CN114957394B - 一种促皮肤修复多肽pm-7及应用 - Google Patents
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Abstract
本发明提供了一种促皮肤修复多肽PM‑7及应用。本发明从斑腿泛树蛙皮肤分泌物中分离纯化得到的多肽PM‑7，氨基酸序列为FLNWRRILFLKVVR。本发明提供的多肽PM‑7能显著的促进皮肤的修复，而且分子量小，可通过固相化学合成实现工业化生产，还能够在制备促皮肤创伤修复治疗药物中应用，可作为治疗体表创伤、组织修复、烧伤、皮肤溃疡、减少疤痕产生、加快疤痕修复的候选药物，促皮肤修复多肽PM‑7的发现和应用在生物医药领域具有重要的意义。
Description
技术领域
本发明涉及生物医学技术领域，具体涉及一种促皮肤修复多肽PM-7及应用。
背景技术
皮肤作为内部和外部环境的物理保护屏障，容易受到意外伤害、微生物感染、皮肤病和代谢功能障碍的损害。皮肤受损机体就会立即开启自我生理修复，修复过程包括四个阶段：出血、炎症、增殖和组织重塑，然而四阶段超出自我修复极限将导致皮肤大面积缺损、溃疡和疤痕，具有严重的危害性。利用药物、干细胞和组织修复技术治疗皮肤修复已越来越常见，其中药物治疗仍发挥着重要作用。临床上，常见的治疗药物包括小分子化合物、生长因子、抗生素、磺胺银和传统软膏。然而，由于合成困难、药物活性不稳定、储存条件严格以及形成肥厚性瘢痕，这些促愈合药物的实用性受到限制。因此，寻找新型促创伤修复药物是非常重要和非常有必要的。天然活性多肽具有高活性、高特异性、高稳定性及低免疫原性，以广泛应用于新型药物筛选。相比于在抗菌，镇痛和抗癌方面的活性多肽研究，促皮肤修复肽仍处于起步阶段。
两栖动物生存环境复杂，长期的自然选择下，导致其皮肤系统拥有抵抗外界损伤和快速修复创伤的能力，是当前挖掘皮肤修复肽的主要来源。斑腿泛树蛙为无尾目、树蛙科、泛树蛙属的两栖动物。体色淡棕色，身体背部为浅棕色，常在水塘边的灌丛和草丛中活动，我国南部分布较广，云南分布于滇中、滇西和滇南地区。开发斑腿泛树蛙皮肤分泌物中的皮肤修复肽具有重要的意义。
发明内容
本发明的目的在于，针对现有技术的上述不足，提供了一种促皮肤修复多肽PM-7及应用。
为实现上述目的，本发明采用如下的技术方案：
本发明的第一目的在于提供一种促皮肤修复多肽PM-7，所述多肽PM-7的氨基酸序列为SEQ ID NO:1FLNWRRILFLKVVR。
进一步的，所述促皮肤修复多肽PM-7的分子量为1860.32Da，等电点为12.3。
本发明的第二目的在于提供所述促皮肤修复多肽PM-7在制备促进伤口愈合药物中的应用，所述促皮肤修复多肽PM-7的分子量为1860.32Da，等电点为12.3，氨基酸序列为SEQ ID NO:1FLNWRRILFLKVVR。
进一步的，所述的促进伤口愈合药物用于制备化妆品或日用品。
本发明的促皮肤修复多肽PM-7的分离、纯化和鉴定步骤如下：
步骤S1，刺激斑腿泛树蛙皮肤产生分泌物并收集；
步骤S2，将步骤S1收集得到的分泌物，通过Sephadex G-50柱层析和C18反相制备型HPLC分离纯化，MALDI-TOF MS和Edman降解分析获得促皮肤修复多肽PM-7。
本发明提供的技术方案带来的有益效果是：本发明从斑腿泛树蛙皮肤分泌物中分离纯化得到的多肽PM-7能显著的促进皮肤的修复，而且分子量小，可通过固相化学合成实现工业化生产，还能够在制备促皮肤创伤修复治疗药物中应用，可作为治疗体表创伤、组织修复、烧伤、皮肤溃疡、减少疤痕产生、加快疤痕修复的候选药物，促皮肤修复多肽PM-7的发现和应用在生物医药领域具有重要的意义。
附图说明
图1为促皮肤修复多肽PM-7的分离纯化鉴定图，A为采用G-50凝胶柱对斑腿泛树蛙皮肤分泌物进行第一次分离纯化，收集得到Ⅰ～Ⅳ四种不同吸收峰组分图；B为对组分Ⅳ采用采用反相HPLC进行第二次分离纯化，收集得到Ⅱ1～Ⅱ4四种不同吸收峰组分图；C为组分Ⅱ4冻干粉的吸收谱图；D为组分Ⅱ4的分子量测定图谱；
图2a为促皮肤修复多肽PM-7对HSF细胞的增殖的促进作用图；
图2b为促皮肤修复多肽PM-7对Huvec细胞的增殖的促进作用图；
图2c为促皮肤修复多肽PM-7对HSF细胞的迁移促进作用图；
图3a为促皮肤修复多肽PM-7对小鼠皮肤损伤模型的小鼠伤口愈合对比图；
图3b为促皮肤修复多肽PM-7对小鼠皮肤损伤模型的修复效果对比数据结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面结合具体实施例和附图，对本发明的具体实施方式作进一步详细描述。
实施例1
(1)促皮肤修复肽PM-7的制备
步骤S1，皮肤分泌物收集
斑腿泛树蛙用蒸馏水洗净、酒精棉擦干，去除杂质。用10V稳压间断性多点刺激蛙耳后腺及背部5-10min，超纯水清洗蛙背部、收集分泌物，并添加少量蛋白酶抑制剂，12000rpm冷冻离心20min，取上清液真空冷冻干燥，收集冻干粉末-20℃低温保存备用。
步骤S2，分离纯化
步骤S21，将步骤S1制得的皮肤分泌物冻干粉用0.1M磷酸缓冲液(pH＝6)溶解成100mg/mL，采用G-50凝胶柱进行第一次分离纯化，收集得到4种不同吸收峰组分，按吸收峰的时间范围收集洗脱液，分别命名为Ⅰ～Ⅳ组分，如图1中A所示；然后对收集的Ⅰ～Ⅳ组分进行促细胞增殖活性测定，其中组份Ⅳ的促细胞增殖活性最高；
步骤S22，选择组分Ⅳ采用反相HPLC进行第二次分离纯化，收集不同吸收峰组分，分别命名为Ⅱ1～Ⅱ4组分，如图1中B所示；然后对Ⅱ1～Ⅱ4组分进行第二次促细胞增殖活性测定，其中组分Ⅱ4的促细胞增殖活性最高。
步骤S3，多肽一级结构鉴定
将步骤S2中获得的组分Ⅱ4再一次进行反相HPLC分离纯化，如图1中C所示，收集28min吸收峰冷冻干燥，冻干粉在PPSQ-31A蛋白质测序仪上通过Edman降解法测出多肽的N端序列，MALDI-TOF检测冻干粉的分子量，通过以构建的斑腿泛树蛙cDNA文库中进行分析比对，最终获得促皮肤修复肽PM-7，其氨基酸序列为FLNWRRILFLKVVR，分子量为1860.32Da，如图1中D所示。
该步骤中，采用1.5×31cm，superfine，GE Healthcare，SwedenSephadex的G-50凝胶柱；流动相：0.1M NaCl的25mM Tris-HCl缓冲液(pH 7.8)；洗脱条件：0.1mL/min的流速，洗脱1h。C18反相制备型色谱采用4.0×300mm，5μm的C18色谱柱；流动相为：水(0.1％TFA)，乙腈(0.1％TFA)，洗脱梯度为：0 70min，5％-70％乙腈梯度洗脱。Edman降解使用PPSQ-31A蛋白质测序仪(日本岛津)按照制造商的标准GFD协议进行Edman降解以确定多肽PM-7的序列信息。
(2)促皮肤修复肽PM-7用于研究对HSF和Huvec细胞的增殖和迁移的促进作用
培养的HSF和Huvec细胞经胰酶消化后，用含10％FBS的培养基重悬，抗迁移实验则用无血清培养基重悬，进行细胞增殖和细胞划痕实验测定。
细胞增殖实验：HSF和Huvec细胞以4×104个细胞/孔的密度种于96孔板中，细胞密度培养至80％左右时，使用不同浓度的PM-7(100、500、100和2000μg/mL，每个浓度重复5次)处理24小时，加10μL的CCK 8孵育2小时，酶标仪测出吸光值，计算细胞增殖率。
测定结果如图2a和2b所示，与对照组相比，对于HSF细胞，在500ug/ml和1000μg/mlPM-7浓度下，HSF细胞增殖率分别增加了150％和180％。对于Huvec细胞，500μg/ml的PM-7可以略微提高细胞增殖率，而1000μg/ml的PM-7显着提高了80％的细胞增殖。而2000μg/ml的PM-7对两种细胞的细胞增殖表现出中等影响。
细胞划痕实验：HSF细胞在24孔板中以2×106个细胞/孔的密度培养并生长过夜。使用无菌的200μL移液器吸头刮擦细胞单层，并通过磷酸盐缓冲盐水处理洗涤细胞3次后，将细胞与具有/不具有不同浓度PM-7(250、500和1000μg/mL)的培养基一起温育16h，倒置显微镜(Motic，AE2000，China)获得不同时间的划痕图像。
测定结果如图2c所示，与对照组相比，PM-7处理16h后显著提高细胞的迁移能力。
(3)促皮肤修复肽PM-7对小鼠皮肤损伤模型的修复作用
小鼠用2％戊巴比妥钠麻醉后，背部两侧皮肤用剃毛器除毛，酒精消毒后，在其后背开两个直径8mm的创口，从第0天到第8天，使用不同的药物(PBS、PM-7和EGF)治疗伤口，每天两次，每次30ul。从第0天到第8天，每隔一天使用数码照片记录伤口状况。使用ImageJ软件计算伤口面积(残留伤口面积占原始面积的百分比)并通过GraphPad Prism进行量化。
结果如图3a和3b所示，与PBS治疗相比，PM-7的应用显着加速了小鼠的伤口闭合，在受伤后第3天，PM-7治疗小鼠的伤口面积比PBS治疗小鼠的伤口面积小50％，这表明PM-7诱导了伤口愈合的快速加速。受伤后第5天，PM-7和表皮生长因子(EGF)治疗的伤口几乎闭合，而对照组小鼠的伤口保持45％未闭合。7天后，PM-7处理的小鼠没有明显的伤口，然而，PBS处理的小鼠的伤口仍然存在。
在不冲突的情况下，本文中上述实施例及实施例中的特征可以相互结合。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 成都佩德生物医药有限公司
<120> 一种促皮肤修复多肽PM-7及应用
<141> 2022-04-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213> Polypedates megacephalus
<400> 1
Phe Leu Asn Trp Arg Arg Ile Leu Phe Leu Lys Val Val Arg
1 5 10
Claims (3)
1.一种促皮肤修复多肽PM-7，其特征在于，所述多肽PM-7的
氨基酸序列为SEQ ID NO:1 FLNWRRILFLKVVR。
2.一种促皮肤修复多肽PM-7在制备促进伤口愈合药物中的应
用，其特征在于，所述促皮肤修复多肽PM-7的分子量为1860.32Da，等电点为12.3，氨基酸序列为SEQ ID NO:1 FLNWRRILFLKVVR。
3.一种促皮肤修复多肽PM-7在制备促进伤口愈合化妆品中的
应用，其特征在于，所述促皮肤修复多肽PM-7的分子量为1860.32Da，等电点为12.3，氨基酸序列为SEQ ID NO:1 FLNWRRILFLKVVR。
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