CN115089698B - 改善皮肤的活性肽与干细胞外泌体在药品或化妆品中的应用 - Google Patents
改善皮肤的活性肽与干细胞外泌体在药品或化妆品中的应用 Download PDFInfo
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Abstract
本发明涉及改善皮肤的活性肽与干细胞外泌体在药品或化妆品中的应用。本发明从生物体内提取获得了具有较好抗氧化活性以及抗菌活性双重功能的活性肽，该活性肽能够提高细胞的抗氧化特性，同时该活性肽与外泌体一起联用后也能够有效的促进皮肤细胞的增殖以及抗衰老作用，具有极好的应用前景。
Description
技术领域
本领域涉及生物领域，更具体的涉及改善皮肤的活性肽与干细胞外泌体在药品或化妆品中的应用。
背景技术
皮肤老化的主要原因为紫外线、年龄老化导致的多种变化，如DNA突变，蛋白质氧化导致的功能损伤，脂质氧化导致的信号传导功能受损，进而导致成纤维细胞活性下降，其合成胶原蛋白等细胞外基质的能力下降，成纤维细胞活性下降，I型和Ⅲ型胶原蛋白的前体减少，导致胶原纤维、弹性纤维含量下降，Ⅲ型/I型比例增加。另一方面，随着皮肤老化，基质金属蛋白酶(MMPs)活性上升将导致胶原蛋白等降解增加。此外，细胞外糖蛋白等异常交联，蛋白多糖含量下降，皮肤保水保湿能力下降，导致皮肤正常的结构和功能受到破坏。随着年龄的增长，胶原蛋白的疏水性氨基酸含量增加，这也可解释为何老年人皮肤容易干燥。
随着人类对健康的关注度提高，也使得人们希望自己外表年轻的需求更加强烈；同时有研究表明，自我感觉好能使人感觉更健康、更快乐、更高效.据估计全球目前美容市场，口服美容产品仅占3％左右的比例，其年增长率为7％～12％，显示了良好的市场前景.活性肽在口服美容和外用美容方面均有大量的应用和研究报道。比如口服胶原蛋白可降解产生二肽和三肽，其在血液中2h后可检测到，体外成纤维细胞实验结果表明200nmol/L的脯氨酸一羟脯氨酸可提高成纤维细胞增殖能力1.5倍，透明质酸合成能力增加3.8倍，透明质酸合成酶mRNA水平提高2.3倍.其主要机理为脯氨酸一羟脯氨酸通过SATA-3的磷酸化，进而提高HAS2的转录水平，促进成纤维细胞的分裂和透明质酸的合成。也有研究表明通过动物实验，无毛老鼠1：3服0.2g/(kg·d)来源于鱼鳞胶原蛋白水解物6周，研究胶原蛋白水解物对抗紫外线对皮肤的损伤的效果，结果为相对紫外线照射模型组，胶原蛋白水解物加紫外照射样品组在表皮层厚度与正常组接近，而模型组显著比样品组和正常组高.皮肤胶原蛋白含量，正常组和样品组均显著高于模型组.结果表明胶原蛋白水解物可增强皮肤对抗紫外线损伤的能力。
外泌体(exosomes)是一种由细胞分泌的直径为40-150nm、密度范围为1.09-1.18g/ml的具有双层磷脂膜结构的囊泡样物质。目前已经从各物种、各类型的细胞培养上清中提取出了外泌体，如神经元细胞、间充质干细胞、成纤维细胞、内皮细胞、巨核细胞等。外泌体也被发现存在于人体的各类体液中，如血液、唾液、尿液、乳汁等。
研究还显示，外泌体携带的miRNA能够部分调节htNSC的抗老化效应，并在htNSC进入脑脊髓液后发挥相应作用。在皮肤抗衰老方面，研究发现，脐带间充质干细胞来源的外泌体可以轻易穿透角质层到达真皮，促进人皮肤组织Ⅰ型胶原和弹力蛋白的生成，改善皮肤质地。这些研究表明外泌体具有抗衰老应用前景。皮肤的创面愈合与再生是一个复杂的生理学过程，需要多种组织、细胞协同作用来替换、修复、重建缺失的细胞结构、组织层次。虽然大多数的皮肤创伤愈合不存在阻碍，但是对于创伤面积大、有慢性疾病影响、合并感染等情况，皮肤的修复再生便会受到影响，有些甚至终身不愈合。生理学中一般将创伤的愈合分为几个阶段：出血期、炎症期、增生期、组织改建期(瘢痕形成期)。目前已有的研究发现，外泌体可以影响其中多个过程、多种细胞促进皮肤的修复与再生。人脐带间充质干细胞(hucMSCs)来源的外泌体，并将其应用于烧伤大鼠模型中。研究发现，该外泌体可以以剂量依赖的方式促进皮肤细胞(真皮成纤维细胞、表皮角质细胞)的增殖。此外，利用该外泌体还可以抑制由热应激引起的皮肤细胞的凋亡，激活AKT信号通路，降低前凋亡蛋白Bax的表达。在体内试验中，研究者采用局部多点注射的方法，将外泌体注人大鼠深二度烧伤模型的伤口周围来评价外泌体的作用。结果显示，脐带问充质干细胞来源的外泌体加速了伤口愈合。
但是目前，针对皮肤抗衰老以及提高皮肤损伤修复能力的外泌体功能组合物的研究还不够多，是值得迫切研究的方向。
发明内容
基于蝗虫体内含有大量的抗氧化剂的研究，发明人从蝗虫体内鉴定并筛选获得了具有较好抗氧化、抗菌活性的活性肽。
所述筛选步骤包括中性蛋白酶和木瓜蛋白酶进行酶解，采用截留Mr 10000的超滤膜4℃下超滤分离，随后采用Sephadex G-50凝胶色谱(1.6cm*60cm)分离，随后采用DEAE阴离子交换树脂进行层析，并采用高效液相色谱进行分析鉴定及质谱分析获得了相应的活性肽。
进一步的，所述活性肽是LOC-D2-3活性肽，其测序结果为AFACRYYDIAMQWDSRNLLKPQRNKK，其分子量为3.2kD。
进一步的，本发明提供了将所述活性肽用于制备皮肤抗衰老及抗菌的药物中的应用。
进一步的，本发明还提供了脂肪间充质干细胞外泌体。所述外泌体具有促进细胞抗衰老的效果。
所述外泌体的制备方法为取4代脂肪间充质干细胞，细胞融合生长至80-90％时，换无血清培养基培养48h，收集细胞上清液。300×g离心10min，去除死细胞和大的细胞碎片，2000×g离心10min，去除死细胞和细胞碎片，10000×g/min离心30min去除细胞碎片较大的囊泡，0.22μm针头滤器过滤，去除微泡及可能存在的凋亡小体。用20mL空针将上清液转移至超速离心管中，106×g离心70min，去除上清液，收集沉淀，得到粗提取的外泌体，106×g离心70min，去除上清液，用100μLPBS溶解沉淀，得到较纯的外泌体。
另外一方面，本发明还提供了活性肽和外泌体在制备抗氧化抗衰老的药物组合物中的用途。
在一般情况下，该组合物包含有效量的至少一种物质，该物质与药学上可接受的添加剂以下述重量百分比的比例组合而成。
作为药物添加剂，可以使用用于稳定组合物的单糖或多糖，氨基酸，低分子蛋白质和随后的冷冻干燥。
该药物组合物以溶液的形式制成用于体内给药，含有0.01-0.5％，优选0.01-0.1％的主要活性物质，在用于治疗创伤表面的形式中或在用于肠内给药的的形式中含有0.1至1.0％的主要活性物质。
为了制备液体介质，使用选自以下的溶剂：a)平衡盐溶液，b)各活性组分及药学上可接受的载体。
为了制备冻干形式，使用已知用于稳定药理学生物活性多肽的单糖和/或多糖。冻干形式可用于制备选自以下的粉末：制备注射用粉末，输液溶液，吸入用粉末，软膏用粉末，凝胶，悬浮液，制备压片形式的粉末。
粉末制剂中多肽的浓度为0.1％至5％，外泌体的浓度为0.1％至10％。
当包含过多肽和外泌体的组合物与至少一种广谱治疗剂联合施用时或施用之后，可以使用多模态治疗。
治疗剂选自i)抗菌剂，抗病毒，抗真菌，抗组胺制剂，ii)提供抗自由基(超氧化物歧化酶，过氧化氢酶，谷胱甘肽过氧化物酶)，iii)能进一步降低细胞内自由基水平的低分子化合物(生育酚，谷胱甘肽，泛醌)，iv)用于器官移植或冷冻保存的制剂，v)生物活性蛋白，例如胰岛素，vi)激素，vii)维生素，viii)细胞因子。
当使用多肽治疗时，所述组合物的引用形式及其使用方法不限制医学或兽医领域已知的其它变体。对本领域普通技术人员来说显而易见的本发明的变型必须被认为是在所提出的本发明的范围内。
正如本文所报道的，多肽和外泌体增加的抗氧化活性或生物活性可用于治疗或预防与氧化损伤相关的疾病或病症。因此，本发明提供了增加抗氧化活性以增强组织中抗氧化活性的治疗组合物。本发明的多核苷酸可以作为药物组合物的一部分给药。该组合物应当是无菌的，并且包含治疗有效量的多肽或核酸分子，其单位重量或体积适于给予受试者。
编码所述多肽的核酸分子可与常规抗氧化剂一起以单位剂型在药学上可接受的稀释剂载体或赋形剂中给药。可以使用常规的药学实践来提供合适的制剂或组合物，以将所述化合物给予患有与氧化损伤相关的疾病的患者。给药可以在患者症状出现之前开始。可以采用任何合适的给药途径，例如可以通过吸入或胃肠外静脉内动脉皮下瘤内，肌内，颅内，眼科，心室内，肝囊内，鞘内，腹膜内，鼻内气雾剂栓剂或口服给药。例如，治疗制剂可以是液体溶液或悬浮液的形式；对于口服给药，制剂可以是片剂或胶囊的形式；用于鼻内的。
本发明提供了治疗疾病和/或其病症或症状的方法，该方法包括给予受试者(例如哺乳动物如人)治疗有效量的包含本文所述多肽的药物组合物。因此，在一个实施方案中是治疗患有或易患与氧化损伤或抗氧化活性降低相关的疾病或病症或其症状的受试者的方法。该方法包括给予哺乳动物治疗量的本文化合物的步骤，该治疗量足以治疗其疾病或病症或症状在疾病或病症得到治疗的条件下。
有益效果
本发明从生物体内提取获得了具有较好抗氧化活性以及抗菌活性双重功能的活性肽，该活性肽能够提高细胞的抗氧化特性，同时该活性肽与外泌体一起联用后也能够有效的促进皮肤细胞的增殖以及抗衰老作用，具有极好的应用前景。
附图说明
图1多肽对H2O2诱导的大鼠表皮角化上皮细胞损伤修复效果图
图2外泌体对HSF的增殖效果图
图3β-半乳糖苷酶检测结果图
具体实施方式
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
实施例1生物活性肽的筛选和鉴定
将鲜蝗虫，洗净后捣碎。向100g的鲜蝗虫捣碎液中加入1.5倍质量的纯净水，于40℃，pH6条件下，加入中性蛋白酶和木瓜蛋白酶进行酶解，酶加量为2500U/g，50r/min条件下搅拌酶解5h，酶解结束后在95℃水浴中灭酶10min，待冷却后，过滤取上清液。
采用截留Mr 10000的超滤膜4℃下超滤分离，收集Mr大于10000和小于10000两部分溶液，采用抑菌圈法筛选有抗菌活性溶液(大肠杆菌和铜绿假单胞菌、白色念珠菌作为试验菌)，采用DPPH清除率来检测有抗氧化活性的溶液，通过鉴定发现Mr小于10000具有抗菌活性以及抗氧化活性。将Mr小于10000的溶液继续0.45um膜过滤后，Sephadex G-50凝胶色谱(1.6cm*60cm)分离，用0.05mol/L的Tris-HCl缓冲液pH7.2平衡色谱柱，样品上样后用同样的缓冲液洗脱，流速为1.5mL/10min，每10min收集1管，测定A280nm，绘制洗脱曲线，收集各峰。共收集五个主峰，其中第三个主峰在46-73管收集液中有明显的抗菌、抗氧化特性。将第46-83管收集液进行冷冻干燥浓缩样品至5mL。
采用DEAE阴离子交换树脂进行层析，用0.02mol/L的Tris-HCl缓冲液(pH8.O)和1.0mol/L的NaCI溶液进行梯度洗脱，洗脱速率为0.5mL/min，分管收集，每管收集时间为8min，检测波长为280nm，合并同一分离峰的洗脱液，共收集得到6组主峰。测定各主峰清除DPPH自由基的能力以及三种抑菌圈抑菌能力。结果如表1所示。
表1DEAE阴离子交换树脂层析分离组分DPPH自由基清除能力及抑菌特性分析
从表1的结果可以看出，D2和D4这两个主峰组分存在着活性多肽能够具有较好的抗氧化性和抑菌能力。
用透析法对D2和D4活性的组分进行脱盐，透析得到的溶液经旋转蒸发浓缩，浓缩液冻干备用。
分别取干燥后的D2和D4样品溶解并用0.45um微孔滤膜过滤。采用岛津2010LC分析型反向高效液相ODS-SP柱(4.6*250mm*5um)进一步进行分离纯化。RP-HPLC采用乙腈-三氟乙酸梯度洗脱，流速为1mL/min，在吸收波长220nm下收集各组分峰，采用DPPH自由基清除能力及抑菌特性分析最终鉴定获得了2个活性肽，分别命名LOC-D2-3和LOC-D4-2，冷冻干燥后备用。
实施例2LOC-D2-3序列鉴定及活性分析
将LOC-D2-3活性肽采用ESI-MS进行质谱测定分析，鉴定其测序结果为AFACRYYDIAMQWDSRNLLKPQRNKK，其分子量为3.2kD。分析该多肽序列发现富含疏水性氨基酸和芳香族氨基酸增强抗氧化能力，同时富含精氨酸和赖氨酸具有抗菌能力。将所述多肽进行人工合成后备用。
(1)抗菌活性鉴定：将大肠杆菌、铜绿假单胞菌采用MHA培养基进行培养，白色念珠菌采用沙氏葡萄糖蛋白胨琼脂培养基进行培养；采用抑菌圈法中央孔加入超纯水作为阴性对照，其中一孔加入相应的替卡西林抗生素作为阳性对照，大肠杆菌和铜绿假单胞菌37摄氏度培养18h，白色念珠菌28摄氏度恒温培养2d，观察抑菌圈大小。抑菌圈10mm作为样品最小抑菌浓度的检测标准，检测获得最小抑菌浓度如表2所示。从表2的结果可以看出，本发明的多肽具有较好的抗菌特性，与阳性对照的抑菌效果接近。
表2多肽对各菌的最小抑菌浓度
| 菌名称 | 最小抑菌浓度(μg/mL) |
| 大肠杆菌 | 5.6±0.2 |
| 铜绿假单胞菌 | 9.7±0.3 |
| 白色念珠菌 | 14.5±0.6 |
(2)抗氧化活性鉴定：
按照5×104/mL的密度将大鼠表皮角化上皮细胞铺板于96孔板，待细胞贴壁完全后，实验组分别加入含有0.05、0.1、0.2、0.4、0.8mmol/L的多肽预孵育大鼠表皮角化上皮细胞24h，模型组和空白对照组分别加入等量的完全培养基进行孵育。阳性对照组设置0.4mmol/L的维生素C；然后，吸取并弃掉培养基，实验组和模型组和阳性对照组加入含有0.4mmol/L的H2O2的完全培养基，空白对照组加入不含H2O2的完全培养基继续孵育细胞6h。弃去培养基，然后重新加入含有10％CCK-8的基础培养基。细胞于37℃细胞培养箱内孵育1h。采用多功能酶标仪于450nm下测定吸光值。并根据公式计算不同浓度H2O2处理后各组细胞的存活率：细胞存活率(％)＝(实验组OD-调零组OD)/(对照组OD-调零组OD)×100％。
从图1中可以看出，相比于空白组，模型组的细胞存活率显著下降(p<0.05)，H2O2的加入导致大鼠表皮角化上皮细胞损伤，以至部分细胞死亡。而实验组却有明显的改善，多肽在高浓度0.2mmol/L、0.4mmol/L的条件下都能提高大鼠表皮角化上皮细胞的存活率，与模型组对比有显著的提升(p<0.05)，0.1mmol/L的多肽已经和阳性对照组的效果基本相同，在0.8mmol/L的多肽作用下，细胞存活率达到了99.3％，表明多肽具有缓解H2O2导致的损伤的能力，具有较强的抗氧化特性。
(3)多肽安全性鉴定
取健康人血液用生理盐水洗涤红细胞至上清无色，3000r/min离心2min，弃去上清，用生理盐水配置成2％红细胞悬液，将多肽用生理盐水稀释成为500，100,50,10μg/mL的溶液，加入等体积的2％红细胞悬液，阳性对照为0.1％TritonX-100，阴性对照为生理盐水，每个样品设置3个平行孔，37摄氏度孵育1h，离心去掉完整的红细胞。取上清，540nm波长处测定各管的吸光度值。结果显示，各浓度的多肽溶液与阴性对照结果基本相同，而阳性对照具有明显的吸光值数值，这表明本发明的多肽具有较好的安全性。
实施例3脂肪间充质干细胞外泌体的制备
取脂肪25mL，用含青霉素-链霉素双抗的PBS冲洗3遍，去除肉眼可见的筋膜及红细胞后，加入等体积0.075％Ⅰ型胶原酶，在37℃、150r/min的条件下消化40min，加入等体积含体积分数10％胎牛血清的DMEM/F12培养基终止消化，15000r/min离心10min，去除上层油脂及中层清液，用5mL红细胞裂解液重悬，室温静置5min，1000r/min离心5min，去除上清液，用含体积分数15％胎牛血清的低糖DMEM培养基重悬，70μm细胞筛网过滤后，移入培养皿行原代培养，每隔两三天换液，细胞浓度融合生长至80％-90％时，用体积分数0.25％胰蛋白酶消化后按1∶2传代培养。取4代细胞，细胞融合生长至80-90％时，换无血清培养基培养48h，收集细胞上清液。300×g离心10min，去除死细胞和大的细胞碎片，2000×g离心10min，去除死细胞和细胞碎片，10000×g/min离心30min去除细胞碎片较大的囊泡，0.22μm针头滤器过滤，去除微泡及可能存在的凋亡小体。用20mL空针将上清液转移至超速离心管中，106×g离心70min，去除上清液，收集沉淀，得到粗提取的外泌体，106×g离心70min，去除上清液，用100μLPBS溶解沉淀，得到较纯的外泌体。
取5μL外泌体溶液，用BCA法测外泌体的蛋白浓度达到1.38g/L；取20μL外泌体溶液，用PBS稀释至200μL，用粒度仪检测直径分布在80-110nm之间。
外泌体的流式细胞学检测：旋涡30s重悬CD81标记的磁珠，取40μL磁珠到1mL离心管中，加入400μLPBS清洗磁珠，混合均匀，将离心管放在磁力架上1min，弃去上清液；用60μL的PBS重悬磁珠，加入40μL外泌体溶液，在2-8℃条件下孵育过夜；第2天，将离心管离心3-5s，收集管底沉淀；加入300μLPBS清洗磁珠结合的外泌体，混合均匀，将离心管放在磁力架上1min，弃去上清液；重复清洗磁珠结合的外泌体1次，800μLPBS重悬；分别向8支1mL离心管加入100μL悬液，依次加入PE-anti-humanCD9，PE-anti-humanCD29，FITC-anti-humanCD34，488-anti-humanCD44，PE-anti-humanCD45，PE-anti-humanCD63，PE-anti-humanCD90，488-anti-humanCD105和PBS各10μL，室温避光孵化40min后，将离心管放在磁力架上1min，弃去上清液；加入400μLPBS清洗磁珠结合的外泌体2次，用100μL PBS重悬，等待上机检测。数据用FlowJoSoftware7.6.1软件分析。结果显示，外泌体的流式细胞学鉴定CD9，CD29，CD44，CD63，CD90及CD105均为阳性表达，阳性率分别为99.3％，99.9％，99.7％，96.2％，99.8％及99.9％，CD34及CD45为阴性表达，表明制备的的外泌体是脂肪间充质干细胞外泌体。
实施例4LOC-D2-3多肽与外泌体联合应用效果验证
取对数生长期的人皮肤成纤维细胞HSF，以每孔5×104/mL个接种于96孔板上，每组设5个重复孔，待其铁壁后将DMEM完全培养基与CCK-8溶液按体积比100:1的比例混合孵育细胞2h，吸出上清，用酶标仪检测波长450nm处的吸光度值，将96孔板内细胞用PBS洗涤1次，按照如下分组进行培养：
实验1组：加入含200ug/mL的外泌体的DMEM完全培养基培养；
实验2组：加入含50ug/mL LOC-D2-3多肽的DMEM完全培养基培养；
实验3组：同时加入含200ug/mL外泌体和50ug/mL LOC-D2-3多肽的DMEM完全培养基培养；
阳性对照组：加入含50ug/mL维生素C的DMEM完全培养基培养；
阴性对照组：加入DMEM完全培养基培养；
分别测定培养96h时波长450nm处的吸光度值，结果如图2所示。
从图2结果可以看出，实验组1-3与与空白对照组对比有显著的促进人皮肤成纤维细胞增殖的效果(p<0.05)，同时促进效果也比阳性对照组维生素C也有显著的提升。LOC-D2-3多肽单独使用具有较好的促进促进增殖的效果，特别是与外泌体联用后，OD450更是达到了(4.9±0.2)，具有显著的协同促进的效果。
将以上各组细胞按照β-半乳糖苷酶染色试剂盒说明书进行染色。由于β-半乳糖苷酶染色是与细胞衰老相关的β-半乳糖苷酶活性水平上调而对衰老细胞进行染色的方法。结果如图3所示。
从图3的结果可以看出，采用实验组1-3与与空白对照组对比有显著的降低衰老相关β-半乳糖苷酶阳性比例的的效果(p<0.05)，同时降低效果也比阳性对照组维生素C也有显著的提升。特别是实验组3，其中的SA-β-gal阳性比例只有(4.0±0.9)％，呈现了较好的抗衰老效果。
虽然已描述并详细举例说明本发明至足以使本领域技术人员制备和使用它，但是在不背离本发明的精神和范围的情况应清楚各种替代方案、修改和改进。本文所提供的实施例代表优选的实施方案，为示例性的，并且不旨在为对本发明的范围的限制。其中的修改和其他用途将是本领域技术人员能够想到的。在本发明的精神内涵盖这些修改并且由权利要求书的范围限定这些修改。
序列表
<110> 北京绎源生物科技有限公司
<120> 改善皮肤的活性肽与干细胞外泌体在药品或化妆品中的应用
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Claims (7)
1.LOC-D2-3活性肽和脂肪间充质干细胞外泌体在制备促进皮肤细胞增殖和抗衰老的药物组合物中的用途；其中，所述活性肽的序列如SEQ ID NO:1所示。
2.LOC-D2-3活性肽和脂肪间充质干细胞外泌体在制备抑制皮肤细胞衰老的化妆品中的用途；其中，所述活性肽的序列如SEQ ID NO:1所示。
3.如权利要求1所述的用途，其特征在于所述药物组合物中还含有药学上可接受的载体。
4.如权利要求3所述的用途，其特征在于所述药物组合物中含有赋形剂。
5.如权利要求1或3所述的用途，其特征在于所述药物组合物中还使用已知用于稳定药理学生物活性多肽的单糖和/或多糖。
6.如权利要求1或3所述的用途，其特征在于所述药物组合物是冻干的形式。
7.如权利要求6所述的用途，其特征在于冻干的形式对应于注射用粉末，软膏用粉末或压片形式的粉末。
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