WO2023125770A1 - 一种多肽在制备用于预防或治疗皮肤损伤疾病产品中的应用 - Google Patents
一种多肽在制备用于预防或治疗皮肤损伤疾病产品中的应用 Download PDFInfo
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Abstract
提供了一种多肽在制备用于预防或治疗皮肤损伤疾病的产品中的应用,该多肽为Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu或其生理学上相容的盐。
Description
本发明涉及一种多肽在制备用于预防或治疗皮肤损伤疾病产品中的应用,本发明的多肽具有显著的预防和/或治疗皮肤损伤疾病,尤其是具有预防和/或治疗体表慢性难愈合创面、急和/或慢性皮肤病的效果。
人类皮肤由表皮、真皮、皮下组织构成,并含有附属器官(汗腺、皮脂腺)以及血管、淋巴管、神经和肌肉等。皮肤是人体最外层的器官,也是在人体器官中属于最大的器官,而且是和外界接触最频繁最密切的,它覆盖在人体表面,除保护机体、调节体温、抵抗外界侵害外,还有感觉功能,对保护人体健康起着重要的作用,所以称皮肤是人体的第一道防线。由于皮肤覆盖于人体表面,易于受到外力损伤、化学性刺激、微生物侵染等。皮肤损伤是指在皮肤黏膜上可以看到或者是摸到的异常表现。皮肤损伤易给患者带来心理和生理的双重打击。
近年来,在由皮肤损伤引起的疾病中,体表慢性难愈合创面疾病受到了较大关注。体表慢性难愈合创面又称难治性溃疡,是一种常见的难治性疾病。因各种原因引起的局部组织缺损、液化、感染、坏死,导致内皮祖细胞增殖受损,引起血管再生障碍,伤口灌注不足、代谢障碍,以及上皮细胞形成延迟,伤口的正常愈合过程遭到破坏,从而形成慢性溃疡。
体表慢性难愈合创面是糖尿病、周围血管病和放疗等常见的并发症。国际伤口愈合学会对其定义为:无法通过正常有序而及时的修复过程达到解剖和功能上完整状态的创面。临床多指各种原因形成的创面,经1个月以上正规治疗未能愈合,也无明显愈合倾向的创面。创面愈合经历三个阶段:①炎症阶段;②增殖阶段;③成熟和重建阶段。慢性创面由于炎症期延长,阻碍了血管内皮细胞和成纤维细胞的增殖以及胶原基质的沉积。
常见的慢性难愈性创面包括糖尿病足溃疡、压迫性溃疡、血管性溃疡、神经营养不良性溃疡、感染性溃疡、创伤性溃疡、自身免疫性溃疡、癌性溃疡、放射性溃疡等,其发病率随着年龄增加而上升,发病机制复杂,病程较长。
糖尿病足溃疡主要是由于糖尿病基础疾病引发了神经和血管病变,导致皮肤组织缺血坏死或者是皮肤组织感染,或者由于承重点的受压造成破损,皮肤不完整,露出皮下脂肪、肌肉组织,甚至骨骼,形成了溃疡。糖尿病足溃疡造成的危害,轻度可导致皮肤出现瘙痒、无汗、干燥、色素沉着,脚部出现微痛、感觉迟钝、间歇性跛行、关节变形、皮肤表面溃疡;进 一步发生发展到中度会出现比较深的溃疡合并感染;严重会累及骨头,造成骨折以及骨头坏死;更严重的有可能要被截肢。糖尿病足溃疡是糖尿病患者致残,甚至致死的重要原因之一,不但给患者造成痛苦,而且使其增添了巨大的经济负担。目前,糖尿病足溃疡多通过控制原发病进行治疗,但由于治疗过程较为缓慢,在治疗过程中局部溃疡易发生感染,从而造成严重后果。
压迫性溃疡,是因机体局部组织受压,血液循环障碍,导致局部皮肤缺血、缺氧、营养缺乏而致使皮肤失去正常功能出现组织的破损和坏死。常发生在卧床老年患者的骨性突出部位,如骶尾部、枕部、足跟部。这些地方软组织少,耐压能力弱,固定体位、较长时间压迫就可产生溃疡。压疮不仅给患者带来痛苦,延缓疾病的康复,严重者可因继发感染和败血症而危及生命。
血管性溃疡是由于下肢静脉曲张、脉管炎引起的下肢溃疡,以小腿远端及踝部多见,是下肢慢性功能不全晚期并发症。静脉回流严重受阻,局部静脉压增高、水肿,导致氧弥散障碍,皮肤营养缺乏。患者有长期的静脉原发病史,创面一般为单个,较为表浅,创基晦暗,创面周围皮肤粗糙且有明显的色素沉着。局部皮温很低。常规创面换药疗效很差,即使行断层皮片移植,疗效也极不可靠。
目前研究认为,皮肤创面修复是角质形成细胞、成纤维细胞、血管内皮细胞、炎性细胞、细胞外基质、细胞因子及生长因子等多因素共同参与、高度协调、相互调控的复杂过程。目前常用于皮肤损伤疾病预防或治疗的药物包括重组人表皮生长因子等,虽具有一定的预防和/或治疗效果,但仍存在吸收较慢,治疗周期较长,治疗效果不明显等缺点,尤其是应用于体表慢性难愈合创面及急和/或慢性皮肤病,治疗效果甚微。
发明内容
为了克服现有技术的不足和缺陷,本发明的目的在于提供一种多肽在制备预防或治疗皮肤损伤疾病的产品中的应用。
第一方面,本发明提供了一种多肽在制备用于预防或治疗皮肤损伤疾病的产品中的应用,该多肽为Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu或其生理学上相容的盐;或者提供了一种预防或治疗皮肤损伤疾病的方法,所述方法包括对皮肤损伤部位局部施用包含Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu或其生理学上相容的盐的产品;或者提供包含Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu或其生理学上相容的盐的产品,其用于预防或治疗皮肤损伤疾病。
进一步地,皮肤损伤疾病包括创面、溃疡、皮炎、湿疹、荨麻疹、多形性日光疹、疱疹、痤疮、脓疱疮、黄褐斑、白癜风、红斑狼疮、皮肌炎、硬皮病、毛囊炎、疥疮、灰指 甲、水痘、幼儿急疹、疣、痈、疖或癣。
进一步地,皮炎包括特应性皮炎、接触性皮炎、神经性皮炎、溢脂性皮炎、激素依赖性皮炎或淤积性皮炎。
进一步地,创面包括体表慢性难愈合创面。
进一步地,体表慢性难愈合创面包括糖尿病足溃疡、压迫性溃疡、血管性溃疡和感染性溃疡。
进一步地,产品包括药物、护肤品或化妆品。
进一步地,产品为外用制剂。
进一步地,外用制剂的剂型包括溶液、乳剂、凝胶、霜剂、喷雾、面膜或敷料。
进一步地,所述多肽是通过对人永生化角质形成细胞、人微血管内皮细胞、成纤维细胞、神经胶质细胞以及组织和/或血管再生产生促增殖作用而起到伤口愈合。
进一步地,所述生理学上相容的盐是指生理学上相容的(即药理学上可接受的)并且对将被施用本发明化合物的个体基本上无毒的盐形式。本发明化合物的生理学上相容的盐包括由适宜的、无毒的有机或无机酸或无机碱形成的常规的和化学计量的酸加成盐或碱加成盐。
本发明提供的多肽在制备预防或治疗皮肤损伤疾病、尤其是体表慢性难愈合创面产品上应用的有益效果:
本发明的上述多肽,相较于现有技术中常用于皮肤损伤疾病治疗的重组人表皮生长因子,不仅对皮肤溃疡和损伤具有明显的伤口促愈合作用,且由于本发明的肽链短,皮肤吸收更快更好,体内外稳定性好,且对人永生化角质形成细胞、人微血管内皮细胞、成纤维细胞、神经胶质细胞以及组织和血管再生均具有显著的促增殖作用,因而对急和/或慢性皮肤病、体表慢性难愈合创面(糖尿病足溃疡、压迫性溃疡、血管性溃疡和感染性溃疡)具有显著的预防和/或治疗效果。本发明的上述多肽适用于制备预防或治疗皮肤损伤疾病产品,可以诱导损伤附近细胞去分化,去分化的细胞重新获得干细胞形态和细胞分裂潜能,形成新的单元来修复损伤创面,并能够产生积极的治疗和预防效果。
图1示出了多肽对HaCaT细胞株促增殖情况;
图2示出了多肽对HMEC-1细胞株促增殖情况;
图3示出了多肽对Balb-3T3细胞株促增殖情况;
图4示出了多肽对RSC96细胞株促增殖情况;
图5示出了多肽对斑马鱼尾鳍再生的影响;
图6示出了多肽对微血管缺失斑马鱼的再生血管面积的影响;
图7示出了多肽对微血管缺失斑马鱼的再生血管分支数的影响;
图8示出了多肽对STZ诱导糖尿病大鼠伤后第3天的新生肉芽组织生成率。
以下是结合具体试验对本发明的说明,并不是对本发明保护范围的限制。
实施例1:多肽的化学合成
多肽的合成采用常规固相合成方法,经过树脂溶胀、取代、脱保护、洗涤、氨基酸溶解、氨基酸活化和缩合过程、洗涤、再脱保护多个循环过程以及最后裂解和侧链脱保护。
缩写:HBTU表示苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐;Methanol表示甲醇;Tert-Butyl methyl ether表示甲基叔丁基醚;Ethanol表示乙醇;AA表示氨基酸;Cl-2-Cl-Resin表示2-氯三苯甲基氯树脂;Fmoc-Aa(n)等表示9-芴基甲氧基羰基的氨基酸;DIPEA为N,N-二异丙基乙胺;DCM为二氯甲烷;PIP为哌啶;DMF为N,N-二甲基甲酰胺;HOBt为1-羟基苯并三唑;DIC为N,N'-二异丙基碳二亚胺;TFA为三氟乙酸;TIPS为三异丙基硅烷。
多肽的合成和纯化的方法如下:
步骤1、全保护肽树脂的制备
(1)树脂溶胀:称取2-Chlorotrityl Chloride Resin 2.0192g(S=0.73mmol/g),加入到有筛板的合成管中,用40ml二氯甲烷溶胀30min,抽滤除去二氯甲烷。
(2)制备Fmoc-Asp(OtBu)-树脂:按树脂、Fmoc-Asp(OtBu)-OH、DIPEA,以1:1.5:1.65的摩尔比分别称取Fmoc-Asp(OtBu)-OH、DIPEA,溶解于二氯甲烷20ml中,加入合成管中。室温N
2鼓泡震荡1~3小时,直接向反应液中加入甲醇2ml封闭30min。然后分别用二甲基甲酰胺洗涤4次,25ml/次,抽干树脂。
(3)脱除Fmoc保护基:向反应器中加入20%哌啶-DMF(v/v)溶液20ml,N
2鼓泡反应20min,抽干;然后用二甲基甲酰胺洗涤6次,25ml/次,3分钟/次,抽干,茚三酮法检测Fmoc脱除结果。
(4)氨基酸预活化:在250ml圆底烧瓶中加入4.38mmol的Fmoc保护的氨基酸、5.26mmol HOBt、4.60mmol DIC,用1:1的DCM-DMF(v/v)20ml溶解,-5-0℃冰浴搅拌下预活化30~60min。
(5)氨基酸连接:将已活化的保护氨基酸溶液倒入反应器中,补加适量的DCM清洗用具。室温下N
2鼓泡反应1~3小时,茚三酮法检测氨基酸连接是否完全,若完全,抽干。树脂用二甲基甲酰胺洗涤4次,25ml/次,3min/次,抽干。每一种氨基酸、缩合剂的用量及具体反应时间见表1。
(6)第一个氨基酸缩合完成后,重复步骤(3)~(5),按照氨基酸顺序延长肽链至最后一个氨基酸偶联完毕。
(7)树脂肽用二氯甲烷洗涤6次,25ml/次,3min/次,抽干。
表1氨基酸、缩合剂的用量
氨基酸名称 | AA/eq | 氨基酸用量/g | HOBt/g | DIPEA/g | DIC/g |
Fmoc-L-Leu-OH | 4.38 | 1.55 | 0.71 | 0.57 | 0.58 |
Fmoc-L-Pro-OH | 4.38 | 1.48 | 0.71 | 0.57 | 1.16 |
Fmoc-L-Val-OH | 4.38 | 1.49 | 0.71 | 0.57 | 0.58 |
Fmoc-L-Pro-OH | 4.38 | 1.48 | 0.71 | 0.57 | 1.16 |
Fmoc-L-Glu(OtBu)-OH·H 2O | 4.38 | 1.94 | 0.71 | 0.57 | 1.16 |
Fmoc-L-Ala-OH·H 2O | 4.38 | 1.44 | 0.71 | 0.57 | 1.16 |
Fmoc-L-Ala-OH·H 2O | 4.38 | 1.44 | 0.71 | 0.57 | 1.16 |
Fmoc-L-Pro-OH | 4.38 | 1.48 | 0.71 | 0.57 | 1.16 |
步骤2、切割、脱保护
(1)在步骤1的合成管中,加入切割剂(TFA:TIPS:H
2O=95:2.5:2.5,v/v)50ml,N
2鼓泡反应1.5-3小时。
(2)切割反应完成后,将切割剂抽滤至250ml圆底烧瓶中。真空浓缩至原切割剂体积的三分之一后,加入10倍现有体积的甲基叔丁基醚,搅拌30min。将所得混合溶剂过滤,并用30ml甲基叔丁基醚分别清洗3次后,将所得粗肽产品置于砂芯漏斗在通风橱中N
2吹干,使溶剂挥发至粗肽为粉末状。
步骤3、纯化(换盐)和冻干
采用以下色谱参数条件A,对步骤2中获得的粗肽进行HPLC纯化。具体而言,用水和/或乙腈溶解步骤2中获得的粗肽,并经0.45μm滤膜过滤;进样;用乙腈-水流动相梯度洗脱;收集目的肽洗脱液;最后进行旋蒸浓缩。
色谱参数条件A:
色谱柱:YMC-Actus Triart C18 30*250mm;
洗脱液A:0.1%(v/v)TFA/H
2O;
洗脱液B:乙腈;
流速25ml/min;
紫外检测波长220nm;
表2梯度洗脱条件
时间min | 洗脱液A(%) | 洗脱液B(%) |
0 | 90 | 10 |
30 | 75 | 25 |
接着采用以下色谱参数条件B利用HPLC法对上述步骤所获得的产品进行换盐:使用95%A1+5%B平衡色谱柱;然后进样;接着采用95%A2+5%B平衡色谱柱;用A1和B梯度洗脱;收集目的肽洗脱液;最后旋蒸浓缩并且冷冻干燥,得到多肽。多肽的结构经MS、
1H-NMR确认。
色谱参数条件B:
色谱柱:YMC-Actus Triart C18 30*250mm
洗脱液A1:0.1M乙酸
洗脱液A2:0.025M乙酸+0.1M乙酸铵
洗脱液B:乙腈
流速25ml/min
紫外检测波长220nm
表3梯度洗脱条件
时间min | 洗脱液A1(%) | 洗脱液B(%) |
0 | 95 | 5 |
5 | 95 | 5 |
35 | 70 | 30 |
多肽的
1H-NMR如下:
1H NMR(600MHz,DMSO)δ8.25(s,1H),8.09(d,J=7.5Hz,1H),7.94(d,J=7.6Hz,1H),7.89(d,J=8.3Hz,2H),4.53–4.46(m,1H),4.39(dd,J=8.3,4.2Hz,1H),4.34(dd,J=8.4,3.8Hz,1H),4.31–4.19(m,3H),4.13(dd,J=15.1,7.7Hz,1H),3.71–3.49(m,5H),2.94–2.77(m,2H),2.33–2.20(m,2H),2.06–1.77(m,13H,AcOH),1.77–1.56(m,6H),1.46(t,J=7.3Hz,2H),1.25–1.11(m,6H),0.95–0.76(m,12H).
多肽的MS:793.4,397.3(双电荷)。
多肽的氨基酸序列:Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu。
本申请实施例中使用以下物质:
多肽,除非另有指出即氨基酸序列为Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu的多肽。
金因肽,即商品名:金因肽(GeneTime),通用名称:重组人表皮生长因子外用溶液(Ⅰ),英文名:Recombinant Human Epidermal Growth Factor Derivative for External Use,Liquid;生产厂家:深圳市华生元基因工程发展有限公司。其成分:活性成分为重组人表皮生长因子(rhEGF),以10%的甘油和1.0的甘露醇为保护剂,金因肽中的rhEGF具有促进皮肤与粘膜创面组织修复过程中的DNA、RNA和羟脯氨酸的合成,加速创面肉芽组织生成和上皮细胞 增殖。从而缩短创面的愈合时间。
康复新液(Kangfuxin Ye);生产厂家:四川好医生攀西药业有限责任公司;其是美洲大蠊干燥虫体提取物的溶液;其用于金疮、外伤、溃疡、瘘管、烧伤、烫伤、褥疮之创面。
实施例2多肽对db/db小鼠皮肤溃疡愈合作用
实验动物:糖尿病造模小鼠(db/db雄性小鼠),正常小鼠(m/m雄性小鼠),SPF级,8-10周,体重40~50g,常州卡文斯实验动物有限公司,实验动物生产许可证号:SCXK(苏)2016-0010,实验动物使用许可证号:SYXK(沪)2020-0038。
实验方法:待db/db小鼠血糖达标后(禁食4h血糖≥16.7mmol/L),开始分组试验。按照随机分组法将动物分成5组,每组10只,分别为正常对照组,模型对照组,康复新液组(40μL/只),金因肽组(40IU/cm
2),受试物多肽组(30μg/cm
2)。小鼠异氟烷吸入麻醉后用切割器在小鼠背部将皮肤全层切割一直径约12mm的圆形创面,深达筋膜层,创面拍照并描记创面面积作为基线值(创伤当日记为Day0)。次日(Day1)开始各组动物按照应给予药物治疗,均皮肤创面处外涂,正常对照组、模型对照组均给予40μL生理盐水,金因肽组(40IU/cm
2)给予40μL生理盐水稀释的重组人表皮生长因子外用溶液(深圳市华生元基因工程发展有限公司),受试物多肽组(30μg/cm
2)分别给予40μL生理盐水配制的相应浓度的多肽溶液,一天1次;康复新液组给予40μL康复新液(四川好医生攀西药业有限责任公司),一天2次。每天连续给药,直至创面基本愈合。每周2次测量皮肤创面并进行拍照,用Image-J软件计算创面面积,并观察各组别动物的创面愈合情况。
实验结果:实验结果见表4所示:
表4创面愈合率(%,Mean±SD,n=10)
注:与模型对照组相比,*:P<0.05;**:P<0.01;
表4研究结果表明,多肽在第14天,有显著促进糖尿病小鼠皮肤伤口愈合的作用,且效果优于康复新液组,且多肽组的效果与金因肽组效果相当。
实施例3多肽对大鼠急性机械皮肤损伤愈合作用
实验动物:SD雄性大鼠,SPF级,6周龄,体重180~220g,北京维通利华实验动物技术有限公司;动物许可证号:SCXK(京)2016-0006;合格证号:NO.110011210104746538。
实验方法:SPF级SD大鼠分别饲养于干净消毒笼内,每天定时给予水、饲料和更换垫料,保持饲养温度20~26℃,湿度40%~70%,饲养一周使其适应环境。按照随机分组法将动物分成3组:模型对照组(生理盐水)、金因肽组(40IU/cm
2,重组人表皮生长因子外用溶液,深圳市华生元基因工程发展有限公司)、受试物多肽组(8μg/cm
2),每组6只。大鼠用3%戊巴比妥钠腹腔注射麻醉成功后,剪去创口边缘1厘米处毛发,先用碘伏消毒创口区,再用75%的酒精局部消毒创口区,于耳连线中间向背部下4cm近颈侧以脊柱为中线位制作一直径是1.5cm圆形全层皮肤创口,深至肌层。用相同大小的橡胶圈固定其周围皮肤,形成急性机械性损伤动物模型。造模后大鼠伤口暴露,单笼饲养。换药时均先用碘伏清创,再以无菌生理盐水冲洗创面并拭干,各组别大鼠再给予40μL相应药物溶液,每日定时创面局部涂抹给药1次。在给药第0、3、7、10、14天,摄取每组大鼠的创面图像,采用图像分析软件(Image J)计算创面面积,根据公式计算创面愈合率。
实验结果:实验结果见表5所示:
表5创面愈合率(%,Mean±SD,n=6)
注:与模型对照组相比,*:P<0.05;**:P<0.01;
表5研究结果表明,多肽组在第14天显示出显著的促进大鼠皮肤机械损伤后伤口愈合的作用,且效果明显优于金因肽组。
实施例4多肽对HaCAT细胞增殖的促进作用
实验方法:将人永生化角质形成细胞(HaCaT细胞)浓度调整为1.0×10
5~5.0×10
5/mL进行传代培养,于37℃,5%CO
2条件下培养24~36小时用于生物学活性检测。用0.25%胰酶消化细胞5min,加入胰酶体积1倍以上的1640全血培养基终止消化,收集细胞悬液,1000RPM离心3min,弃掉上清,加入2mL的1640全血培养基重悬细胞,取20uL细胞悬液,用AOPI染色,再用细胞计数仪检测悬液中细胞的浓度,用10%血清浓度的1640培养基配成浓度5×10
4/mL接种于96孔细胞培养板中,每孔100μL,即5000个细胞/孔,于37℃,5%CO
2 条件下培养过夜。
24h后弃掉原培养基,加入100μL 1%血清浓度的1640培养基配制的不同浓度多肽溶液;同时设置EGF对照组,即加入100μL用1%血清浓度的1640培养基配制的重组人表皮生长因子(EGF)溶液,终浓度为100ng/mL;模型对照组,即加入等体积的1%血清浓度的1640培养基。于37℃,5%CO
2条件下培养72h,采用CCK8试剂盒检测HaCaT细胞株增殖情况。
实验结果:实验结果见图1所示(*:P<0.05;**:P<0.01)。图1研究结果表明,多肽作用72小时后对参与创面修复过程中的主要细胞类型之一的表皮细胞:HaCaT细胞(人永生化角质形成细胞)具有显著的促增殖作用。
实施例5多肽对HMEC-1细胞增殖的促进作用
实验方法:将人微血管内皮细胞(HMEC-1细胞)用10%血清培养基于37℃,5%CO
2条件下培养,每1~2d换液,控制细胞浓度为4×10
6细胞/mL进行传代。收集细胞,用无血清培养基配成4×10
4细胞/mL;接种于96孔细胞培养板中,每孔100uL,即4000个细胞/孔,于37℃,5%CO
2条件下培养至细胞贴壁。
多肽溶于PBS溶液配置成浓度为1mg/mL的母液,将多肽母液用0%血清培养基配制成受试浓度作为实验组,同样的方法配置重组人血管内皮生长因子VEGF(100ng/mL)加入作为阳性对照,空白对照组加入等量0%血清不含药物培养基,5复孔加入,于37℃,5%CO
2条件下孵育48小时。使用CCK-8检测细胞的增殖情况:每孔加入含10%CCK-8的无血清培养基,在培养箱内孵育2h后用酶标仪在450nm处测量吸光度。
实验结果:实验结果见图2所示(*:P<0.05;**:P<0.01)。图2研究结果表明,多肽对参与创面修复过程中的主要细胞类型之一的内皮细胞:HMEC-1细胞(人微血管内皮细胞)具有显著的促增殖作用。
实施例6多肽对Balb-3T3细胞增殖的促进作用
实验方法:将小鼠胚胎成纤维细胞(Balb-3T3细胞)用10%血清培养基于37℃,5%CO
2条件下培养,每1~2d换液,控制细胞浓度为4×10
6细胞/mL进行传代。收集细胞,用2.5%FBS维持培养基配成3×10
4细胞/mL;接种于96孔细胞培养板中,每孔100uL,即3000个细胞/孔,于37℃,5%CO
2条件下培养至细胞贴壁。
多肽溶于2.5%FBS维持培养基配置成浓度为1mg/mL的母液,将多肽母液用2.5%FBS维持培养基配制成受试浓度作为实验组,同样的方法配置重组人碱性成纤维细胞生长因子FGF(50ng/mL),重组人血小板衍生生长因子PDGF-BB(30ng/mL)加入作为阳性对照,空白对照组加入等量2.5%FBS维持培养基,5复孔加入,于37℃,5%CO
2条件下孵育48小时。使用CCK-8检测细胞的增殖情况:每孔加入含10%CCK-8的无血清培养基,在培养箱 内孵育2h后用酶标仪在450nm处测量吸光度。
实验结果:实验结果见图3所示(*:P<0.05;**:P<0.01)。图3研究结果表明,多肽对参与创面修复过程中的主要细胞类型之一的成纤维细胞:Balb-3T3细胞(小鼠胚胎成纤维细胞)具有显著的促增殖作用。
实施例7多肽对RSC96细胞增殖的影响
实验方法:将大鼠雪旺细胞(RSC96细胞)浓度调整为1.0×10
5~5.0×10
5/mL进行传代培养,于37℃,5%CO
2条件下培养24~36小时用于生物学活性检测。用胰酶消化并收集细胞,用无血清培养基配成浓度5×10
4/mL接种于96孔细胞培养板中,每孔100μL,即8000个细胞/孔,于37℃,5%CO
2条件下培养过夜。
多肽溶于PBS溶液配置成浓度为400ug/ml的母液,将多肽母液用0%血清培养基配制成受试浓度作为实验组,对照组加入等量0%血清不含药物培养基,4复孔加入,于37℃,5%CO
2条件下孵育48小时。使用CCK-8检测细胞的增殖情况:除去旧培养基,每孔加入含10%CCK-8的无血清培养基,在培养箱内孵育2h后用酶标仪在450nm处测量吸光度。
实验结果:实验结果见图4所示(*:P<0.05;**:P<0.01)。图4研究结果表明,多肽对RSC96细胞具有显著的促增殖作用。
实施例8多肽促进斑马鱼组织再生作用
实验动物:野生型AB品系斑马鱼,饲养于28℃的养鱼用水中(水质:每1L反渗透水中加入200mg速溶海盐,电导率为450~550μS/cm;pH为6.5~8.5;硬度为50~100mg/L CaCO
3),年龄为受精后3天(3dpf)的斑马鱼。由杭州环特生物科技股份有限公司养鱼中心繁殖提供,实验动物使用许可证号为:SYXK(浙)2012-0171,饲养管理符合国际AAALAC认证(认证编号:001458)的要求。
实验方法:随机选取3dpf野生型AB品系斑马鱼,切除斑马鱼的尾鳍建立斑马鱼尾鳍损伤模型。将模型斑马鱼随机分配至6孔板中,每孔(每实验组)均放置30尾斑马鱼。分别水溶给予不同浓度多肽(终浓度为250、500、1000μg/mL),同时设置正常对照组(斑马鱼尾鳍无损伤)和模型对照组,每孔容量为3mL。置于28℃处理3天后,每个实验组随机选取10尾斑马鱼置于解剖显微镜下拍照,用NIS-Elements D 3.20高级图像处理软件分析并采集数据,分析斑马鱼尾鳍再生面积,以该指标的统计学分析结果评价样品促进组织再生功效。
实验结果:实验结果见图5所示(与模型对照组比较,*:P<0.05,**:P<0.01)。研究结果表明,多肽具有显著促进斑马鱼组织再生的功效。
实施例9多肽促斑马鱼血管再生作用
实验动物:血管绿色荧光转基因斑马鱼,饲养于28℃的养鱼用水中(水质:每1L反渗 透水中加入200mg速溶海盐,电导率为450~550μS/cm;pH为6.5~8.5;硬度为50~100mg/L CaCO
3),年龄为受精后1天(1dpf)的斑马鱼。由杭州环特生物科技股份有限公司养鱼中心繁殖提供,实验动物使用许可证号为:SYXK(浙)2012-0171,饲养管理符合国际AAALAC认证(认证编号:001458)的要求。
实验方法:随机选取1dpf血管绿色荧光转基因斑马鱼于6孔板中,每孔(每实验组)30尾。正常对照组用标准稀释水处理斑马鱼,其余各实验组均水溶给予60nM辛伐他汀诱导3h建立斑马鱼微血管缺失模型,每孔容量为3mL。3h后,终止辛伐他汀诱导。然后阳性对照组水溶液用5.90μg/mL黄芪甲苷替换,其余各组别水溶液替换为标准稀释水,每孔容量为3mL。受试药物组静脉注射给予不同剂量多肽,即浓度分别为12.5、25.0、50.0mg/mL,注射体积均为10nL。置于28℃处理2天,每个实验组随机选取10尾斑马鱼置于荧光显微镜下拍照,用NIS-Elements D 3.20高级图像处理软件分析并采集数据,分析肠下血管面积和肠下血管分支数,以该指标的统计学分析结果评价样品促血管再生功效。
实验结果:实验结果见图6和图7所示(与模型对照组比较,*:P<0.05,**:P<0.01)。研究结果表明,多肽能够显著促进斑马鱼肠下血管面积增加,肠下血管分支数增加,具有促进血管再生的功效。
实施例10多肽对链脲佐菌素(Streptozotocin,STZ)诱导糖尿病大鼠皮肤溃疡的愈合作用
实验动物:SPF级雄性健康SD大鼠(体重180~200g)经高脂、高糖的饲料喂养2周后,禁食6h,每只大鼠一次性快速腹腔注射STZ溶液(50mg·kg
-1),48h后2次随机血糖值>16.7mmol/L,认为Ⅱ型糖尿病大鼠模型造模成功。实验动物购自北京维通利华实验动物技术有限公司,实验动物质量合格证号SCXK(京)2017-0033;实验动物使用许可证:SCXK(京)2017-0033。
实验方法:选取90只糖尿病大鼠随机分成6组,每组15只,分别为模型对照组(生理盐水),多肽高剂量组(30μg/cm
2),多肽中剂量组(10μg/cm
2),多肽低剂量组(3μg/cm
2),金因肽组(40IU/cm
2,重组人表皮生长因子外用溶液,深圳市华生元基因工程发展有限公司),康复新液组(36μL/cm
2,康复新液,四川好医生攀西药业有限责任公司)。每只实验大鼠用2%戊巴比妥钠(0.2mL/100g)腹腔注射麻醉后,于背部脊柱两侧各制备1个2cm
2的全层皮肤缺损创面,并于创面外缘处加缝直径2.3cm的橡胶圈。创面止血后按照组别进行创面局部药物治疗。除康复新液组给药2次/天,其余组给药均为1次/天,给药体积均为每次72μL/创面,连续14天。创面给药后覆盖无菌凡士林纱布(5cm×5cm),以及多层无菌纱布包扎。创面手术后对创面拍照并描记创面面积作为基线值(创伤当日记为Day0),给药期间每周3次对创面面积拍照测量,并每天观察各组别创面的肉芽组织生长及愈合情况。
实验结果:见表6和图8(注:与模型对照组相比,*:P<0.05;**:P<0.01)。
由表6可以看出,在Day10时,多肽低剂量组、中剂量组愈合率稍高于模型对照组,说明多肽低、中剂量对糖尿病大鼠创面具有促进愈合的趋势;在创面愈合全过程,而多肽高剂量组愈合率均较高,在Day10显著高于模型对照组,且稍高于金因肽组,在Day7-Day12天,愈合率稍高于康复新液组,说明多肽高剂量组对糖尿病大鼠创面具有显著促愈合作用,且在该实验中效果稍优于金因肽。由图8可以看出,多肽低、中、高剂量组促进新生肉芽组织生长率相较于模型对照组均具有显著性差异。因此,多肽对STZ诱导的糖尿病大鼠创面具有促进新生肉芽组织生长以及创面愈合的作用。
表6各组伤后不同天数糖尿病大鼠创面愈合率(%,Mean±SD,n=15)
实施例11多肽对SD大鼠全皮层缺损创面的愈合作用
实验动物:SD雄性大鼠,SPF级,6周龄,体重180~220g,北京维通利华实验动物技术有限公司;实验动物质量合格证号:NO.110011220109610345;实验动物使用许可证:SCXK(京)2021-0011。
实验方法:SD大鼠按照体重随机分组法将动物分成8组,每组8只;分别为溶媒组、多肽组(50μg/cm
2)、对比多肽1组(41.90μg/cm
2)、对比多肽2组(52.66μg/cm
2)。对比多肽1的氨基酸序列为:Glu-Pro-Val-Pro-Leu;对比多肽2的氨基酸序列为:Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu-Val-Lys-Gln-Glu。
每只实验大鼠用2%戊巴比妥钠(0.2mL/100g)腹腔注射麻醉后,于背部脊柱两侧各制备1个2cm
2的全皮层缺损创面。创面止血后按照组别在创面局部给予相应药物。各组给药均为1次/天,给药体积均为每次72μL/创面,连续14天。创面给药后覆盖无菌凡士林纱布(5cm×5cm),以及多层无菌纱布包扎。创面手术后对创面拍照并描记创面面积作为基线值(创伤当日记为Day0),给药期间每周3次对创面面积拍照测量及观察愈合情况。
实验结果:实验结果见表7所示:
表7创面愈合率(%,Mean±SD,n=8)
Day0 | Day3 | Day5 | Day7 | Day10 | |
溶媒组 | 0.00±0.00 | 11.01±23.19 | 38.94±18.95 | 61.81±11.97 | 87.77±5.65 |
多肽组 | 0.00±0.00 | 29.70±19.21* | 52.21±11.54* | 74.12±5.95*** | 94.55±2.40*** |
对比多肽1组 | 0.00±0.00 | 21.13±17.43 | 40.18±17.43 | 68.84±7.64 | 88.90±8.40 |
对比多肽2组 | 0.00±0.00 | 17.21±22.78 | 50.08±16.55 | 65.26±14.46 | 90.34±3.04 |
注:与溶媒组相比,*:P<0.05;**:P<0.01;
研究结果表明,与溶媒组相比,本发明多肽组(50μg/cm
2)在术后第3~10天愈合率较高且具有显著性差异;并且,本发明多肽的愈合效果均优于对比多肽1组和对比多肽2组。
综上所述,本发明的上述多肽,相较于现有技术中常用于皮肤损伤疾病治疗的重组人表皮生长因子,不仅对皮肤溃疡和损伤,尤其是糖尿病足溃疡、压迫性溃疡、血管性溃疡和感染性溃疡等体表慢性难愈合创面、急和/或慢性皮肤病、糖尿病足溃疡等具有明显的伤口促愈合作用,且由于本发明的肽链短,皮肤吸收更快更好,体内外稳定性好,且对人永生化角质形成细胞、人微血管内皮细胞、成纤维细胞、神经胶质细胞以及组织和血管再生均具有显著的促增殖作用。本发明的上述多肽适用于制备预防或治疗皮肤损伤疾病产品,并能够产生积极的治疗和修复效果。
尽管本发明披露了上述实施例,但本发明的实施方式并不限于上述实施例,其他的任何未背离本发明的改变、修饰、替代、组合、简化,均应为等效的置换方式均包含在本发明的保护范围之内。
Claims (9)
- 一种多肽在制备用于预防或治疗皮肤损伤疾病的产品中的应用,其特征在于,所述多肽为Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu或其生理学上相容的盐。
- 根据权利要求1所述的应用,其特征在于,所述皮肤损伤疾病包括创面、溃疡、皮炎、湿疹、荨麻疹、多形性日光疹、疱疹、痤疮、脓疱疮、黄褐斑、白癜风、红斑狼疮、皮肌炎、硬皮病、毛囊炎、疥疮、灰指甲、水痘、幼儿急疹、疣、痈、疖或癣。
- 根据权利要求2所述的应用,其特征在于,所述皮炎包括特应性皮炎、接触性皮炎、神经性皮炎、溢脂性皮炎、激素依赖性皮炎或淤积性皮炎。
- 根据权利要求2所述的应用,其特征在于,所述创面包括体表慢性难愈合创面。
- 根据权利要求4所述的应用,其特征在于,所述体表慢性难愈合创面包括糖尿病足溃疡、压迫性溃疡、血管性溃疡或感染性溃疡。
- 根据权利要求1所述的应用,其特征在于,所述产品包括药物、护肤品或化妆品。
- 根据权利要求1所述的应用,其特征在于,所述产品为外用制剂。
- 根据权利要求7所述的应用,其特征在于,所述外用制剂包括溶液、乳剂、凝胶、乳剂、霜剂、凝胶、喷雾、面膜或敷料。
- 根据权利要求7所述的应用,其特征在于,所述多肽是通过对人永生化角质形成细胞、人微血管内皮细胞、成纤维细胞、神经胶质细胞以及组织和/或血管再生产生促增殖作用而起到伤口愈合。
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WO2013098743A1 (en) * | 2011-12-27 | 2013-07-04 | Biodue S.P.A. | Composition for a medical device or for a cosmetic or pharmaceutical preparation comprising a decapeptide derived from deinococcus radiodurans |
CN104524544A (zh) * | 2014-12-05 | 2015-04-22 | 重庆市畜牧科学院 | 一种多肽在治疗糖尿病中的应用 |
NZ711281A (en) * | 2013-02-14 | 2017-12-22 | Helix Biomedix Inc | Short bio-active peptides for promoting wound healing |
CN111748015A (zh) * | 2020-05-25 | 2020-10-09 | 昆明医科大学 | 一种活性多肽os-ll11及其应用 |
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WO2013098743A1 (en) * | 2011-12-27 | 2013-07-04 | Biodue S.P.A. | Composition for a medical device or for a cosmetic or pharmaceutical preparation comprising a decapeptide derived from deinococcus radiodurans |
NZ711281A (en) * | 2013-02-14 | 2017-12-22 | Helix Biomedix Inc | Short bio-active peptides for promoting wound healing |
CN104524544A (zh) * | 2014-12-05 | 2015-04-22 | 重庆市畜牧科学院 | 一种多肽在治疗糖尿病中的应用 |
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