CN104602699B - 含c‑肽的用于预防或治疗糖尿病性血管生成损伤的组合物 - Google Patents
含c‑肽的用于预防或治疗糖尿病性血管生成损伤的组合物 Download PDFInfo
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Abstract
本发明涉及使用C‑肽预防或治疗糖尿病性血管生成障碍或糖尿病性伤口愈合障碍的方法,以及用于该方法的组合物。根据本发明的C‑肽能够通过诱导内皮细胞的趋化性迁移、促进细胞迁移至受伤区域中、促进毛细管样网络的形成和阻止NO形成的诱导及ERK1/2和Akt的磷酸化,促进诱导血管生成。因此,本发明可以广泛用于预防或治疗由糖尿病性血管生成障碍引起的各种糖尿病并发症。
Description
技术领域
本发明涉及使用C-肽预防或治疗糖尿病相关疾病。更具体的,本发明涉及使用C-肽预防或治疗糖尿病性血管生成损伤的方法,使用C-肽预防或治疗糖尿病性伤口愈合损伤的方法,和包括C-肽的用于预防或治疗损伤的组合物。
背景技术
糖尿病是一组具有多种病因的代谢性疾病,特征在于由胰岛素活性的完全或功能性缺乏引起的慢性高血糖。维持很长时间的高血糖水平会引起慢性代谢性障碍并且引起血管的损伤,并伴随各种并发症的随后发病。这些典型地在10年糖尿病之后显现,因为几乎身体所有的器官都受到了损伤。在糖尿病患者中尤其观察到不足的血管生成。血管生成是伤口愈合和治疗缺血性组织损害的关键步骤,但是在糖尿病患者中血管生成不能正常进行。相应的,需要一种糖尿病性血管生成障碍的疗法,但是迄今为止还没有报道有引人注目的成果。
这些糖尿病并发症与VEGF(血管内皮生长因子)相关。已知VEGF诱导ERK1/2和Akt的磷酸化以及NO的产生,促进血管生成,并且用作血管渗漏因子以引发肌肉水肿。
人类的C-肽是从胰岛素原中切下的短肽,并且以与胰岛素等摩尔的浓度被胰腺β细胞分泌到循环中。伴随胰岛素的C-肽缺乏是I型糖尿病和II型糖尿病晚期的典型特征,并且随后出现各种并发症的发病,包括受损的伤口愈合。在糖尿病中,受损的伤口愈合是严重的并发症,导致系统性感染、疼痛、溃疡和截肢,并且甚至可能是致命的。
C-肽用于诊断糖尿病,但是还没有报道C-肽在治疗糖尿病性血管生成障碍中的应用的引人注目的成果。
在此背景下,本发明人对治疗糖尿病性血管生成损伤进行了深入且彻底的研究,并且发现C-肽可以用于在糖尿病中保护生理血管生成损伤或刺激伤口愈合,从而得到了本发明。
发明内容
因此,本发明的一个目的是提供一种使用C-肽预防或治疗糖尿病性血管生成损伤的方法,或用于该方法的组合物。
本发明的另一个目的是提供一种使用C-肽预防或治疗糖尿病性伤口愈合损伤的方法,或用于该方法的组合物。
根据本发明的一个方面,本发明提供了一种预防或治疗糖尿病性血管生成损伤的方法,所述方法包括给予有需要的动物有效量的C-肽,也就是说,给予易于受糖尿病性血管生成损伤影响或受到糖尿病性血管生成损伤影响的动物有效量的C-肽。
根据本发明的另一方面,本发明提供了一种预防或治疗糖尿病性血管生成损伤的药物组合物,该药物组合物包括作为活性成分的C-肽。
根据本发明的又一方面,本发明提供了一种预防或治疗糖尿病性伤口愈合损伤的方法,所述方法包括给予有需要的动物有效量的C-肽,也就是说,给予易于受糖尿病性伤口愈合损伤影响或受到糖尿病性伤口愈合损伤影响的动物有效量的C-肽。
根据本发明的再一方面,本发明提供了预防或治疗糖尿病性伤口愈合损伤的药物组合物,该药物组合物包括作为活性成分的C-肽。
根据本发明的被发现能够诱导血管生成的包括C肽的药物组合物在治疗需要血管生成的广谱的症状中,诸如糖尿病性伤口愈合损伤,以及包括糖尿病性血管生成损伤的各种糖尿病并发症中,都具有预防或治疗应用。
附图说明
图1提供了如通过转板迁移测定法(A、B)和通过伤口愈合测定法(C、D)分析,示出C-肽在HUVEC中诱导细胞迁移和伤口愈合的图像和曲线图。HUVEC被接种在可渗透支撑物的上孔上,并且在下孔中存在10ng/ml VEGF或有预定浓度的C-肽(C-Pep)的情况下,孵育12h:
图1A示出在过滤器的下表面上的迁移的细胞的图像;
图1B是柱状图,其中通过对迁移细胞进行计数量化了趋化性迁移(n=3);
图1C示出了融合的细胞层的共聚焦显微图像,该融合的细胞层是受伤的并且与10ng/ml VEGF或0.5nM C-肽孵育24h;以及
图1D是柱状图,其中通过对迁移至刮擦区的细胞进行计数量化了细胞迁移(n=3),并且结果表示为三次独立实验的平均值±S.D.。*p<0.05,**p<0.01。CON,对照。
图2提供了根据剂量依赖性的小管形成(A、B)、小管形成(C)和体内基质胶塞测定(D),示出C-肽在体外和体内诱导血管生成的图像和曲线图。将HUVEC在具有示出浓度的C-肽(C-Pep)的基质胶层上培养24h(n=3)(A,B):
图2A示出小管形成的共聚焦显微图像;
图2B是曲线图,其中通过测量来自图2A中的图像的小管长度确定相对小管长度(%),以检验C-肽诱导的、剂量依赖性的小管形成;
图2C是示出通过10ng/ml VEGF或0.5ng/ml C-肽诱导的小管形成的柱状图;以及
图2D示出在进行体内基质胶塞测定之后的基质胶的图片,在该体内基质胶塞测定中,小鼠经含100ng/ml VEGF或5nM C-肽的0.5ml基质胶皮下感染7d(n=8/组)。
图3提供了示出C-肽在HUVEC中诱导ERK1/2和Akt磷酸化以及NO产生的图像和柱状图。图3A和3B示出在使用10ng/ml VEGF或0.5nM C-肽处理HUVEC预定时间之后的结果:
图3A示出ERK1/2磷酸化的蛋白印记分析的结果,其中,使用蛋白的总量对蛋白磷酸化的水平进行标准化,并且相对于未经刺激的对照水平(CON)进行表示;
图3B示出Akt磷酸化的蛋白印记分析的结果,其中,使用蛋白的总量对蛋白磷酸化的水平进行标准化,并且相对于未经刺激的对照水平(CON)进行表示;以及
图3C示出在使用10ng/ml VEGF或0.5nM C-肽处理HUVEC 2h之后获得的结果,以及使用本领域已知的方法通过共聚焦显微检查确定的细胞内NO的水平。结果表示为来自三次独立实验的平均值±S.D.。*p<0.05,**p<0.01。
图4提供了示出各种抑制剂防止由C-肽和VEGF诱导的小管形成的柱状图:
图4A是在将HUVEC接种在基质胶层上,并且在所示浓度的L-NAME、PD98058和渥曼青霉素的存在下与0.5nM C-肽(C-pep)孵育24至30h之后,通过共聚焦显微检查量化的小管形成的柱状图,并且结果表示为来自三次独立实验的平均值±S.D.;
图4B是在将HUVEC接种在基质胶层上,并且在所示浓度的L-NAME、PD98058和渥曼青霉素的存在下与10ng/ml VEGF孵育24至30h之后,通过共聚焦显微检查量化的小管形成的柱状图,并且结果表示为来自三次独立实验的平均值±S.D.。
图5提供了示出C-肽修复糖尿病受损的伤口愈合的柱状图:
图5A是在皮肤受损之后4d和12d,在健康小鼠(健康;n=12/组)、糖尿病小鼠(糖尿病;n=12/组)和给予C-肽的糖尿病小鼠(糖尿病+C-pep;n=12/组)的伤口愈合之间进行对比的柱状图。**,p<0.01,N.S.没有显著性差异,以及
图5B是使用数码照相观察4mm直径的皮肤伤口愈合之后的柱状图。
图6提供了通过转板迁移测定法(A、B)和通过伤口愈合测定法(C、D)进行测量,示出C-肽在NIH3T3细胞中诱导细胞迁移和VEGF表达的图像和曲线图。在图6A和图6B中,将NIH3T3细胞接种在可渗透支撑物的上孔上,并且在下孔中存在1ng/ml FGF-2或者所示浓度的C-肽(C-Pep)的情况下孵育12h:
图6A示出在过滤器的下表面上的迁移细胞的图像;
图6B是通过对迁移细胞进行计数量化了趋化性迁移(n=3)的柱状图;
图6C示出了融合的细胞层的共聚焦显微图像,该融合的细胞层是受伤的并且与1ng/ml FGF-2或0.5nM C-肽孵育24h;以及
图6D是通过对迁移到刮擦区的细胞进行计数量化了细胞迁移(n=3)的柱状图。
图7提供了示出C-肽增加了受伤部位周围毛囊再生和血管形成的图像:
图7A示出来自移除正常小鼠(n=6)、糖尿病小鼠(n=6)和糖尿病+C-肽小鼠(n=6)的后肢的背部皮肤的代表性照片,其中正方形区域显示为每一图像的底部的放大图像。毛囊和血管在正常小鼠和糖尿病+C-肽小鼠中得到发育,但在糖尿病小鼠中没有发育(由箭头所示);以及
图7B示出经PECAM-1抗体可视化的血管的图像(上半部分),及在背部皮肤中可视化的毛囊(中间部分)。C-肽增加了毛囊和血管的数量。比例尺=200mm。
图8是示出C-肽诱导的血管生成和防止糖尿病受损的伤口愈合的可能的信号传导通路,暗示C-肽经由涉及ERK1/2、Akt和NO生成的通路激活血管生成,并且通过提高血管生成改进延迟的伤口愈合。
具体实施方式
本发明对预防和治疗在糖尿病患者中的血管生成失调和随后受损的伤口愈合的深入且彻底的研究,最终发现了:通过诱导内皮细胞迁移并且刺激毛细管样网络的形成(对于血管生成都是不可或缺的),C-肽可以在体外和体内激活血管生成;并且,C-肽通过引起ERK1/2和PI3K/AKT磷酸化和NO生成的信号传导通路刺激血管生成,并且这些信号传导通路参与C-肽诱导的血管生成。还发现了,C-肽的预防性或治疗性效果在糖尿病性伤口愈合损伤上诱导血管生成。
因此,本发明涉及使用C-肽预防或治疗糖尿病性血管生成损伤的方法或组合物;以及预防或治疗糖尿病性伤口愈合损伤的方法或组合物。
根据本发明的一个方面,本发明解决了一种使用C-肽预防或治疗糖尿病性血管生成损伤的方法,或用于该方法的组合物。
更具体的,本发明提供了一种预防或治疗糖尿病性血管生成损伤的方法,该方法包括给予有需要的动物有效量的C-肽。另外,本发明提供了预防或治疗糖尿病性血管生成损伤的药物组合物,该药物组合物包括作为活性成分的C-肽。
在本发明中,观察到C-肽引起血管内皮细胞和成纤维细胞的趋化性迁移中剂量依赖性的升高(图1A、图1B、图6A和图6B),刺激细胞迁移至受伤区域(图1C、图1D、图6C和图6D),并且以剂量依赖的方式诱导毛细管样网络的形成(图2A和图2B),并且诱导在基质胶中的新的血管的形成(图2D)。此外,实验证明C-肽在HUVEC中通过ERK1/2和Akt的磷酸化和NO的生成,刺激血管生成和VEGF(图3)。因此,根据本发明,C-肽或包括C-肽的组合物可以用于预防或治疗糖尿病性血管生成损伤的方法或药物组合物中。C-肽或本发明的组合物对血管生成损伤的预防性或治疗效果,可以应用于可能遭受糖尿病性血管生成损伤的任何动物,包括人类。
如本文所使用的,术语“C-肽”指在哺乳动物和鸟中发现的胰岛素原的短的蛋白成分,该胰岛素原由胰岛的胰腺β细胞生成。它的长度依赖于它的来源在21个氨基酸至31个氨基酸之间变化。来自各种哺乳动物(包括狗、猫、大鼠、黑猩猩、小鼠、母牛等)的哺乳动物的C-肽,以及人类的C-肽,和来自例如鸫的鸟类C-肽,都在本发明的范围内。
例如,来源于人类(Homo sapiens,SEQ ID NO:1)、大鼠(Rattus norvegicus,SEQID NO:2)和黑猩猩(Pan troglodytes,SEQ ID NO:3)的所有C-肽都由31个氨基酸构成,同时发现C-肽在小鼠(Mus musculus,SEQ ID NO:4)中为29-mer肽,在牛(Bos taurus,SEQ IDNO:5)中为26-mer肽,在鸫(Turdus merula,SEQ ID NO:6)和红脚鲣鸟(Sula sula,SEQ IDNO:7)中为21-mer肽。本发明的C-肽可以具有选自由SEQ ID NO:1至SEQ ID NO:7组成的组中的氨基酸序列。
为了本发明的目的,C-肽在本发明提供的方法或组合物中用作活性成分。所述方法或组合物可以应用于可能受糖尿病性血管生成损伤引起的疾病影响的任何动物,以及人类。在此上下文中,C-肽优选应用于它的来源的动物。例如,如果它应用于人类,则本发明的方法或组合物优选包括人来源的C-肽(SEQ ID NO:1)。
在本发明的一个实施方式中,发现C-肽在糖尿病的模型小鼠中诱导血管生成,这样C-肽或包括C-肽的组合物可应用于预防或治疗糖尿病性血管生成损伤。如本文所使用的,术语“糖尿病性血管生成损伤”指糖尿病引起的自身受损的血管生成,或者由糖尿病性血管生成损伤引起的疾病,该疾病可以选自由中风、肾脏疾病、心脏疾病、足部溃疡,及它们的组合组成的组中。
详细来讲,本发明的方法或组合物对糖尿病性血管生成损伤引起的疾病的预防性或治疗性效果,可以归因于C-肽的如下活性中的至少一种:诱导内皮细胞的趋化性迁移,诱导细胞迁移至受伤区域,诱导毛细管样网络的形成,及诱导ERK1/2和Akt的磷酸化和NO的生成。
因此,在各种糖尿病并发症的发病之后,本发明的方法或组合物可以应用于预防或治疗在任何糖尿病患者(动物或人类)中出现的糖尿病性血管生成损伤。
本发明的组合物是药物组合物,该药物组合物可进一步包括药学上可接受的递送剂、赋形剂或稀释剂。如本文所使用的,术语“药学上可接受的递送剂、赋形剂或稀释剂”旨于包括任何和所有的溶剂,分散介质,包衣剂,佐剂,稳定剂,防腐剂,抗菌剂和抗真菌剂,等渗剂,和吸收延迟剂。在本发明中有用的递送剂、赋形剂或稀释剂的实例包括乳糖、右旋葡萄糖、蔗糖、山梨醇、甘露醇、木糖醇、麦芽糖醇、葡萄糖、甘油、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、甲基羟基苯甲酸盐、丙基羟基苯甲酸盐、滑石、硬脂酸镁、矿物油等。
使用常规方法,可以将本发明的药物组合物配制成口服剂型(诸如粉剂、颗粒剂、片剂、胶囊、悬浮液、乳液、糖浆和喷雾剂),或配制成无菌的注射剂。为了配制根据本发明的组合物,通常使用稀释剂或赋形剂,诸如填充剂、增稠剂、粘合剂、湿润剂、崩解剂和表面活性剂。用于口服剂量的固体制剂包括片剂、丸剂、粉剂、颗粒剂和胶囊。使用卵磷脂样的乳化剂与诸如淀粉、碳酸钙、蔗糖、乳糖或明胶的至少一种赋形剂的组合,制备这些固体制剂。
除了赋形剂之外,可以使用润滑剂,诸如镁、硬脂酸盐、滑石等。用于口服给药的液体制剂包括悬浮液、内部溶液、乳液和糖浆。在这些液体制剂中,可以含有各种赋形剂(诸如湿润剂、甜味剂和防腐剂),以及简单的稀释剂(诸如水和液体石蜡)。用于非口服剂量的制剂的代表可以是无菌水溶液、非水溶液、悬浮液、乳夜、冻干剂和栓剂。对于非水溶液和悬浮液,可以使用植物油(诸如丙二醇、聚乙二醇和橄榄油),或可注射的酯(诸如油酸乙酯)。
只要导向靶组织,则根据本发明的使用C-肽的方法或组合物可采用任何口服或非口服的给药途径。优选的是使用渗透泵的皮下注射,皮内注射,静脉注射,腹膜内注射或玻璃体内注射。
在本发明中,C-肽可以以“有效量”或“药学上的有效量”给予。如本文所使用的,术语“有效量”或“药学上的有效量”指在不引发显著或过度的免疫应答的情况下,足以提供预防或治疗效果的量。它是依赖于药学或药物领域中已知的各种因素确定的,该已知的各种因素包括疾病的种类和严重程度,药物活性,给药途径,流量比,给药时间段,共给予或组合的其它药物,患者的年龄、体重、性别、饮食习惯、健康状态等。在确定“有效量”或“药学上的有效量”时考虑到的各种因素对于本领域技术人员来说是已知的,并且例如在Gilman等人编辑的Goodman And Gilman's:The Pharmacological Bases of Therapeutics(第8版,培格曼出版社(Pergamon Press),1990)和Remington's Pharmaceutical Sciences(第17版,马克出版有限公司(Mack Publishing Co.),伊斯顿(Easton),Pa.,1990)中进行了解释。
本发明的组合物可以单独给予,或者与其它疗法组合给予。本发明的组合物和其它疗法的共给予可以同时进行或顺序进行。可以有单一剂量或多种剂量。重要的是,以足以获得最大治疗效果且没有副作用的尽可能最小的量使用组合物,本领域技术人员可以容易地确定这样的量。此外,可以使用装置进行组合物的给予,该装置帮助活性成分导向靶细胞。
根据本发明的另一方面,本发明解决了一种使用C-肽预防或治疗糖尿病性伤口愈合损伤的方法和组合物。更具体的,本发明提供了一种预防或治疗糖尿病性伤口愈合损伤的方法,该方法包括给予有需要的动物有效量的C-肽。本发明还提供了预防或治疗糖尿病性伤口愈合损伤的药物组合物,该药物组合物包括作为活性成分的C-肽。
如上所述,发现了C-肽有效治疗糖尿病性伤口愈合损伤(如在下面的工作实施例2中的糖尿病模型小鼠中证实的),从而C-肽或包括C-肽的组合物适用于预防或治疗糖尿病性伤口愈合损伤。C-肽对糖尿病性伤口愈合损伤的预防性或治疗效果可以应用于人类,以及会受到糖尿病影响的任何动物。如本文所使用的,术语“糖尿病性血管生成损伤”指糖尿病引起的受损的血管生成自身,或者由糖尿病性血管生成损伤引起的疾病,该疾病可以选自由中风、肾脏疾病、心脏疾病、足部溃疡,及它们的组合组成的组中。
详细来讲,本发明的方法或组合物对糖尿病性血管生成损伤的预防性或治疗性效果,可以归因于C-肽的如下活性中的至少一种:诱导内皮细胞的趋化性迁移,诱导细胞迁移至受伤区域,诱导毛细管样网络的形成,及诱导ERK1/2和Akt的磷酸化和NO的生成。
在糖尿病性血管生成损伤或糖尿病性伤口愈合损伤的预防或治疗的背景中使用的术语“C-肽”、“剂量”、“给药”和“有效量(药学上的有效量)”如在本发明的在先方面中所述。
通过以下实施例可以获得对本发明的更好的理解,其中给出以下实施例以例示本发明,但不应理解为限制本发明。
实例1:实验的准备
实例1-1:实验动物
六周龄的雄性C57BL/6小鼠购自奈良生物科技(首尔,韩国)。在温度受控的清洁的架子上饲养这些小鼠,该架子具有12-h的光/暗周期。所有实验都依据江原大学校的实验动物管理与使用委员会的指标进行。
实例1-2:细胞培养
根据公知的方法从人类的脐带静脉中分离HUVEC,并且在以下的实验中使用来自传代3至6次的细胞。将细胞接种在2%明胶包被的盖玻片、盘或板中的M199培养基(补充有20%FBS,3ng/ml bFGF,5U/ml肝素,100U/ml青霉素,和100μg/ml链霉素)中,并且在37℃潮湿的5%CO2的恒温箱中生长。
从ATCC(美国模式培养物保藏所)获得的NIH3T3成纤维细胞生长在补充有10%小牛血清、100U/ml青霉素和100μg/ml链霉素的DMEM培养基中。
对于实验,通过将细胞培养在低血清的培养基(如上补充的M199,但仅具有1%FBS)中,对细胞血清饥饿处理6h,并且在存在或不存在各种浓度的C-肽的情况下使用10ng/ml VEGF处理适当的时间。
在低血清的培养基(如上补充的DMEM,但仅具有0.1%的小牛血清)中,对NIH3T3进行血清饥饿处理12h。
实例1-3:统计学分析
使用Origin 6.1(OriginLab,北安普敦(Northampton),MA,USA)对在每一实例中获得的数据进行处理,并用图表表示出来。使用t-检验和ANOVA确定统计学的显著性。认为小于0.05的p-值是有统计学显著性的。
实例2:C-肽对血管生成的效果
实例2-1:转板迁移测定(Transwell migration assay)
血管生成伴随有内皮细胞的迁移和毛细管样网络的形成。为了通过检验C-肽是否刺激内皮细胞的迁移来评价C-肽诱导血管生成的能力,使用可渗透支撑物(科斯塔(Costar),科宁(Corning),NY)测定HUVEC和NIH3T3细胞的趋化性运动。
详细来讲,使过滤器的下表面包被有5μl 2%的明胶。将含VEGF、FGF-2或C-肽的低血清培养基放置在下孔中。在每一个上孔中,以每200μl低血清培养基1x 105个细胞的密度接种HUVEC或NIH3T3细胞,并且在37℃培养12h。使用100%甲醇固定细胞15min,使用苏木精和曙红进行染色,并且干燥。使用荧光倒置显微镜(奥林帕斯(Olympus)),获得每种添加物的三个随机选择的视野中的迁移细胞的平均数量,以量化迁移程度。
结果示于图1A和图1B中。如从结果中所理解的,C-肽在内皮细胞的趋化性迁移中诱导了剂量依赖型的增加,并且在0.5nM具有最大效果(图1A和图1B)。
实例2-2:伤口愈合测定
通过伤口愈合测定,对C-肽对血管生成的效果进行检验。
将HUVEC和NIH3T3细胞培养在包被有明胶的6孔培养板上。在获得之后,用低血清培养基使融合的细胞层饥饿6h,经1μM钙黄绿素-AM染色30min,并且用塑料刮刀使融合的细胞层受伤。去除低血清培养基和细胞碎片,然后为细胞培养重新装满含VEGF或C-肽的2ml低血清培养基。然后在37℃培养细胞24h。根据使用共聚焦显微镜(FV-300,Olympus)获得的图像,对迁移的细胞进行计数。
结果示于图1C和图1D中。如在图1C和图1D中所示,C-肽(0.5nM)显著刺激了细胞迁移至受伤区域(p<0.01)。
如之前所报道的,VEGF也刺激内皮细胞中的趋化性迁移和伤口愈合(p<0.01;图1)。
因此,C-肽刺激内皮细胞的迁移,而内皮细胞的迁移对血管生成是必要的。
实例3:小管形成测定
为了检验C-肽是否能够体外激活血管生成,使用HUVEC在基质胶层中进行了小管形成测定。
使用本领域已知的方法,实现了HUVEC在生长因子减少的基质胶(BDBiosciences,富兰克林湖(Franklin Lakes),NJ)上形成毛细管样网络。简单来讲,根据制造商的说明书,将24孔培养板包被有基质胶。HUVEC以4x 105个细胞/孔的密度接种在基质胶层上,并且在37℃使用VEGF或C-肽处理24h至30h。使用FV-300软件,通过由图像测量小管的长度来量化小管形成的程度。结果示于图2A、图2B和图2C中。
与实例2的细胞迁移测定的结果一致,C-肽以剂量依赖的方式刺激毛细管样网络的形成,并且在0.5nM具有最大效果(图2A和图2B)。与C-肽的效果类似,VEGF诱导毛细管样网络的形成(图2C)。
因此,证实了C-肽激活内皮细胞的迁移和毛细管样网络的形成,这对体外血管生成是必要的。
实例4:体内基质胶塞测定
使用基质胶塞测定,以检验C-肽是否能够诱导体内的血管生成。在这一方面,根据本领域已知的方法进行体内基质胶塞测定。
简单来讲,小鼠经皮下注射含100ng/ml VEGF或5nM C-肽的0.5ml基质胶,和10U肝素。7天之后,从皮下区移出基质胶塞,并且使用相机(索尼(Sony),日本(Japan))进行拍照。结果示于图2D中。
在基质胶塞植入后一周,含C-肽的基质胶显示出显著高于对照的新血管形成的诱导,并且该结果与VEGF处理期间看到的结果类似(图2D)。
相应的,这些结果表明C-肽激活体外和体内的血管生成。
实例5:C-肽相关的细胞内信号传导通路
实例5-1:C-肽对ERK1/2和Akt磷酸化的影响
因为ERK1/2和Akt的活化是诱导细胞迁移和小管形成的关键,因此检验了在HUVEC中C-肽对ERK1/2和Akt磷酸化的影响。
通过蛋白印迹分析,确定ERK1/2和Akt的磷酸化。简单来讲,使用冰冷的裂解缓冲液(50mM Tris-HCl,pH 7.5,1%Triton X-100,150mM NaCl,1mM EDTA,0.1mM PMSF,10μg/mL抑肽酶(aprotinin),和10μg/mL亮抑酶肽(leupeptin))刮掉HUVEC,并且在4℃,18,000g离心10min。得到的细胞裂解物通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,并且转移至聚偏氟乙烯膜上。
通过曝光于x射线胶片和化学发光底物(Pierce,罗克福(Rockford),IL),使蛋白条带可视化。在图3A和图3B中,使用蛋白总量对蛋白磷酸水平进行标准化,并且相对于未经刺激的对照水平(CON)表示蛋白磷酸化水平。
可以看出,C-肽以时间依赖的方式增加了ERK1/2的磷酸化,并且在15min具有最大的刺激,并且该ERK1/2的活化维持到60min(图3A)。
类似的,C-肽以时间依赖的方式诱导Akt磷酸化,并且在15min具有最大效果(图3B)。
VEGF也以时间依赖的方式诱导ERK1/2和Akt的磷酸化(图3A和图3B)。
实例5-2:C-肽对NO产生的作用
因为Akt的磷酸化通过VEGF(是介导血管生成的重要因子)增加了NO的产生,因此进行了检验,以确定C-肽是否能够刺激NO的产生。为此,根据本领域已知的方法,使用DAF-FM DA(二氨基荧光素-FM双乙酸盐,分子探针(Molecular Probes),尤金市(Eugene),OR)测量了细胞内的NO水平。
简单来讲,在不含酚红的低血清培养基中,使用VEGF或C-肽处理HUVEC预示的时间,并且使用2μM DAF-FM DA培养最后的1h。之后,在共聚焦显微观察之前用低血清培养基对细胞进行洗涤。通过对比经处理的细胞与未经处理的对照细胞的荧光强度来测定细胞内NO的水平,其中测定的值表示为倍数差异。
使用C-肽对HUVEC的处理显著提高了细胞内NO的水平(图3,p<0.05)。基于VEGF的处理,也产生了较高水平的细胞内NO(p<0.01)。
实例5-3:ERK1/2、PI3K/Akt和NO产生在C-肽诱导的血管生成中的作用
使用PD98059、渥曼青霉素(wortmannin)和L-NG-硝基精氨酸甲酯(L-NAME)分别检验ERK1/2、PI3K/Akt和NO在C-肽诱导的血管生成中的作用。
PD98059(ERK1/2的抑制剂)以剂量依赖的方式抑制了HUVEC的C-肽刺激的小管形成,并且在10μM具有最大的抑制。PI3K/Akt的抑制剂渥曼青霉素和NOS的抑制剂L-NAME也显示出对C-肽诱导的小管形成的剂量依赖的抑制(图4A)。
通过PD98059、渥曼青霉素和L-NAME的处理,防止了VEGF诱导的小管形成(图4B)。
总之,上面获得的数据表明C-肽经由涉及ERK1/2、PI3P/AKT和NO产生的信号传导通路刺激血管生成,并且涉及ERK1/2、Akt和NO产生的信号传导通路可能参与到C-肽诱导的血管生成中。
实例7:C-肽对糖尿病性伤口愈合损伤的治疗效果
通过以150mg/kg体重的量,单次腹膜内注射含链脲霉素的100mM柠檬酸盐缓冲液(pH 4.5),生成糖尿病小鼠。在注射之后,为小鼠过夜供给10%的蔗糖,以防止发生突然的低血糖休克。如通过Accu-Chek活力血糖监测仪(罗氏诊断(Roch Diagnostics),德国)测量血糖,并且通过Uriscan(TD诊断(TD Diagnostics),Young-In,韩国)测量糖尿,在注射之后观察到足够的高血糖症。在1周之后,将具有高于16mM的非空腹血糖水平、多尿和糖尿的小鼠定义为糖尿病,并且用于实验。在链脲霉素注射之后2周,为一组糖尿病小鼠皮下移植Alzet微型渗透泵2004(DIRECT,库比蒂诺(Cupertino),CA),该Alzet微型渗透泵2004含有在PBS中的C-肽,以35pmol/kg/min的递送速率递送C-肽两周。其它糖尿病组和正常组进行假手术。
在造成伤口之前,使用薇婷(Veet)乳膏剃去脊柱的背部皮肤上的毛。使用活检穿孔器在后肢的背部表面上造成直径为4mm的全层性皮肤伤口,并且使用数码照相机监测闭合。使用游标卡尺以两天的有规律的时间间隔追踪伤口边缘,测量开放的伤口尺寸,并且使用数码相机进行监测。
为了确定C-肽是否能够在糖尿病小鼠中诱导伤口闭合,经移植在糖尿病小鼠中的皮下渗透微型泵,以35pmol/kg/min的递送速率持续给予C-肽。在正常小鼠、糖尿病小鼠和给予C-肽的糖尿病小鼠(STZ诱导的糖尿病4周)中,在后肢的背部表面上造成直径4mm的伤口。糖尿病小鼠的伤口已经显示出比正常小鼠的伤口显著低的愈合率。但是,在皮肤损害之后的4天和10天,在使用渗透泵补充C-肽的糖尿病小鼠中的伤口面积中没有观察到显著差异(图5A)。在第4天和第10天,正常小鼠、糖尿病小鼠和给予C-肽的糖尿病小鼠的伤口面积之间的对比示出显著差异(图5B)。
实例8:成纤维细胞迁移的C-肽活化
为了解释C-肽在趋化性成纤维细胞迁移中的作用,使用C-肽处理NIH3T3细胞12h。C-肽在成纤维细胞的趋化性迁移中诱导剂量依赖的增加,并且在1nM具有最大效果(图6A和图6B)。
通过伤口愈合测定,进一步研究C-肽诱导的细胞迁移。C-肽(0.5nM)显著刺激细胞迁移至受伤区域(p<0.01;图6C和图6D)。FGF-2也在成纤维细胞中刺激趋化性迁移和伤口愈合(p<0.01;图6)。因此,C-肽刺激成纤维细胞的迁移,这对受损皮肤中的伤口愈合过程是必要的。
实例9:毛囊再生和血管形成的C-肽刺激
通过整体(whole-mount)免疫染色,观察C-肽对毛囊再生和血管形成的效果。在这方面,在受伤之后14天,处死小鼠,并且移除背部皮肤以观察毛囊和血管。为了避免破坏它的愈合边缘,使用剪刀切割来自所有死亡小鼠的皮肤组织,随即在PBS中的4%多聚甲醛中固定过夜,并且在-20℃的甲醇中储存过夜。对于免疫组化,使用在TBS中的0.1%TritonX-100中的2%BSA封闭组织样品,并且与山羊多克隆血小板内皮细胞粘附分子-I的抗体孵育2d,接着用Alexa-546缀合的兔抗山羊IgG的抗体探查,并且然后进行共聚焦显微检查。
结果示于图7中。依据从图片(图7A)以及通过PECAM-1染色(图7B)进行的评价,在环绕糖尿病小鼠的伤口的皮下组织中的毛囊和血管的数量显著低于正常小鼠。与那些糖尿病小鼠相比,给予C-肽的小鼠皮肤示出显著增加了毛囊再生和血管形成,并且同时加速了伤口闭合。
这些结果暗示C-肽对治疗糖尿病延迟的伤口愈合,尤其在患有受损的微循环的糖尿病患者中对治疗糖尿病延迟的伤口愈合的潜在的治疗价值。
工业实用性
根据本发明的被发现能够诱导血管生成的包括C-肽的药物组合物在治疗需要血管生成的广谱的症状中,诸如糖尿病性伤口愈合损伤,以及包括糖尿病性血管生成损伤的各种糖尿病并发症中,都具有预防或治疗应用。
Claims (2)
1.包括C-肽的组合物在制造预防或治疗糖尿病性血管生成损伤的药剂中的应用,其中所述糖尿病性血管生成损伤是足部溃疡,其中所述C-肽具有选自由SEQ ID NO:1至SEQ IDNO:7组成的组中的氨基酸序列,其中所述糖尿病性血管生成损伤的预防或治疗归因于C-肽的如下活性:诱导内皮细胞的趋化性迁移,诱导细胞迁移至受伤区域,诱导毛细管样网络的形成,及诱导ERK1/2和Akt的磷酸化和NO的生成。
2.根据权利要求1所述的应用,其中,所述药剂用于治疗人类。
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WO2017010673A2 (ko) | 2015-07-16 | 2017-01-19 | 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 | 혈관누수 증후군의 예방 또는 치료용 조성물 |
US10155948B2 (en) | 2016-05-12 | 2018-12-18 | Kangwon National University University-Industry Cooperation Foundation and | Pharmaceutical composition for preventing or treating diabetic complications and screening method for preventive or therapeutic agent for diabetic complications |
KR101881662B1 (ko) * | 2016-05-12 | 2018-07-26 | 강원대학교산학협력단 | 당뇨성 합병증 예방 및 치료용 약제학적 조성물 및 당뇨성 합병증 예방 또는 치료제 스크리닝 방법 |
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WO2014025146A1 (ko) | 2014-02-13 |
KR20150035713A (ko) | 2015-04-07 |
CN104602699A (zh) | 2015-05-06 |
KR20150035714A (ko) | 2015-04-07 |
WO2014025127A1 (ko) | 2014-02-13 |
EP2881119B1 (en) | 2019-09-18 |
US9457063B2 (en) | 2016-10-04 |
KR101646054B1 (ko) | 2016-08-05 |
EP2881119A1 (en) | 2015-06-10 |
CN104640559A (zh) | 2015-05-20 |
EP2881120A1 (en) | 2015-06-10 |
US9119816B2 (en) | 2015-09-01 |
EP2881120A4 (en) | 2015-08-19 |
US20140148386A1 (en) | 2014-05-29 |
CN104640559B (zh) | 2017-05-17 |
US20140128323A1 (en) | 2014-05-08 |
EP2881119A4 (en) | 2015-08-19 |
KR101620942B1 (ko) | 2016-05-13 |
EP2881120B1 (en) | 2019-08-07 |
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