、缓冲剂,为化妆品原料提供一种新型生物相容性良好、降解产物有益的水凝胶。
本申请中,术语“三维培养”是指将具有三维结构不同材料的载体与各种不同种类的细胞在体外共同培养,使细胞能够在载体的三维立体空间结构中迁移、生长,构成三维的细胞-载体复合物,改变或减少细胞在培养的过程贴壁的特性,使细胞在空间上获得更多的生存空间,减少细胞接触抑制,细胞附着与自组装短肽上,在显微镜下一般呈现为圆形。
本申请中,术语“类器官”是指类器官属于三维(3D)细胞培养物,包含其代表器官的一些关键特性。此类体外培养系统包括一个自我更新干细胞群,可分化为多个器官器官特异性的细胞类型,与对应的器官拥有类似的空间组织并能够重现对应器官的部分功能,从而提供一个高度生理相关系统。含有成体干细胞的组织样本、单一成体干细胞或者通过多能干细胞的定向诱导分化都能够产生类器官其中类器官的种类包含且不限于:扁桃体、肾上腺、胆管、膀胱、骨、骨髓、脑、胸、子宫颈、结肠直肠、食管、眼睛、头部和颈部、肾、肝、肺、淋巴结、神经系统、卵巢、胰腺、前列腺、皮肤、软组织、胃、睾丸、胸腺、甲状腺、子宫等其中至少一种。包含的肿瘤类器官主要有且不限于:结肠癌、肾上腺癌、软组织肉瘤、淋巴瘤、神经癌、脑癌、皮肤癌、胆管癌、膀胱癌、骨癌、乳腺癌、宫颈癌、乳腺癌、骨癌眼内黑色素瘤、视网膜母细胞瘤、输卵管癌、胆囊癌、胃癌、软组织肉瘤、中枢神经系统细菌细胞肿瘤(脑癌)、儿童颅外细菌细胞肿瘤、外角细菌细胞肿瘤、卵巢细菌细胞肿瘤、睾丸癌、心脏肿瘤、肝细胞癌、眼内黑色素瘤、胰岛细胞肿瘤,胰腺神经内分泌肿瘤、肾细胞癌症、喉癌、白血病唇癌和口腔癌、肝癌、肺癌(非小细胞、小细胞、肺泡肿和气管支气管肿瘤)、淋巴瘤、男性乳腺癌、黑色素瘤、皮肤癌、间皮瘤、转移性鳞状颈部癌症(头颈部癌症)、口腔癌、多发性骨髓瘤/等离子细胞肿瘤、骨髓增殖性肿瘤、旁鼻窦癌、神经母细胞瘤、口腔癌、唇癌、咽癌、胰腺癌、胰岛细胞肿瘤、鼻腔癌、甲状旁腺癌、咽癌、垂体肿瘤、原发性腹膜癌、前列腺癌、直肠癌、唾液腺癌、皮肤癌、T细胞淋巴瘤睾丸癌、鼻咽癌、食道癌、低咽癌、胸腺瘤、甲状腺癌、肾骨盆癌、尿道癌的过渡性细胞癌、尿道癌、子宫癌、子宫内膜癌、阴道癌、血管肿瘤等的癌症所表现的肿瘤的其中至少一种。
本申请中,术语“抗肿瘤”,其中“肿瘤”所包含的器官有且不限于:结肠癌、肾上腺、胆管、膀胱、骨、骨髓、脑、胸、子宫颈、结肠直肠、食管、眼睛、头部和颈部、肾、肝、肺、淋巴结、神经系统、卵巢、胰腺、胸膜、前列腺、皮肤、软组织、胃、睾丸、胸腺、甲状腺、子宫等其中至少一种。
本申请中,术语“抗肿瘤”,其中“肿瘤”通常理解为包含的类型有且不限于:结肠癌、肾上腺癌、软组织肉瘤、淋巴瘤、神经癌、脑癌、皮肤癌、胆管癌、膀胱癌、骨癌、乳腺癌、宫颈癌、乳腺癌、骨癌眼内黑色素瘤、视网膜母细胞瘤、输卵管癌、胆囊癌、胃癌、软组织肉瘤、中枢神经系统细菌细胞肿瘤(脑癌)、儿童颅外细菌细胞肿瘤、外角细菌细胞肿瘤、卵巢细菌细胞肿瘤、睾丸癌、心脏肿瘤、肝细胞癌、眼内黑色素瘤、胰岛细胞肿瘤,胰腺神经内分泌肿瘤、肾细胞癌症、喉癌、白血病唇癌和口腔癌、肝癌、肺癌(非小细胞、小细胞、肺泡肿和气管支气管肿瘤)、淋巴瘤、男性乳腺癌、黑色素瘤、皮肤癌、间皮瘤、转移性鳞状颈部癌症(头颈部癌症)、口腔癌、多发性骨髓瘤/等离子细胞肿瘤、骨髓增殖性肿瘤、旁鼻窦癌、神经母细胞瘤、口腔癌、唇癌、咽癌、胰腺癌、胰岛细胞肿瘤、鼻腔癌、甲状旁腺癌、咽癌、垂体肿瘤、原发性腹膜癌、前列腺癌、直肠癌、唾液腺癌、皮肤癌、T细胞淋巴瘤睾丸癌、鼻咽癌、食道癌、低咽癌、胸腺瘤、甲状腺癌、肾骨盆癌、尿道癌的过渡性细胞癌、尿道癌、子宫癌、子宫内膜癌、阴道癌、血管肿瘤等的癌症所表现的肿瘤的其中至少一种。
本申请中,术语“抗原”和其语法等同的表述(例如,“抗原性”)指可以由特异性体液免疫或细胞免疫的产物(如抗体分子或T细胞受体)特异性结合的化合物。抗原可以是任何类型的分子,例如包括半抗原、简单的中间代谢物、糖(例如,低聚糖)、脂质和激素以及大分子如复杂糖(例如,多糖)、磷脂和蛋白质。常见类别的抗原包括但不限于病毒性抗原、细菌性抗原、真菌性抗原、原虫和其他寄生虫性抗原、肿瘤抗原、参与自身免疫疾病、过敏和移植排异的抗原、毒素和其他杂项抗原其中至少一种。
本发明能够在通常的基剂例如且不限于水、多元醇、凡士林、石蜡、植物油、硅油等中溶解、分散或混合使用。另外,也能够根据需要并用各种添加剂。作为能够使用的添加剂,只要是用于得到所期望的剂型所通常使用的添加剂即可,没有特别限制,可以适当选择赋型剂、着色剂、增粘剂、结合剂、崩解剂、分散剂、稳定化剂、凝胶化剂、抗氧化剂、表面活性剂、保存剂、保湿剂和p H调整剂等公知的添加剂来使用。或者,作为发挥所期望的效果的有效成分,可以作为医药品或化妆品的一个成分配合给药。医药品的情况下,能够在片剂、胶囊剂、颗粒剂、散剂、液剂、悬浊剂等口服剂;皮外用剂、贴剂、滴眼剂、滴鼻剂、口腔剂、栓剂等外用剂;点滴剂、注射剂等非口服剂中配合。化妆品的情况下,能够在化妆水、护肤液、凝胶、乳液、美容液、霜、面膜、洗面奶、身体清洁剂等皮肤化妆品;粉底、口红、唇膏、睫毛液等彩妆化妆品;洗发水、润丝精、护发素、头发喷雾、发蜡、造型乳液、染色乳液、美发剂、生发剂等头发化妆品中配合。本发明的美白剂、胶原蛋白产生促进剂、透明质酸产生促进剂、细胞内活性氧清除剂、激缓解剂和皱纹改善剂中,还能够并用具有同样的生理活性的成分、血循环促进剂至少其中一种等。通过并用这样的成分,能够协同地发挥本发明的效果。
本发明能与美白祛斑剂共用从而起到美白的效果。能够并用的美白祛斑剂,具体而言,比如且不限于:β-熊果苷、α-熊果苷等氢醌配糖体及其酯类熊果苷;熊果苷、硫辛酸、白藜芦醇、曲酸、曲酸二棕榈酸酯、羟基乙酸、苹果酸、酒石酸、柠檬酸、光甘草定、根皮素、烟酰胺、凝血酸、阿魏酸乙基己酯、十一碳烯酰基苯丙酸氨、抗坏血酸、抗坏血酸磷酸酯钠盐和抗坏血酸磷酸酯镁盐等抗坏血酸磷酸酯盐;抗坏血酸单硬脂酸酯、抗坏血酸单棕榈酸酯、抗坏血酸二棕榈酸酯、抗坏血酸四异棕榈酸酯等抗坏血酸脂肪酸酯类;3-O-乙基抗坏血酸、2-O-乙基抗坏血酸、鲸蜡基抗坏血酸酯、甘油抗坏血酸、己基甘油基抗坏血酸等抗坏血酸醚类;抗坏血酸-2-葡糖苷等抗坏血酸葡糖苷及其脂肪酸酯类;抗坏血酸硫酸酯、磷酸抗坏血酸生育酚酯等抗坏血酸衍生物;氨甲环酸、氨甲环酸鲸蜡酯、氨甲环酸酰胺等氨甲环酸衍生物;褐藻多酚、姜黄素、花青素、原花青素、儿茶素、鞣花酸、苹果多酚等多酚类;烟酰胺、曲酸、鞣花酸、4-甲氧基水杨酸、亚油酸、阿魏酸、胎盘提取物、谷胱甘肽、谷维素、丁基间苯二酚、光甘草定、葡糖基神经酰胺、麦角硫因、叶绿醇、桧木醇、半胱氨酸、龙胆酸、甲戊二羟酸、没食子酸、鸟氨酸、葡糖基芸香苷;以及母菊、甘草、虎耳草、桑、当归、地榆、芍药、黄芩、金丝桃、虎杖、茶、紫苏、桃叶、雪见草、日本百合叶、芦荟、蓍(西洋蓍草)、贯叶泽兰、葛根、山金车、熊果、细辛、玫瑰果、芦笋、桔梗、匐枝百里香、七叶树、金银花、艾蒿、杨梅、木莓、杨桃叶、洋蓟叶、杏核、荔枝核、西兰花芽、金鸡纳树、聚合草、杏仁、栀子、苦参、云杉、麦门冬、粉花绣线菊、针叶樱桃、丁香、蔷薇、薏仁、欧洲椴、芝麻、当药、地黄、辣薄荷、龙胆、杨桃、菜心、生姜、蜀葵、莨菪(天仙子)、迷迭香、葡萄籽、木槿、月见草籽、猕猴桃籽等具有美白作用的植物提取物至少其中一种。
本发明能作为胶原蛋白促进剂与胶原蛋白促进剂联用,具体而言如且不限于:抗坏血酸、抗坏血酸磷酸酯钠盐和抗坏血酸磷酸酯镁盐等抗坏血酸磷酸酯盐;抗坏血酸单硬脂酸酯、抗坏血酸单棕榈酸酯、抗坏血酸二棕榈酸酯、抗坏血酸四异棕榈酸酯等抗坏血酸脂肪酸酯类;3-O-乙基抗坏血酸、2-O-乙基抗坏血酸、鲸蜡基抗坏血酸、甘油抗坏血酸、己基甘油基抗坏血酸12等抗坏血酸醚类;抗坏血酸-2-葡糖苷等抗坏血酸葡糖苷及其脂肪酸酯类;抗坏血酸硫酸酯、磷酸抗坏血酸生育酚酯等抗坏血酸衍生物;视黄醇、视黄醇乙酸酯、视黄醇棕榈酸酯、氢化视黄醇等维生素A类;烟酰胺、谷胱甘肽、半胱氨酸、藏花酸、丝胶蛋白、牻牛儿醇(香叶醇)、甘油葡糖苷、乳铁蛋白、原花青素、泛酸、泛醇、大豆皂苷、白藜芦醇、异黄酮、辅酶Q10、硫酸软骨素、乙酰葡糖胺、甘油磷脂酰胆碱、水解透明质酸、胶原蛋白肽、壳基质水解物、5'一磷酸腺苷、脯氨酸、甘氨酸、精氨酸、天冬氨酸、丙氨酸;以及朱槿、甘草叶、罗马甘菊、甘茶、山楂、凤仙花、莲叶、葛根、绣球菌、小球藻、维氏熊竹、牛蒡、山槐根、菜心、枇杷叶、紫苏、豌豆、桑叶、百里香、云杉、踊子草、鸭跖草、柏子仁、决明子、瓜蒌仁、地榆、苦参、柿、芍药、紫苏、陈皮、锦葵、生姜、母菊、草莓籽、当药、黄豆、小麦、人参、薏仁、防葵、小豆蔻、迷迭香、洋苏(鼠尾草)等具有胶原蛋白产生促进作用的植物提取物至少其中一种。
本发明可作为抗粉刺剂的制备,联合制备的原料有且不限于:核黄素、吡哆素、吡哆醇而棕榈酸酯、壬二酸、黄芩素等至少其中一种。
本发明可促透明质酸生成组装与透明质酸联合使用作为皮肤填充剂、抗皱,具体而言可联合的促生成剂如且不限于:透明质酸、胶原、抗坏血酸、抗坏血酸磷酸酯钠盐和抗坏血酸磷酸酯镁盐等抗坏血酸磷酸酯盐;抗坏血酸单硬脂酸酯、抗坏血酸单棕榈酸酯、抗坏血酸二棕榈酸酯、抗坏血酸四异棕榈酸酯等抗坏血酸脂肪酸酯类;3-O-乙基抗坏血酸、2-O-乙基抗坏血酸、鲸蜡基抗坏血酸、甘油抗坏血酸、己基甘油抗坏血酸等抗坏血酸醚类;抗坏血酸-2-葡糖苷等抗坏血酸葡糖苷及其脂肪酸酯类;抗坏血酸硫酸酯、磷酸抗坏血酸生育酚酯等抗坏血酸衍生物;视黄醇、视黄醇乙酸酯、视黄醇棕榈酸酯、氢化视黄醇等维生素A类;烟酰胺、胡萝卜素、生育酚、生育三烯酚、硫酸软骨素、乙酰葡糖胺、甘油磷脂酰胆碱、甘油葡糖苷、水解透明质酸、胶原蛋白肽、谷甾醇、肌肽、肌酸、植酸、N-甲基丝氨酸、3-甲基环十五酮、皂苷、染料木黄酮、大豆黄酮、叶绿醇;以及墨角兰、辣薄荷、苹果薄荷、紫苏、白苏、柠檬、蒙桑、面包果、楮树、构树、无花果、石莼、欧锦葵、待雪草、黄柏、鱼腥草、蓍、杏仁、山楂、亚麻籽、栀子、荨麻、草莓籽、杨桃、百香果、海葡萄、藏红花、山茶、南瓜、丝瓜、芦笋、绞股蓝、苦参、陈皮、芍药、柿、丹参、积雪草、普洱茶、舞茸、莼菜、木贼、木梨、母菊等具有透明质酸产生促进作用的植物提取物至少其中一种。
本发明可作为细胞抗氧化剂,作为能够并用的细胞内活性氧清除剂,如且不限于:抗坏血酸、抗坏血酸磷酸酯钠盐和抗坏血酸磷酸酯镁盐等抗坏血酸磷酸酯盐;抗坏血酸单硬脂酸酯、抗坏血酸单棕榈酸酯、抗坏血酸二棕榈酸酯、抗坏血酸四异棕榈酸酯等抗坏血酸脂肪酸酯类;3-O-乙基抗坏血酸、2-O-乙基抗坏血酸、鲸蜡基抗坏血酸、甘油抗坏血酸、己基甘油抗坏血酸等抗坏血酸醚类;抗坏血酸-2-葡糖苷等抗坏血酸葡糖苷及其脂肪酸酯类;抗坏血酸硫酸酯、磷酸抗坏血酸生育酚酯等抗坏血酸衍生物;视黄醇、视黄醇乙酸酯、视黄醇棕榈酸酯、氢化视黄醇等维生素A类;生育酚、生育酚乙酸酯、生育三烯酚等维生素E类;胡萝卜素、番茄红素、虾青素、叶黄素等类胡萝卜素类;褐藻多酚、姜黄素、花青素、原花青素、儿茶素、鞣花酸、苹果多酚等多酚类;辅酶Q10、硫辛酸、乳铁蛋白、芝麻素、木脂素、绿原酸、芸香苷;以及黄芩、迷迭香、枇杷、啤酒花、野蔷薇、博士茶、金缕梅、百里香、龙胆、牡丹、壳基质、地榆、菊花、胡萝卜、鼠尾草(一串红)、连翘、桑葚、胡椒薄荷、火棘、柠檬、橄榄叶、金丝桃、葡萄叶、明日叶、儿茶钩藤、银杏、荨麻、茵陈蒿、锦葵、乌龙茶、玫瑰果、香茶菜、艾蒿、母菊、甘草、金银花、苦参、中日老鹳草、红茶、聚合草、肥皂草、洋苏、山楂、华东椴、芍药、鱼腥草、白桦、常春藤、欧刺柏、积雪草、茶、丁香、陈皮、甜茶、金盏花、接骨木、杨桃、菜心、忍冬、辣薄荷、蜜蜂花、桃叶、矢车菊、虎耳草、柚子、桑等具有细胞内活性氧清除作用的植物提取物至少其中一种。
本发明能与以下物质联用作为清洁剂的制备且不限于:月桂醇硫酸酯钠、月桂醇硫酸酯钠铵、月桂醇硫酸酯TEA盐、月桂醇聚醚硫酸酯钠、月桂醇聚醚硫酸酯铵、月桂醇聚醚硫酸酯TEA盐、十二烷基苯磺酸钠、C12~14烯烃磺酸钠、2-磺基月桂酸甲酯钠、月桂酰肌氨酸钠、椰油酰甘氨酸钾、甲基椰油酰胺基牛磺酸钠、月桂酸钾、硬脂酸钾、月桂醇聚醚-4羧酸钠、PEG-7橄榄油羧酸钠、脂肪醇聚氧乙烯磷酸单酯盐、磺基琥珀酸酯盐、酰胺基磺基琥珀酸酯钠、月桂基甜菜碱、月桂基羟基磺基甜菜碱、月桂酰胺丙基甜菜碱、月桂酰两性基乙酸钠、月桂酰两性基二乙酸二钠、辛基葡糖苷、葵基葡糖苷、肉豆蔻基葡糖苷、月桂基葡糖苷、椰油基葡糖苷、C12~18葡糖苷、C12~20葡糖苷、椰油酰胺、椰油酰胺MEA、硬脂胺氧化物、椰油胺氧化物、月桂基胺氧化物、肉豆蔻胺氧化物、二月桂基胺氧化物、PEG-7甘油椰油酸酯、PEG-30甘油椰油酸酯、PEG-80甘油椰油酸酯、PEG-6辛酸甘油酯类、鳄梨油PEG-11酯类、橄榄油PEG-8酯类、霍霍巴油PEG-150酯类。
本发明能作为皱纹改善剂且并用皱纹改善剂,具体而言,如且不限于:透明质酸、胶原、三氟化异丙基氧代丙基氨基羰基吡咯烷羰基甲基丙基氨基羰基苯甲酰基氨基乙酸Na、视黄醇、烟酰胺、氨甲环酸、3-O-乙基抗坏血酸等。作为血循环促进剂,能够例示辣椒素、烟酸、烟酰胺、生育酚烟酸酯、生育酚、生育酚乙酸酯、谷维素、dl-樟脑、人参提取物、银杏叶提取物、当归提取物、白桦提取物、桂皮提取物、牡丹提取物、藏红花提取物、小麦胚芽提取物、生姜根茎提取物、辣椒提取物、胡萝卜提取物、洋苏提取物、艾蒿提取物、明日叶提取物等至少其中一种。
本发明能作为增稠剂且并用增稠剂,具体而言如且不限于:透明质酸酸、胶原、聚丙烯酸、聚丙烯酰胺、聚乙烯氧化聚合物和乙烯氧化物-丙烯氧化共聚物、聚乙烯醇、聚乙二醇、聚乙烯吡咯烷酮,水溶性增稠剂:透明质酸、汉生胶、淀粉、羧甲基纤维素、羟乙基纤维素、PEG-120甲基葡糖二油酯酯、PEG-120甲基葡糖二油酸酯、卡波、聚乙烯醇、聚乙烯二醇、硅酸镁铝、二氧化硅、膨润土、改性气相二氧化硅、司拉氯铵水辉石、微晶纤维素、淀粉、黄原胶、小核菌胶、瓜儿豆胶、果胶、褐藻酸钠、鹿角菜胶、结冷胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠、羟乙基纤维素、卡波姆、聚丙烯酸钠、丙烯酸类、丙烯酸共聚物、丙烯酸类、丙烯先二甲基牛黄铵、丙烯酸羟乙酯、聚丙烯酸酯交联聚合物-6、聚乙二醇、PEG-150二硬脂酸酯、硅酸镁锂至少其中一种。
本发明能与香料制备成为化妆品,具体而言如且不限于:精油、浓缩精油、除单萜、精馏精油、浸膏、净油、超临界流体萃取物、浸剂、提取液、花香脂、芳香水、香膏、树脂、油树脂、香树脂。具体有:花香天然香料、乙酸芳樟酯、薰衣草醇、芳樟醇、松油醇、香叶醇、橙花醇、樟脑、龙脑、乙酸大松油脂、乙酸薰衣草酯、壬醛、蒜烯、月桂烯、罗勒烯、松油烯、石竹烯、姜黄烯、檀香烯、香柠檬烯、杜松烯、金合欢烯、洋甘菊薁、异丁酯、天蓝烃左旋、香茅醇、香叶醇、橙花醇、芳樟醇、苯乙醇、甲基丁香酚,以及它们的酯类、洋甘菊精油、德国洋甘菊精油、罗马洋甘菊提取物及罗马洋甘菊精油、α-紫罗兰酮、桂醛、柠檬醛、紫罗兰酮、葛缕酮、丁香酚、玫瑰醚、橙花醚、乙酸香茅酯、乙酸橙花酯、大花茉莉净油主要含有乙酸苄酯、苯甲于茉莉等花香型高级香水、香皂、化妆品的香精中,并广泛用于各种类型的香精配方。酸苄酯、苯甲酸叶醇酯、茉莉内酯、茉莉酮酸甲酯、顺式茉莉酮、芳樟醇、苯甲醇、吲哚、在茉莉香精中,有笼罩全局的作用,而用于丁香酚、植醇、异植醇等。小花荣莉净油主要其他花香香精中也有添鲜增清的效能。小花茉莉可用于窨茶,少量用于食品加香。含有乙酸苄酯、苯甲酸苄酯、苯甲酸叶醇酯、苯莉酮酸甲酯、亚麻酸甲酯、棕榈酸甲酯、茉莉内酯、芳樟醇、叶醇、苄醇、橙花叔醇、茉莉酮、丁香酚、吲哚、金合欢烯、白兰花油主要成分为松油醇、橙花醇、丁香酚甲醚、异丁香酚甲醚、月桂烯、柠檬烯、石竹烯、芳樟醇、橙花叔醇、丁香酚甲醚、桉叶油素、石竹烯、β-罗勒烯、1-茨烯、α-旅烯、二萜烯、癸醛、芳樟醇、乙酸芳樟酯、苯乙醇、α-松油醇、橙花醇和乙酸橙花酯、香叶醇、茉莉酮、橙花叔醇、金合欢醇,以酯化形式存在的醋酸、苯乙酸、苯甲酸、十六酸(微量)、苯酚(微量)、邻氮基苯甲酸甲酯、吲哚、石蜡、芳樟醇、香叶醇、橙花醇、松油醇、化妆品等用香精。卡南加精油价格上比依兰精油苯甲醇、苯乙醇、叶醇、丁香酚、对甲酚、对甲酚醚、黄樟油素、异黄樟油素、甲基庚、烯酮、戊酸、苯甲酸、水杨酸、乙酸香叶酯、水杨酸甲酯、苯甲酸苄酯、邻氨基苯甲酸甲酯、蒸烯、金合欢烯、石竹烯异丁香酚甲醚、苯甲酸苄酯、金合欢用及禁用规定,但香叶醇、异丁香酚甲醚、烯、香叶烯、丁香酚、金合欢醇、芳樟醇、松丁香酚、苯甲酸苄酯、金合欢烯是致敏性物质,使用时注意在不同类型产品中的最大使油醇、苯乙醇、橙花醇、香叶醇、叶醇、苯乙醇、水杨酸甲酯、水杨酸异戊酯、乙酸苄酯、乙酸苯乙酯、苯甲酸甲酯、邻氨基苯甲中的至少一种。
本发明可作为持久保湿剂,并于保湿剂联用,具体如且不限于:水、透明质酸、胶原、赤藓醇、木糖醇、山梨醇、甘露醇、透明质酸、PCA纳、泛醇、乳酸、乳酸钠、聚谷氨酸、甜菜碱、芦荟提取物、甘油、1,2-丙二醇、双甘油、1,3-丙二醇、双丙甘醇、1,3-丁二醇、聚甘油-10、羟乙基脲、尿素、牛油树脂、鳄梨油、橄榄油等、相对分子质量线性脂肪酸酯类、如花生醇丙酸酯、鲸蜡醇棕榈酸酯、乳酸钾、烷基聚甲基硅氧烷、成膜聚合物、丙三醇,俗称甘油(Glycerin)、丁二醇(Butyleneglyol)、聚乙二醇(Polyethyleneglycol,PEG)、丙二醇(Propyleneglycol)、已二醇(2-Methyl-2,4-pentanediol)、木糖醇(Xylitol)、聚丙二醇(Polypropyleneglycol,PPG)、山梨糖醇(Sorbitol)、PCA钠、尿素、羟乙基尿素、甲基葡糖聚醚、甲壳质衍生物、海藻糖、银耳多糖、透明质酸钠、糖类同分异构体泛醇、神经酰胺、天然植物提取物等其中至少一种。
本发明能与防腐剂联合制备为新型防腐剂且不限于:双氧水、甲醛、甲醛苄醇半缩醛、咪唑烷基脲、双咪唑烷基脲、DMDM乙内酰脲、戊二醛、4-羟基苯甲酸酯类、4-羟基苯甲酸酯类、碘丙炔醇丁基氨甲酸酯IPBC、苯扎氯铵、卞索氯铵、西曲氯铵、硬脂基三甲基氯化铵、氯乙定、海克替叮、己脒定二磺酸盐、聚氨丙基双胍、甲酸、丙酸、山梨酸、苯甲酸、水杨酸、脱氢乙酸、卞氯酚、p-氯-m-甲酚、氯二甲酚、本基苯酚、溴氯芬、o-散花烃-5-醇、苯甲醇、苯氧乙醇、苯氧异丙醇、2-溴-硝基-1,3-丙二醇、二氯苯甲醇、三氯叔丁醇、氯苯甘醚、三氯生、5-溴-5-硝基-1,3-二噁烷、硫柳汞、沉积二氧化钛上的氯化银、三氯卡班、甲基异噻唑啉酮、N-羟甲基甘氨酸钠等其中至少一种。
本发明可作为持久抗菌剂,并于抗菌剂联用,具体如且不限于:1,2-戊二醇、1,2-己二醇、辛甘醇、1,2-葵二醇、乙基己基甘油、p-茴香酸、乙酰丙酸、阿魏酸、地衣酸、辛酰羟肟酸、甘油辛酸酯、甘油癸酸酯、山梨坦辛酸酯、桃柁酚、麝香草酚、苯乙醇、茶树精油香草醛或乙基香草醛类化合物、酰基苯胺类、咪唑类、噻唑类、异噻唑酮衍生物、季铵盐类、双呱类、酚类、甲壳素、芥末、蓖麻油、山葵、四甲基秋兰姆二硫化物、氯己定、吡啶硫酮锌、甘宝素、谷甾醇、松香酸、松萝酸、茜草素、柠檬烯黄芪黄素、愈创木薁、蜂胶等其中至少一种。
本发明可抗衰老且与抗衰老剂联合应用作为抗衰老剂的制备,具体如且不限于:生育酚(维生素E)、维生素E磷酸酯二钠、生育酚乙酸酯、富勒烯、泛醌、葡萄籽提取物、茶提取物、视黄醇、视黄醇乙酸酯、银杏提取物、植物甾醇类、白藜芦醇、神经酰胺、人参根提取物、葛根素、大豆异黄酮等其中至少一种。
本发明可作为抗水成膜剂且能与成膜剂联用作为成膜剂的制备,具体如且不限于:乙基纤维素、乙烯基吡咯烷酮烯烃类共聚物等其中至少一种。
本发明可作为防晒剂,且与防晒剂联合应用,具体如且不限于:二氧化钛、氧化锌、PEG-25对氨基苯甲酸、二甲基PABA乙基己酯、水杨酸乙基己酯、胡莫柳酯、甲氧基肉桂酸乙基己酯、对甲基肉桂酸异戊酯、3-亚苄基樟脑、4-甲基苄亚基樟脑、亚编辑樟脑磺酸、樟脑苯扎氨甲基硫酸盐、聚丙烯酰胺甲基亚苄基樟脑、对苯二亚甲基二樟脑磺酸、双-乙基己氧苯酚甲基氨基三嗪、二乙基己基丁酰胺基三嗪酮、乙基己基三嗪酮、亚甲基双-苯并三唑基四甲基丁基酚、苯基二苯并咪唑四磺酸酯二钠、聚硅氧烷-15、甲酚曲唑三硅氧烷、奥克立林、芸香苷等其中至少一种。
在含有本发明的复合物的化妆品或皮肤外用剂中,可以根据需要,以不损及本发明的效果的程度含有通常在化妆品中配合的添加成分,例如且不限于油性基剂、表面活性剂、醇类、保湿剂、增粘剂、抗氧化剂、防腐剂、杀菌剂、螯合剂、pH调整剂、紫外线吸收剂、美白剂、溶剂、止痒剂、消炎剂、止汗剂、清凉剂、抗组胺剂、收敛剂、刺激剂、生发用药剂、血循环促进剂、还原剂-氧化剂、高分子粉体、羟基酸、维生素类及其衍生物类、糖类及其衍生物类、有机酸类、酶类、核酸类、激素类、无机粉体类、香料、和色素等。
附图说明
图1是本发明自组装短肽RCC5和RCC6的圆二色谱图;
图2是本发明自组装短肽RCC5和RCC6的透射电镜图;
图3是本发明自组装短肽RCC5和RCC6的原子力显微镜图;
图4是本发明自组装短肽RCC5和RCC6的冷冻扫描电镜图;
图5是本发明自组装短肽RCC5和RCC6的刚果红染色图;
图6是本发明自组装短肽RCC5和RCC6的苯胺蓝染色图;
图7是本发明自组装短肽RCC5和RCC6对成纤维细胞HFF-1第七天的三维培养中的AO/EB染色图;
图8是本发明自组装短肽RCC5和RCC6对兔韧带组织损伤修复的HE染色;
图9是本发明自组装短肽RCC5和RCC6对抗原的控释图;
图10是本发明自组装短肽RCC5和RCC6对C57小鼠的抗肿瘤作用淋巴细胞的流式分选图;
图11是本发明自组装短肽RCC5、RCC6对C57的抗肿瘤作用小鼠图;
图12是本发明自组装短肽RCC5、RCC6对C57的抗肿瘤作用小鼠肿瘤图;
图13是本发明自组装短肽RCC5和RCC6对C57小鼠的抗肿瘤作用安全性的HE染色图;
图14是本发明自组装短肽RCC5和RCC6对肿瘤细胞因子的调控作用;
图15是本发明自组装段太RCC5和RCC6对紫外灼伤的治疗作用的SD大鼠图;
图16是本发明自组装短肽RCC5组装前和组装后与透明质酸酸混合后的力学测试图;
图17是本发明自组装短肽RCC6组装前和组装后与透明质酸酸混合后的力学测试图;
图18是本发明本发明自组装段太RCC5和RCC6小鼠肝脏的止血图;
图19是本发明短肽RCC5和RCC6对皮肤创伤修复的HE染色图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此,以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。基于本发明中的实施例,本领域普通技术人员在没有作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
本实施例提供由L-氨基酸构成的自组装短肽RCC5、RCC6的制备
材料:
按照氨基酸序列备好原料如:Fmoc-L-Arg-OH(9-芴甲氧羰酰基-L-精氨酸-γ-叔丁氧羰酰基)、Fmoc-L-Lys-OH(9-芴甲氧羰酰基-L-赖氨酸-ε-叔丁氧羰酰基)、Fmoc-L-Asp(OtBu)-OH(芴甲氧羰酰基-L-天冬氨酸-ε-叔丁氧羰酰基)、Fmoc-L-Val-OH(9-芴甲氧羰酰基-L-缬氨酸)、Fmoc-L-Phe-OH(9-芴甲氧羰酰基-L-苯丙氨酸-γ-叔丁氧羰酰基)、Fmoc-L-Ile-OH(9-芴甲氧羰酰基-L-异亮氨酸-γ-叔丁氧羰酰基)、Fmoc-L-Gln-OH(9-芴甲氧羰酰基-L-谷酰氨酸-γ-叔丁氧羰酰基)、Fmoc-L-Thr-OH(9-芴甲氧羰酰基-L-苏氨酸-γ-叔丁氧羰酰基)、Fmoc-L-Tyr-OH(9-芴甲氧羰酰基-L-酪氨酸-γ-叔丁氧羰酰基)、Fmoc-L-Cys-OH(9-芴甲氧羰酰基-L-半胱氨酸)、Fmoc-L-Glu(OtBu)-OH(9-芴甲氧羰酰基-L-谷氨酸-ε-叔丁氧羰酰基)、Fmoc-L-Leu-OH(9-芴甲氧羰酰基-L-亮氨酸)、TBTU(O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯)、HBTU(O-苯并三唑-1-基-N、N、N、N-四甲基尿六氟磷酸脂)和HOBT(1-羟基苯并三氮唑)、哌啶、六氢吡啶、醋酸酐、二氯甲烷;溶剂:DMF(N、N-二甲基甲酰胺)、TFA(三氟乙酸)、ACN(乙腈)、冰乙醚、NMM(N-甲基吗啡啉)。
采用Fmoc(芴甲氧羰基)保护的固相合成法,以RCC5为列,工艺步骤简述如下:
(1)称取0.5mmol/g Rink amide resin 20g于肽合成器中,用200mlDCM(二氯甲烷)浸泡树脂30分钟后,再用400inIDMF分三次清洗树脂,每次清洗时间为3min,抽滤干洗涤液。经过10-20分钟用100ml20%哌啶/DMF震荡反应30分,反应结束后,抽滤干洗涤液,用400mIDMF清洗树脂5次,每次清洗3min,洗完后取少许树脂做前三酮检査,树脂呈阳性,然后向反应器中加入原料:
Fomc-L-Arg(Boc)-OH 15.455g
HBTU 15.l6g
HOBT 8.91g
NMM 7.26ml
DMF 264ml
加入后震荡反应30分钟,用400mlDMF清洗5次,每次清洗3min,取少许树脂做背三酮检査,树脂呈阴性。
上述原料加完后,反应40min,抽滤,用30ml DMF洗涤树脂4次,每次3分钟,取少许树脂做前三酮检査,树脂呈阴性。
(2)向合成器皿中加入5m I 20%哌啶/DMF震荡反应30分钟,反应结束后,抽滤出
反应液,再用40mlDMF分4次洗涤树脂,然后在取少许树脂做前三酮检查,树脂呈阳性,向反应器皿中加入以下原料:
(a)Fomc-L-Leu(Boc)-OH 15.455g
(b)HOBT 25.01g
(c)NMM 7.26ml
(d)DMF 264ml
上述原料加完后,震荡反应40分钟,反应结束后,用30mlDMF分4次洗涤,每次3分钟,取少许树脂做前三酮检査,树脂呈阴性。
(3)变换步骤⑵中(a)原料,(b)(c)(d)原料及加入量不变,按照以RCC5为例,重复步骤⑵的操作:步骤(2)中(a)原料替换为对应的一级结构序列的氨基酸):Fmoc-L-Asp(OtBu)-OH(16.35)、Fmoc-L-Ile-OH(16.33)、Fmoc-L-Lys-OH(13.55)、Fmoc-L-Val-OH(14.32)、Fmoc-L-Glu(OtBu)-OH(15.33)、Fmoc-L-Phe-OH(15.33)、Fmoc-L-Cys-OH(16.32)、Fmoc-L-Cys-OH(16.32)等步骤(合成时按照对应短肽的一级结构中的氨基酸序列合成即可);
再重复一次⑴⑵⑶步骤的操作,各步骤的原料及用量不变,根据RCC5序列合成;最后一个结朿后,脱出Fmoc-保护基,20%哌啶/DMF(体积浓度)反应30分钟,洗净树脂,加入160ml 50%醋酸酐/DMF(醋酸酐的体积浓度)反应30分钟,用40mlDMF洗净树脂,再用甲醇洗涤树脂4次,抽滤干,真空干燥8小时。将50ml 90%TFA/DCM(TFA的体积浓度)加入盛有肽树脂的容器中,反应3小时,抽滤,浓缩滤液,向残留液中加乙醚,析出白色固体,抽滤固体,即得到粗肽,通过HPLC(高效液相色谱法)纯化,经冷冻干燥。合成RCC6时,基本步骤和工艺同RCC5,但原料需要依据RCC6的序列,按照对应原料和序列添加,补充对应的如Fmoc-L-Gln-OH(15.75)、Fmoc-L-Thr-OH(15.42)、Fmoc-L-Tyr-OH(15.45)、Fmoc-L-Leu-OH(15.45)等。即得本发明所述短肽RCC5、RCC6氨基酸序列为序列表中所述。
更进一步,若需要合成RCC5、RCC6手性氨基酸时,采用D氨基酸替换L型时,工艺大致如前,在此不赘述。
实施例2
如图1所示,自组装短肽RCC5和RCC6自组装24h的圆二色谱(CD)
在37℃环境下自组装24小时后的自组装短肽在圆二色谱仪中显示,在组装24小时之后,短肽RCC5和RCC6均可自组装形成纳米纤维交织的膜状结构,两者的二级结呈镜像相关的β折叠结构,如图1所示,图1是自组装短肽RCC5、RCC6组装24h时的圆二色谱图。
实施例3
自组装短肽RCC5和RCC6自组装24h的透射电镜(TEM)
1、实验材料
RCC5、RCC6
主要溶液有:去离子水H2O;PBS溶液(Na+、K+、PO4 3-、HPO4 2-、H2PO4 -等)。
2、主要的实验仪器
透射电子显微镜(TEM,H-200,Hitachi)
3、实验方法
(1)以去离子水或PBS配制RCC5、RCC6至终浓度为100μM的工作液,用于透射电子显微镜的观察;(2)取少量工作液用1%磷钨酸负染:用洁净的吸头吸取一滴约(10-30μl)工作液滴于洁净的载玻片表面,用镊子小心地夹取一块由Formvar膜覆盖TEM的铜网,用铜网蘸取载玻片上的工作液,静置数秒,待工作液与铜网充分结合后,再用铜网蘸取少量1%的磷钨酸,对已吸附的工作液进行负染色;(3)用滤纸吸干铜网上多余的液体,在空气中静置数分钟待铜网干燥。
以TEM扫描铜网,直接观察铜网上的短肤自组装结构。
4、实验结果
在37℃环境下自组装24小时后的自组装短肽在透射电镜中显示,在组装24小时之后,短肽RCC5和RCC6均可自组装形成纳米纤维交织的膜状结构,两者的二级结呈镜像相关的β折叠结构,如图2所示,图2是自组装短肽RCC5、RCC6组装24h时的透射电子显微镜图。
实施例4
如图3所示,
1、实验材料
RCC5、RCC6
主要溶液
无菌去离子水H2O;Millipore Milli-Q system,高压灭菌4℃保存备用。
2、主要仪器
原子力显微镜AFM(multimode8)
3、实验方法
用去离子水配置RCC5、RCC6的工作液,最终浓度为100μM;分别将配置的RCC5、RCC6的工作液5μl滴在新剥光的云母片表面;当涂片完成后约30s,以1000μl去离子水冲洗除去未附着的短肽;将上述各短肽工作液涂片在室温中空气干燥;在气相中对云母片进行AFM扫描,用SPI4000的记录模式收集AFM图像;使用20μm扫描器(400)、Olympus Si-DF20微悬臂,及弹簧常量为12N/M的针(Si,半径10nm,矩形基底200μm);悬臂的自由共振频率为127KHz;相位图以512×512的像素解析度记录;为显示自组装短肽的纳米纤维结构,以600nm×600nm,200nm×200nm的范围进行扫描。
4.实验结果
在37℃环境下自组装24小时后的自组装短肽在原子力显微镜中显示,图3是自组装短肽RCC5、RCC6组装24h时的原子力显微镜图,在组装24小时之后,短肽形成密集规则的纳米棒状纤维结构,棒状纳米纤维相互集结成致密的纳米纤维网状支架。更进一步表明,可广泛用于生物医学领域,为制备医美产品,化妆品或保健品提供物理上的纳米支架等。
实施例5
如图4所示,自组装短肽RCC5和RCC6的冷冻扫描电镜
在37℃环境下自组装24小时后的自组装短肽在冷冻扫描电镜中显示,图4是自组装短肽RCC5、RCC6组装24h时的冷冻扫描电镜图,在组装24小时之后,短肽形成密集规则的纳米棒状纤维结构,棒状纳米纤维相互集结成致密的纳米纤维网状支架。更进一步表明,可广泛用于生物医学领域,为细胞培养,细胞治疗,提供物理上的纳米支架等。
实施例6
如图5所示,自组装短肽RCC5和RCC6自组装24h刚果红染色
1、实验材料
短肽:RCC5、RCC6
混合液成分有:1)配制定浓度混合液体(苯扎氯铵、卞索氯铵、西曲氯铵、樟脑苯扎氨甲基硫酸盐),其中组分中最低浓度为1ppM;2)染色液:刚果红染色。
2、实验过程
将10mg/ml的短肽溶液母液用PBS溶液稀释为2.5mg/ml置于37℃环境中分别自组装0小时、4小时、12小时、24小时后进行刚果红染色检测。吸取15μl短肽溶液于载玻片上,滴加刚果红染色液染色约30s,于光学显微镜下观察、拍照。
3、实验结果
加入配制定浓度混合液体,刚果红染色结果显示RCC5、RCC6在显微镜下呈现纤维凝胶状,在12h基本完成组装、24h完全组装成功、48小时组装稳定,图5是自组装短肽RCC5、RCC6组装24h时的刚果红染色。更进一步表明,可用于制备医美产品,化妆品或保健品等领域。
实施例7
如图6所示,自组装短肽RCC5和RCC6自组装24h苯胺蓝染色
苯胺蓝染色液是Masson三色染色试剂盒的组成之一,苯胺蓝染色液主要有苯胺蓝、弱酸等组成,呈酸性,常与丽春红品红染色液等配合使用对胶原纤维进行染色,染色后肌纤维呈红色,胶原纤维呈蓝色,主要用于区分胶原纤维和肌纤维。
1、实验材料
短肽:RCC5、RCC6
混合液成分有:1)配制定浓度混合液体(含熊果苷、硫辛酸、透明质酸、白藜芦醇、烟酰胺、抗坏血酸、谷胱甘肽、胶原、儿茶素、胡萝卜素、椰油基葡糖苷、薰衣草醇、积雪草提取物、薄荷提取物、地衣酸),其中组分中最低浓度为1ppM;2)染色液:苯胺蓝染色液、95%乙醇、二甲苯、磷钼酸。
2、实验方法
切片常规脱蜡至水;用配制好的Weigert铁苏木素染色液染色5-10min;酸性乙醇分化液分化1-2s,蒸馏水洗;Masson蓝化液返蓝1min,蒸馏水洗;丽春红品红染色液染色5-10min,弱酸工作液洗3-5s,磷钼酸溶液分化1-2min;倒掉分化液,直接放入苯胺蓝染色液中染色1-2min,用弱酸工作液洗1min;95%乙醇快速脱水,无水乙醇脱水3次,每次5-10s;二甲苯透明3次,每次1-2min,中性树胶封固。
3、实验结果
图6是自组装短肽RCC5、RCC6组装24h时的苯胺蓝染色,混合含一定浓度的混合溶液(熊果苷、硫辛酸、透明质酸、白藜芦醇、烟酰胺、抗坏血酸、谷胱甘肽、胶原、儿茶素、胡萝卜素、椰油基葡糖苷、薰衣草醇、积雪草提取物、薄荷提取物、地衣酸),苯胺蓝染色显示RCC5、RCC6在显微镜下呈现纤维凝胶状,在12h基本完成组装、24h完全组装成功、48小时组装稳定。更进一步表明,可用于某些生物医学领域,还可用于制备医美产品,化妆品或保健品等。
实施例8
选取HFF-1细胞作为通用细胞培养的模型或者代表,在RCC5和RCC6构建的三维环境中的培养。
将HFF-1细胞,分别置于37℃水浴中迅速溶解;再加入RPMI-1640(Gibco公司)培养液,悬浮离心沉淀的细胞;然后接种于25cm培养瓶中,再加入培养液为RPMI-1640的完全培养基。
其主要成份为1%双抗溶液(青链霉素-链霉素)、8-10%(体积浓度)胎牛血清(Gibco公司),把培养瓶置于37℃、体积分数为5%二氧化碳,培养箱中培养;
每2天换液一次,待细胞贴壁生长铺满培养瓶后即可将细胞分瓶传代;
传代时在超净台内先将培养瓶内培养液用吸管吸出,培养瓶内加入0.25%的胰酶1ml使贴壁细胞游离,可以适当振荡;显微镜下观察见细胞不再贴壁已经悬浮,加入1~2ml培养液为RPMI-1640的完全培养基终止胰酶作用;将培养瓶中液体移入离心管中,1000转/分,8分钟离心沉淀细胞,弃去上清液,用RPMI-1640的完全培养基培养液悬浮细胞,分瓶接种;当接种细胞生长状态良好后,备用;
三维培养步骤如下:(1)细胞贴壁生长到约80%进行三维培养;(2)胰酶(0.25%)消化前PBS清洗细胞两次;(3)1000rpm离心,计数;(4)加入短肽等溶液,混合均匀,形成三维混悬细胞液;置96孔板于恒温培养箱(37℃,5%CO2)进行培养,观察,分析;在二维环境中,HFF-1为贴壁生长状态,呈长梭形;
在RCC5和RCC6所构建的三维环境中培养时,细胞呈球形镶嵌于短肽水凝胶中;细胞透亮并且边界清晰可见,表现为多层生长,在培养5天后,二维环境中细胞已长满,出现接触抑制,部分细胞死亡。
细胞的生长与增殖受到抑制,而在RCC5和RCC6构建的三维培养环境中,细胞透亮且细胞数量仍有增长。证明HFF-1细胞作为细胞培养的代表或者模型可在短肽水凝胶构建的三维培养环境中生长、增殖且状态良好。更进一步表明,可用于生物医学领域如细胞培养和干细胞领域治疗,也可对制备某些特殊个性化医美产品提供了支持。
实施例9
如图7所示,RCC5、RCC6三维培养细胞吖啶橙/溴化乙锭(AO/EB)染色
1)PBS缓冲液配置:8.5g氯化钠,0.68g磷酸二氢钾,0.15g氢氧化钠,100ml蒸馏水混匀,使用时稀释10倍;2)AO/EB染料:精确称取AO(吖啶橙)、EB(溴化乙锭)各1mg,分别溶于10ml PBS中使之配成100μg/ml的储备液,滤过,4℃保存,用前等量混合,备用;3)AO/EB染色及观察结果:加入AO/EB染料2-4μl/100μl细胞悬液混匀,室温避光染色30s,荧光显微镜下观察结果并计数200个细胞。
由于细胞坏死时,细胞膜的完整性早期即被破坏,线粒体明显肿胀,细胞体积明显增大,所以AO/EB染色就会呈不均匀的橙红色荧光;图7表示RCC5和RCC6在HFF-1三维培养中的AO/EB染色,图中细胞大小均匀,橙红色细胞少,表明HFF-1细胞生长情况良好,短肽RCC5、RCC6可以很好地支撑细胞生长。说明RCC5、RCC6能用于的细胞三维培养。再进一步表明,可为他们能应用于再生物医学领域如细胞工程、组织工程等提供了物理条件。
实施例10
在制备软骨、韧带、子宫、心肌、血管、神经或皮肤的再生和损伤修复材料中的应用中,以软骨修复为器官修复为代表模型之一说明RCC5和RCC6的在再生医学领域中的用途。如图8所示,短肽RCC5和RCC6对兔膝关节软骨损伤的修复情况
为考察短肽RCC5和RCC6对骨(软骨)损伤的修复及生长情况,选取新西兰大白兔的膝关节软骨作为骨组织中软骨组织部分的软骨损伤修复模型,作为研究对象,考察它对软骨损伤的修复情况等。
本实施例中,实验用动物重量在2.5kg左右的成年新西兰大白兔,随机分布,由重庆医科大学动物实验中心提供。
新西兰大白兔分为6组,每组6只,进行下面的实验:(1)取新西兰大白兔,用3%的戊巴比妥钠腹腔麻醉;(2)待完全麻醉后,碘酒消毒膝关节;(3)剪开膝关节内侧皮肤约2cm,逐层剥离皮肤组织直至暴露关节囊,用电动手钻在膝关节软骨正中心钻出直径为4mm,深度为5mm的全层软骨损伤模型;(4)适量骨蜡止血后,分别加入生理盐水、短肽RCC5、短肽RCC6填满软骨缺损伤口;(5)将膝关节复位,缝合伤口,术后用生理盐水冲洗伤口2次,碘伏消毒;(6)做好标记,待其麻醉苏醒后,放回动物房;(7)术后连续一周每天注射8万单位青霉素,以防伤口感染;(8)在1月时处死实验动物,截取膝关节,置于4%多聚甲醛溶液中,过夜;(9)用10%的EDTA脱钙液进行软骨脱钙处理,脱钙完成后,流水冲洗24h,常规石蜡包埋切片,HE染色后镜下拍片。
图8是RCC5和RCC6兔膝盖修复后的HE染色图,图8说明自组装短肽RCC5,RCC6对兔膝关节软骨的修复作用良好。该实施例中,短肽均为短肽水凝胶,浓度均为2.5mg/ml。再进一步表明,可为他们能应用于再生物医学领域特别是困惑人类多年的重大疾病提供了潜在康复的机会,具有重大社会应用价值。
实施例11
在制备软骨、韧带、子宫、心肌、血管、神经或皮肤的再生和损伤修复材料中的应用中,以皮肤创伤修复为器官修复为代表模型之二说明RCC5和RCC6的在再生医学领域中的用途。如图19所示,短肽RCC5和RCC6对皮肤创伤修复的HE染色
实验分组为:PBS组;RCC5组;RCC6组
实验对象:
SD大鼠(每组6只,共18只,由重庆医科大学动物实验中心提供)
SD大鼠皮肤创伤快速修复模型建立:(1)选取体重约为250g的SD大鼠,用7%水合氯醛(0.3ml/100g)进行麻醉。消毒完全后将大鼠固定;(2)使用圆形印章在大鼠后背做标记后沿印章痕迹在大鼠背上剪4个直径约0.8cm的圆形创面,深度到达皮下筋膜层,分别给药:PBS、RCC5、RCC6;(3)分别于术后第1、3、5、7、11和14天随机测量伤口面积,并进行取样;(4)使用多聚甲醛溶液(4%)浸泡组织样本,48h后采用石蜡包埋并切片。为接下来的HE染色做准备;HE染色步骤如下:二甲苯(Ⅰ)15min;二甲苯(Ⅱ)15min;无水乙醇(Ⅰ)5min;无水乙醇(Ⅱ)5min;95%乙醇3min;80%乙醇2min;70%乙醇2min;蒸馏水5min;苏木精染色液6min;自来水冲洗1min;1%盐酸乙醇1-2s;自来水冲洗10-30s;0.2%氨水返蓝6min;自来水冲洗10-30s;0.5%伊红染色液1-3min;蒸馏水冲洗1-2s;70%乙醇1min;80%乙醇1min;95%乙醇2min;无水乙醇(Ⅰ)3min;二甲苯(Ⅰ)10min;二甲苯(Ⅱ)10min。
HE染色结果显示:
第1天时,各组均有较为严重的炎症因子浸润情况,炎症细胞大量增多,且聚集在伤口附近,伤口处部分组织有崩解的情况;第5天时,各组炎症情况减少,RCC5和RCC6组炎症小,而PBS组较其他各组炎症情况较为严重;RCC5和RCC6组毛细血管增生较快;第7天时,各组炎症情况基本消退,肉芽组织增多且开始纤维化,表皮厚度增加,短肽组与PBS组比较表皮厚度增加较多,原位激活胶原快速新生,肉芽组织有表皮化的趋势;第14天时,除PBS组表皮仍在修复外原位激活胶原大量快速形成,其他组基本已完成修复过程。短肽组在第11天时,肉芽组织上皮化覆盖创面,基本完成创伤修复。
如图19所示,HE染色表明RCC5、RCC6在SD大鼠皮肤创伤修复14天时相较于PBS修复更好。
表明,RCC5和RCC6修复皮肤创面时,炎症小,毛细血管增生较快,原位激活胶原快速大量生成,速度快,质量好。该实施例中RCC5和RCC6自组装短肽RCC5和RCC6水凝胶,浓度分别2.5mg/ml。再进一步表明,可为他们能应用于临床医学领域特别是创伤的快速修复,减少医疗费用提高人类健康具有重大社会价值。
实施例12
如图9所示,RCC5、RCC6对抗原OVA体外释放
检测RCC5、RCC6水凝胶中OVA的释放情况,将含有200μlOVA置于200μl水凝胶中,然后在凝胶顶部加入200μlPBS溶液。
每天吸取100μlPBS并补充等体积的PBS溶液。通过BCA测定法提取样品中的OVA浓度,并计算累计的OVA释放,并绘制曲线。
图9结果显示RCC5能持续释放OVA的时间在7天左右,RCC6能持续释放OVA的时间在14天左右。进一步表明,可为他们能应用于临床医学领域特别是疫苗、特别是广谱疫苗领域多场景应用,提供了真实可能。
实施例13
如图10,图11,图12和图14所示,水凝胶负载DC疫苗治疗肿瘤
肿瘤的造模:以结肠癌为模型开展实验说明在疫苗领域用途。
将购买的MC38结肠癌细胞进行传代培养,将细胞制备成浓度为1×106个/ml,每只老鼠注射200μl的MC38细胞悬液,每过2天观察老鼠,在第7天左右长出肿瘤。
肿瘤裂解物的制备
将MC38细胞在-140℃的液氮下反复冻融5次制成肿瘤裂解物TCL。
为了探讨水凝胶负载DC疫苗对肿瘤的杀伤能力,将6-8周龄C57小鼠,每组10只,分组如下:(1)PBS组;(2)RCC5;(3)RCC6;(4)DC-TCL;(5)RCC5-DC;(6)RCC6-DC;(7)RCC5-DC-TCL
(8)RCC6-DC-TCL;(9)RCC5-DC-TCL-anti-PD-1;(10)RCC6-DC-TCL-anti-PD-1;DC细胞分别与RCC5与RCC6混合得到DC-RCC5和DC-RCC6制剂,DC的浓度为5×106个/ml,RCC5、RCC6的浓度为2.5mg/ml。
DC疫苗采用OVA(鸡卵白蛋白)刺激培养24小时后,免疫磁珠分选获得CD11+MHCⅡ+DCs,DC与TCL等体积混合,DC的浓度为5×106个/ml,TCL的浓度为100ug/ml,获得DC-TCL制剂。将DC-TCL制剂与等体积的水凝胶混合,在注射前通过温和的漩涡处理,获得RCC5-DC-TCL,RCC6-DC-TCL制剂;
通过将抗体PD-1加入制剂(RCC5-DC-TCL,RCC6-DC-TCL制剂)中得到RCC5-DC-TCL-anti-PD-1,RCC6-DC-TCL-anti-PD-1。将上述(PBS,RCC5,RCC6,DC-TCL,RCC5-DC,RCC6-DC,RCC5-DC-TCL,RCC6-DC-TCL,RCC5-DC-TCL-anti-PD-1,RCC5-DC-TCL-anti-PD-1)按每只小鼠注射量为200μl的量注射,并分别在第7和14天、21天分别接种三次,其中,DC的浓度为5×106个/ml,TCL和PD-1的剂量分别为20μg和200μg。最终观察疫苗对肿瘤的杀伤情况。图11为小鼠肿瘤图,DC-TCL、RCC5、RCC6、RCC5-DC、RCC6-DC相较于PBS组C57小鼠的肿瘤小,抗肿瘤效果明显,RCC5-DC、RCC6-DC相较于RCC5和RCC6抗肿瘤效果更好;图12为小鼠单个肿瘤大小,DC-TCL、RCC5、RCC6、RCC5-DC、RCC6-DC相较于PBS组C57小鼠的单个肿瘤小,抗肿瘤效果明显,RCC5-DC、RCC6-DC相较于RCC5和RCC6抗肿瘤效果更好。
图10为小鼠脾细胞的流式分选图结果通过流式细胞分选表明,RCC5和RCC 6负载的DC疫苗效果显著,联合PD-1负载的DC疫苗效果更显著。图14是用ELISA试剂盒检测小鼠脾细胞中IFN-γ的含量,结果表明:DC疫苗、RCC5、RCC6、RCC5-DC、RCC6-DC相较于PBS组IFN-γ水平显著升高,抗肿瘤效果更明显。进一步表明,本发明在疫苗领域的多场景应用,特别是肿瘤疫苗领域的有效预防和治疗等,具有重大战略价值。
实施例14
如图13所示,基于实施例13,短肽RCC5和RCC6对肿瘤治疗的心、脾HE染色,
HE染色步骤如下:二甲苯(Ⅰ)15min;二甲苯(Ⅱ)15min;无水乙醇(Ⅰ)5min;无水乙醇(Ⅱ)5min;95%乙醇3min;80%乙醇2min;70%乙醇2min;蒸馏水5min;苏木精染色液6min;自来水冲洗1min;1%盐酸乙醇1-2s;自来水冲洗10-30s;0.2%氨水返蓝6min;自来水冲洗10-30s;0.5%伊红染色液1-3min;蒸馏水冲洗1-2s;70%乙醇1min;80%乙醇1min;95%乙醇2min;无水乙醇(Ⅰ)3min;二甲苯(Ⅰ)10min;二甲苯(Ⅱ)10min。
图13表明,所有的组分(PBS,RCC5,RCC6,DC-TCL,RCC5-DC,RCC6-DC,RCC5-DC-TCL,RCC6-DC-TCL,RCC5-DC-TCL-anti-PD-1,RCC5-DC-TCL-anti-PD-1)对小鼠体内没有产生毒性影响或未观察到毒性影响。进一步表明,本发明在疫苗领域使用安全,若在后期药物开发中通过严格的临床前、后评价,对肿瘤疫苗领域的预防和治疗等,具有极其重要的临床价值、社会价值和商业价值。
实施例15
如图15所示,自组装短肽RCC5和RCC6对SD大鼠紫外灼伤的修复
1.实验动物
采用6周SD大鼠18只,每组6只(购买于重庆医科大学动物实验中心),鼠房恒温恒湿,并且明暗交替各24小时,定期更换垫料,用营养饲料和无菌水饲养。
2.动物水平紫外照射模型
将SD大鼠背部剃除大约3cm×3cm的毛发锡纸覆盖,暴露出直径三厘米的圆形区域。
用144J的UVA加UVB联合照射连续3天。
每天观察SD大鼠。
第三天发现大鼠背后有明显的紫外损伤。
将10mg/ml的短肽溶液母液用PBS溶液稀释为5mg/ml,采用无针注射进大鼠的真皮层,空白组则采用PBS注射。本实验采用无针注射。
3.结果
三天后观察治疗效果,如图15,PBS组损伤严重,恢复不明显;RCC5损伤基本恢复,且无疤痕,组织恢复良好;RCC6损伤基本恢复,且无疤痕,组织恢复良好。治疗组较PBS组有明显治疗效果。更进一步表明,使用方法简单,且无疤痕,可广泛应用于医美产品,化妆品或保健品等领域。
实施例16
自组装短肽三维培养扁桃体类器官
1.实验材料
(人)扁桃体组织,来源于重庆医科大学附属第一医院医疗废弃处置物,符合相应的国家及伦理法规。
2.实验方法
将手术新鲜取下的扁桃体组织浸泡于含有10%双抗的1640培养基中1h;将扁桃体组织取出放入培养皿中,将扁桃体组织剪碎为大小约为1mm×1mm的组织块;用消化酶在37℃烘箱中消化细胞2h。
将消化完的细胞制备成为扁桃体细胞悬液;将细胞悬液调整为浓度1×107个/毫升,转入24孔板中,每孔1ml;每孔加入200μl浓度为5mg/ml的自组装短肽,构建三维培养体系。
转入细胞培养箱中培养。
3.实验结果
该体系下显微镜观察到细胞及组织生长,表明支持组织及器官培养。
为扁桃体为类器官模型,说明本申请的自组装短肽能应用于三维培养类器官培养领域。
实施例17
如图16-17所示,自组装短肽RCC5、RCC6和透明质酸酸混合后的力学测试
自组装前混合溶液测试
1.测试目的:明确短肽和透明质酸的物理力学变化;明确美体填充中短肽和玻尿酸的物理力学变化;
2.试剂及仪器
1)仪器:HAAKE旋转流变仪;型号:RS6000;2)耗材:移液枪、枪头(200μl、1000μl),电子天平,称量纸,双蒸水,1.5ml EP管,10ml离心管;3)试剂:玻尿酸微球,交联剂,短肽(RCC5、RCC6)。
3.检测条件
在(25±0.2)℃,采用P20Ti L转子在0.01Hz-100Hz下进行频率扫描。
4.溶液配制两份,一份作为自组装前使用,一份作为自组装后使用
将纳米自组装短肽(RCC5、RCC6)10mg/瓶,配制成10mg/ml母液。
自组装前:分别加入1ml超纯水,
样品1:1mg/ml的RCC5+0.5mg/ml的透明质酸;样品2:1mg/ml的RCC6+0.5mg/ml的透明质酸自组装后:分别加入1mlPBS溶液,
样品1:1mg/ml的RCC5+0.5mg/ml的透明质酸(1mlPBS);样品2:1mg/ml的RCC6+0.5mg/ml的透明质酸(1mlPBS)
结果:如图16所示,1.RCC5在组装前弹性模量随赫兹的增长而增长,粘性模量无明显变化。RCC5在组装后弹性模量随赫兹的增长而增长,粘性模量有一定变化。在同一赫兹下,RCC5组装后的弹性模量小于组装前的弹性模量;如图17所示,1.RCC6在组装前弹性模量随赫兹的增长而增长,粘性模量物明显变化。RCC6在组装后弹性模量随赫兹的增长而增长,粘性模量有一定变化。在同一赫兹下,RCC6组装后的弹性模量大于组装前的弹性模量。更进一步表明,有一定的物理力学强度,为设计各种产品迭代体系的医美用品,化妆品或保健品等提供了可能。
实施例18
如图18所示,自组装短肽RCC5、RCC6在C57小鼠肝脏快速止血的应用
为考察自组装短肽水凝胶RCC5、RCC6在快速止血中的应用,本实施例选用大小为6-8周的C57小鼠的肝脏人为出血作为实验模型(动物由重庆医科大学动物实验中心提供),每组5只小鼠。
1.实验材料
RCC5、RCC6水凝胶
2.实验方案
(1)将C57小鼠脱颈处死;(2)将处死的小鼠由腹部解剖找出肝脏的位置;(3)用医用手术刀将小鼠的肝脏切开为深度5mm,长度10mm的伤口,将水凝胶缓慢滴至伤口处,直到出血停止为止;(4)实验分组为:①RCC5组、②RCC6组、③空白组。
3.实验结果
结果表明:空白组的小鼠出血后平均止血时间为1分07秒,RCC5组小鼠出血后平均止血时间约为17秒,RCC6小鼠出血后平均止血时间约为25秒。结果表示RCC5、RCC6均具有快速止血的作用,RCC5的效果更显著。图18是RCC5和RCC6对C57小鼠肝脏的止血图。
上述实施例中自组装短肽RCC5和RCC6水凝胶浓度分别5.0mg/ml;更进一步表明,短肽的浓度不同,在生物医学领域应用场景会有一定差异。可针对不同应用场景,选择合适浓度应用于不同领域。
上述实施例中,图1、图2、图3、图4、图5、图6、图7、图8、图10中A对应RCC5,B对应RCC6。
为本发明一类自组装短肽,通过对RCC5和RCC6合成、理化表征、多种类型细胞培养、多种不同组织修复、多种器官的修复、肿瘤疫苗的预防和治疗、多物质联合及使用、多场景应用等,表明本发明其在疫苗、广谱疫苗及生物医学等领域中具有重要理论意义和社会价值,对提高美好品质生活和人类医学健康具有重大战略价值。
序列表:
SEQUENCE LISTING
SEQUENCE LISTING
<110> 成都赛恩贝外科学研究院
<120> 一类自组装短肽,其在广谱疫苗及生物医学中的用途
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> 人工序列
<400> 1
Arg Leu Asp Ile Lys Val Glu Phe Cys Cys
1 5 10
<210> 2
<211> 10
<212> PRT
<213> 人工序列
<400> 2
Arg Gln Asp Thr Lys Thr Glu Tyr Cys Cys
1 5 10
Claims (20)
1.一类自组装短肽,其特征在于,包括两个短肽,其氨基酸序列分别:
RCC5:Arg Leu Asp Ile Lys Val Glu Phe Cys Cys,
RCC6:Arg Gln Asp Thr Lys Thr Glu Tyr Cys Cys。
2.根据权利要求1所述的一类自组装短肽,其特征在于,每个短肽的中的氨基酸为L型,D型或DL型中的一种或多种。
3.根据权利要求1所述的一类自组装短肽,其特征在于,每个短肽的中的氨基酸均为L型。
4.根据权利要求1所述的一类自组装短肽,其特征在于,每个短肽的碳端酰胺化。
5.根据权利要求1所述的一类自组装短肽,其特征在于,每个短肽形成二级结构,二级结构包括α螺旋、β折叠、β蜷曲和无规则卷曲中的一种或多种。
6.如权利要求1-5任一项所述的一类自组装短肽在制备细胞或类器官三维培养纳米支架材料中的应用。
7.如权利要求1-5任一项所述的一类自组装短肽在制备软骨、韧带、子宫、心肌、血管、神经或皮肤的再生和损伤修复材料中的应用。
8.如权利要求1-5任一项所述的一类自组装短肽作为主要成分用于制备医美产品,化妆品或保健品。
9.如权利要求1-5任一项所述的一类自组装短肽作为疫苗佐剂的应用。
10.根据权利要求9所述的应用,其特征在于:所述疫苗包括DC短肽疫苗。
11.根据权利要求9所述的应用,其特征在于:作为疫苗佐剂的短肽的水混合物制备成短肽水凝胶,然后与DC混合,静置后形成的水凝胶用作疫苗。
12.以权利要求9-11任一项所述应用中短肽作为疫苗佐剂的短肽疫苗。
13.如权利要求12所述的短肽疫苗,其特征在于:所述作为疫苗佐剂的短肽与DC物理混合得到疫苗。
14.含权利要求1-5任一项所述的一类自组装短肽的水凝胶。
15.如权利要求14所述的水凝胶的制备方法,其特征在于,包括如下步骤:
步骤1、将两个短肽之一加入水中配制成母液;
步骤2、向母液中加入离子或PBS制备成自组装短肽水凝胶。
16.根据权利要求15所述的制备方法,其特征在于:制备成的自组装短肽水凝胶的浓度为1ppM及以上。
17.如权利要求14所述的含一类自组装短肽的水凝胶在制备抗肿瘤靶向药物中的应用。
18.如权利要求14所述的含一类自组装短肽的水凝胶作为敷料的应用。
19.如权利要求14所述的含一类自组装短肽的水凝胶作为止血材料的应用。
20.一种生物医学材料,它包含权利要求14所述的含一类自组装短肽的水凝胶。
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