CN113827706B - Gip及其衍生肽作为制备皮肤创伤治疗药物中的应用 - Google Patents
Gip及其衍生肽作为制备皮肤创伤治疗药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物医学技术领域,具体涉及GIP及其衍生肽作为制备皮肤创伤治疗药物中的的应用,包括:GIP作为制备皮肤创伤治疗药物中的应用,GIP衍生肽作为制备皮肤创伤治疗药物中的应用,所述GIP衍生肽为GIP活性肽段或连有胶原结合域的GIP融合多肽。本发明通过合成GIP活性多肽以及连接有CBD结构域的GIP融合多肽,辅以凡士林或明胶或甲基丙烯酰化明胶构成皮肤损伤治疗药物,利用小鼠皮肤全层损伤模型,观察GIP及其衍生肽对皮肤损伤的恢复作用,结果显示GIP及其衍生肽能够促进损伤区域的神经及血管新生,降低胶原沉积、减少疤痕形成,有利于损伤皮肤的有效愈合。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及GIP及其衍生肽作为制备皮肤创伤治疗药物中的应用。
背景技术
皮肤是覆盖在人体表面的最大器官,具有屏障、感知、分泌排泄、调节体温等功能,是人体的最外层,其与外界环境持续接触,极易受到损伤。作为日常生活中的常见损伤形式,皮肤损伤是一个对公共健康和卫生经济的重要威胁。皮肤损伤往往不可避免地发生在各种事故中,尤其在某些情况下(糖尿病、感染等)伤口愈合过程会延迟且大都转为慢性创伤。一旦受到损伤后不能快速愈合,皮肤对有害刺激的防御能力减弱甚至丧失,继而会导致感染、休克甚至死亡的严重后果。因此,伤口的快速有效愈合至关重要。深二度创伤所致的皮肤全层缺损创面必须进行自体皮肤移植才能修复,但由于患者供皮区有限,自体皮肤移植修复创面一直受到限制。因此,使用其他类型的皮肤替代物来促进创面愈合已成为迫切的需要。目前尽管有关皮肤创伤愈合的研究很多,但在临床上皮肤创伤的治疗仍然是一大挑战。如皮肤替代品的融合不良(大都情况下是血管化不足的直接结果)、移植边缘疤痕的形成、缺乏完全分化,大面积植皮过程中表皮及真皮厚度不均等,创口的愈合很难达到完整的皮肤功能恢复。对于皮肤损伤的愈合及后续功能的恢复如毛发,汗腺等的再生而言,神经纤维的再支配是一个极为重要的前提条件,而目前以促进皮肤损伤后为切入点,并以此达到促进皮肤损伤修复的产品极少。我们发现了一种短肽,抑胃肽(Gastric InhibitoryPeptide,GIP)具有促神经轴突再生的功能,并且发现该短肽及功能衍生物具有很好的促皮肤损伤修复、减少疤痕的效果。
抑胃肽(GIP)是肠道分泌的响应营养摄入的重要多肽,最先被发现命名是因为其具有抑制胃酸分泌的作用,此后大量的研究发现GIP参与餐时胰岛素分泌的调节,因此又被称为葡萄糖依赖性胰岛素释放肽。研究发现其受体GIPR在人体的许多组织如大脑、胰腺、脂肪组织中表达,GIP在目的器官与其受体结合从而在不同系统中发挥多种作用,例如糖代谢、食欲控制以及其他重要的生理过程。GIP是肠促胰岛素的主要成员,文献报道肠促胰岛素能够促进细胞生长、分化,抑制凋亡,减轻氧化应激、内质网应激,促进血管内皮细胞增殖、血管新生发挥神经保护作用。同时本申请研究发现GIP能够有效刺激皮层神经元的轴突生长,另外本申请还发现GIP还有促进细胞迁移的效应,这些效应使得GIP可以作为新的外用药物在皮肤损伤修复中得到应用。
发明内容
针对以上问题,本发明提供了GIP及其衍生肽作为制备皮肤创伤治疗药物中的的应用。
为了实现上述目的,本发明采用的技术方案如下:
GIP作为制备皮肤创伤治疗药物中的应用。
本发明还提供了GIP衍生肽作为制备皮肤创伤治疗药物中的应用。
优选地,所述GIP衍生肽为GIP活性肽段或连有胶原结合域的GIP融合多肽。
优选地,所述皮肤创伤治疗药物为GIP活性多肽或连接有CBD结构域的GIP融合多肽,辅以凡士林或明胶或甲基丙烯酰化明胶构成的药物。
优选地,所述皮肤创伤治疗药物能够促进损伤区域的神经及血管新生,降低胶原沉积、减少疤痕形成,有利于损伤皮肤的有效愈合。
采用上述技术方案,通过合成GIP活性多肽以及连接有CBD结构域的GIP融合多肽,辅以凡士林或明胶或甲基丙烯酰化明胶构成皮肤损伤治疗药物,利用小鼠皮肤全层损伤模型,观察GIP及其衍生肽对皮肤损伤的恢复作用,结果显示GIP及其衍生肽能够促进损伤区域的神经及血管新生,降低胶原沉积、减少疤痕形成,有利于损伤皮肤的有效愈合。
本发明有益效果:
1、本发明中所用到的主要材料为GIP活性肽段或连有胶原结合域的GIP融合多肽,免疫原性低,辅以凡士林或明胶或甲基丙烯酰化明胶,在加工过程中基本未添加有毒、副作用的物质,因此具有良好的生物相容性。
2、本发明中所述的GIP活性肽段或连有胶原结合域的GIP融合多肽都能起到促进皮肤损伤修复的功效,其中GIP融合多肽能够结合明胶长时间发挥功能,克服了GIP半衰期短的缺点。
3、本发明中所述GIP及其衍生肽,在小鼠皮肤损伤模型中均显示出良好的促伤口愈合效果,有效促进伤口处神经再生,减少疤痕形成。
附图说明
图1是本发明中GIP-L-CBD组新生皮肤的效果图;
图2是本发明中GIP-L-CBD促进伤口的血管再生示意图;
图3是本发明中GIP-L-CBD促进损伤区域神经再生示意图;
图4是本发明中GIP-L-CBD减少损伤区域胶原沉积示意图。
具体实施方式
下面结合附图将对本发明实施例中的技术方案进行清楚、完整地描述,以使本领域的技术人员能够更好的理解本发明的优点和特征,从而对本发明的保护范围做出更为清楚的界定。本发明所描述的实施例仅是本发明一部分实施例,而不是全部的实施例,基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的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。
GIP及其衍生肽氨基酸序列如下所示:
GIP:YAEGTFISDYSIAMDKIRQQDFVNWLLAQK
GIP-CBD:YAEGTFISDYSIAMDKIRQQDFVNWLLAQKTKKTLRT
GIP-L-CBD:YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGGGGSTKKTLRT
实施例1:GIP直接涂抹促进小鼠皮肤损伤的恢复
1.小鼠全皮层损伤模型的构建
选择体重25g左右的SPF级ICR小鼠进行实验。腹腔注射1%的戊巴比妥钠进行麻醉,剃除背部毛发,暴露皮肤。使用75%酒精消毒,利用活检凿孔器在背部切出直径约为1cm的圆形伤口,损伤到达筋膜层。将标尺置于伤口附近拍照,利用Image J软件计算各实验组小鼠伤口的准确大小,为后续实验做准备。术后将小鼠放于加热毯上待其清醒后放回饲养室正常饲养。
2.分组及给药
将模型小鼠随机分为两组,空白对照组及GIP组,每组10只小鼠。GIP多肽使用生理盐水溶解至浓度为100nM-10μM,建立小鼠全皮层损伤模型后每天固定时间给药,空白对照组每天在伤口处涂生理盐水,GIP组涂布GIP溶液。损伤后14天收集伤口附近皮肤组织,使用4%多聚甲醛4℃固定过夜。
3.GIP的功能验证
1)HE染色观察伤口恢复情况
组织固定后更换30%蔗糖脱水24h,切片机内预冷冻台,加入OCT包埋剂,修平,包埋组织后将冷冻组织进行修整,切片厚度12-14μm,37℃过夜后可保存在-80℃。冰冻切片放置于室温复温30min,1×PBS洗10min后置于苏木精染色2.5min,随后用水洗5min。组织切片于分化液上分化30s,用水洗两次,每次10min。接下来伊红染色30s,水洗10min后进行脱水。使用75%、95%、100%、100%乙醇依次脱水1min。无水乙醇:二甲苯(1:1)、二甲苯(Ⅰ)、二甲苯(Ⅱ)各透明2min,中性树胶封片,风干后显微镜明场采集图像并观察伤口恢复情况。
2)免疫荧光检测新生皮肤的血管、神经再生及胶原沉积
制备12-16μm厚度的组织冰冻切片,结束后,37℃过夜。次日使用组织固定液37℃固定2h,加入适当浓度的CD34抗体标记血管内皮细胞、Tuj1标记神经元、COL1A1标记Ⅰ型胶原,4℃孵育过夜,次日室温复温0.5h,1×PBST洗3次,每次10min。加含有一定比例的相应二抗于组织上室温避光孵育2h。加一定比例的Hoechst于组织上,室温10min。1×PBST洗4×15min。荧光封片剂封片,使用荧光显微镜观察结果并采集图像。
实施例2:GIP混合凡士林制备软膏促进皮肤损伤修复
1.敷料的制备
将适量的GIP溶解于水中,配置浓度为100μM-10mM的GIP溶液,吸取100μL溶液,与10g热的液态白凡士林混合均匀,待其冷却形成半透明的粘稠膏剂,空白对照组将100μL的水与10g液态凡士林混匀作为治疗用软膏。
2.分组及给药
小鼠皮肤损伤模型同实施例1所述,造模成功后模型小鼠随机分为两组,每组10只,每天固定时间进行药物治疗,治疗组以GIP软膏涂抹,对照组涂抹凡士林软膏,涂抹时使膏剂覆盖伤口,并在损伤后14天收集伤口附近皮肤,检测GIP软膏对皮肤损伤的作用。
3.GIP的功能验证
1)HE染色观察伤口恢复情况
组织固定后更换30%蔗糖脱水24h,切片机内预冷冻台,加入OCT包埋剂,修平,包埋组织后将冷冻组织进行修整,切片厚度12-14μm,37℃过夜后可保存在-80℃。冰冻切片放置于室温复温30min,1×PBS洗10min后置于苏木精染色2.5min,随后用水洗5min。组织切片于分化液上分化30s,用水洗两次,每次10min。接下来伊红染色30s,水洗10min后进行脱水。使用75%、95%、100%、100%乙醇依次脱水1min。无水乙醇:二甲苯(1:1)、二甲苯(Ⅰ)、二甲苯(Ⅱ)各透明2min,中性树胶封片,风干后显微镜明场采集图像并观察伤口恢复情况。
2)免疫荧光检测新生皮肤的血管、神经再生及胶原沉积
制备12-16μm厚度的组织冰冻切片,结束后,37℃过夜。次日使用组织固定液37℃固定2h,加入适当浓度的CD34抗体标记血管内皮细胞、Tuj1标记神经元、COL1A1标记Ⅰ型胶原,4℃孵育过夜,次日室温复温0.5h,1×PBST洗3次,每次10min。加含有一定比例的相应二抗于组织上室温避光孵育2h。加一定比例的Hoechst于组织上,室温10min。1×PBST洗4×15min。荧光封片剂封片,使用荧光显微镜观察结果并采集图像。
实施例3:GIP及其衍生多肽结合明胶促进皮肤损伤的修复
1.涂料的制备
称取适量明胶,加入水于60℃溶解,配成20%-30%(w/v)的明胶溶液,将适量的GIP、GIP-CBD及GIP-L-CBD溶解于明胶溶液中,配置浓度为100nM-10mM的混合溶液制成涂剂,空白对照组使用未添加GIP及其衍生肽的明胶溶液。GIP-CBD及GIP-L-CBD具有胶原结合域,能够结合明胶发挥缓释效果,延长作用时间。
2.分组及给药
小鼠皮肤损伤模型同实施例1所述,造模成功后模型小鼠随机分为四组,每组10只,每天固定时间进行药物治疗,治疗组以GIP、GIP-CBD或GIP-L-CBD涂剂涂抹,对照组涂抹明胶,涂抹时覆盖伤口,并在损伤后14天收集伤口附近皮肤,检测GIP衍生肽对皮肤损伤的作用。
3.GIP衍生肽的功能验证
1)HE染色观察伤口恢复情况
组织固定后更换30%蔗糖脱水24h,切片机内预冷冻台,加入OCT包埋剂,修平,包埋组织后将冷冻组织进行修整,切片厚度12-14μm,37℃过夜后可保存在-80℃。冰冻切片放置于室温复温30min,1×PBS洗10min后置于苏木精染色2.5min,随后用水洗5min。组织切片于分化液上分化30s,用水洗两次,每次10min。接下来伊红染色30s,水洗10min后进行脱水。使用75%、95%、100%、100%乙醇依次脱水1min。无水乙醇:二甲苯(1:1)、二甲苯(Ⅰ)、二甲苯(Ⅱ)各透明2min,中性树胶封片,风干后显微镜明场采集图像并观察伤口恢复情况。
2)免疫荧光检测新生皮肤的血管、神经再生及胶原沉积
制备12-16μm厚度的组织冰冻切片,结束后,37℃过夜。次日使用组织固定液37℃固定2h,加入适当浓度的CD34抗体标记血管内皮细胞、Tuj1标记神经元、COL1A1标记Ⅰ型胶原,4℃孵育过夜,次日室温复温0.5h,1×PBST洗3次,每次10min。加含有一定比例的相应二抗于组织上室温避光孵育2h。加一定比例的Hoechst于组织上,室温10min。1×PBST洗4×15min。荧光封片剂封片,使用荧光显微镜观察结果并采集图像。
实施例4:GIP及其衍生肽结合光固化改性明胶促进皮肤损伤修复
1.改性明胶的制备
称取10g明胶加入100mLpH 7.5的PB缓冲液中,于60℃加热溶解,随后加入8mL甲基丙烯酸酐与明胶溶液在50℃条件下搅拌反应3h,加入400mLPB缓冲液终止反应。使用截留10KD分子量的透析袋在蒸馏水中透析7天,每隔12h换液。冻干后保存在-80℃备用。
2.水凝胶的制备
用水将光引发剂Irgacure 2959配置为0.5%(w/v)的稀释液,用此稀释液配置含有GIP及其衍生肽的水凝胶溶液,其中甲基丙烯酰化明胶浓度为5%-30%,GIP及其衍生肽浓度为1-10mM,具体以伤口大小及严重程度进行调整。
3.实验分组及给药处理
将模型小鼠分为三组,分别为单明胶空白对照组、GIP组、GIP-CBD组及GIP-L-CBD组。按照实施例1建立小鼠全皮层损伤模型后将含有或不含GIP衍生肽的水凝胶溶液涂布于伤口表面,用发射405nm的紫外手电筒照射10-30秒使溶液成胶,隔天给药,损伤后14天收集伤口附近皮肤组织,制备冰冻组织切片。
4.GIP衍生肽的功能验证
1)HE染色观察伤口恢复情况
组织固定后更换30%蔗糖脱水24h,切片机内预冷冻台,加入OCT包埋剂,修平,包埋组织后将冷冻组织进行修整,切片厚度12-14μm,37℃过夜后可保存在-80℃。冰冻切片放置于室温复温30min,1×PBS洗10min后置于苏木精染色2.5min,随后用水洗5min。组织切片于分化液上分化30s,用水洗两次,每次10min。接下来伊红染色30s,水洗10min后进行脱水。使用75%、95%、100%、100%乙醇依次脱水1min。无水乙醇:二甲苯(1:1)、二甲苯(Ⅰ)、二甲苯(Ⅱ)各透明2min,中性树胶封片,风干后显微镜明场采集图像并观察伤口恢复情况。
2)免疫荧光检测新生皮肤的血管、神经再生及胶原沉积
制备12-16μm厚度的组织冰冻切片,结束后,37℃过夜。次日使用组织固定液37℃固定2h,加入适当浓度的CD34抗体标记血管内皮细胞、Tuj1标记神经元、COL1A1标记Ⅰ型胶原,4℃孵育过夜,次日室温复温0.5h,1×PBST洗3次,每次10min。加含有一定比例的相应二抗于组织上室温避光孵育2h。加一定比例的Hoechst于组织上,室温10min。1×PBST洗4×15min。荧光封片剂封片,使用荧光显微镜观察结果并采集图像。HE染色结果如图1所示,GIP-L-CBD组新生皮肤厚度明显低于对照组,免疫荧光结果如图2、3、4所示,GIP-L-CBD能够有效促进伤口处血管及神经再生,减少胶原沉积,促进皮肤损伤的有效恢复。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (3)
1.GIP衍生肽在制备皮肤创伤治疗药物中的应用,其特征在于:GIP衍生肽氨基酸序列如下所示:
YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGGGGSTKKTLRT。
2.如权利要求1所述的应用,其特征在于:所述皮肤创伤治疗药物为GIP衍生肽辅以凡士林或明胶或甲基丙烯酰化明胶构成的药物。
3.如权利要求1-2任一项所述的应用,其特征在于:所述皮肤创伤治疗药物能够促进损伤区域的神经及血管新生,降低胶原沉积、减少疤痕形成,有利于损伤皮肤的有效愈合。
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