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CN118557704A - Application of barnacle Cp19k protein in the preparation of preparations for treating difficult-to-heal wound repair diseases - Google Patents

Application of barnacle Cp19k protein in the preparation of preparations for treating difficult-to-heal wound repair diseases Download PDF

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CN118557704A
CN118557704A CN202410803289.4A CN202410803289A CN118557704A CN 118557704 A CN118557704 A CN 118557704A CN 202410803289 A CN202410803289 A CN 202410803289A CN 118557704 A CN118557704 A CN 118557704A
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barnacle
protein
cp19k
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肖健
邬芬赞
聂辰瑶
张政
刘秘
邓连成
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Cixi Institute Of Biomedicine Wenzhou Medical University
Xishan Institute Of Applied Biotechnology Nanjing University Wuxi
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Xishan Institute Of Applied Biotechnology Nanjing University Wuxi
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/1825Fibroblast growth factor [FGF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/02Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

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Abstract

本发明涉及藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病的制剂中的应用,本发明首次发现藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病中的应用,特别是与人成纤维生长因子2(FGF2)配合使用,不仅可以延长人成纤维生长因子2的半衰期,还可以进一步提高治疗难愈合的创面修复疾病的效果。The present invention relates to the use of barnacle Cp19k protein in preparing a preparation for treating difficult-to-heal wound repair diseases. The present invention is the first to discover the use of barnacle Cp19k protein in preparing a preparation for treating difficult-to-heal wound repair diseases, especially when used in combination with human fibroblast growth factor 2 (FGF2), it can not only prolong the half-life of human fibroblast growth factor 2, but also further improve the effect of treating difficult-to-heal wound repair diseases.

Description

藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病的制剂中 的应用Application of barnacle Cp19k protein in the preparation of preparations for treating difficult-to-heal wound repair diseases

技术领域Technical Field

本发明属于医药技术领域,具体涉及藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病的制剂中的应用。The invention belongs to the field of medical technology, and particularly relates to the application of barnacle Cp19k protein in preparing a preparation for treating difficult-to-heal wound repair diseases.

背景技术Background Art

皮肤是人体直接与外界环境接触的最大器官,常易受到多种理化和机械因素的影响从而造成不同程度的损伤,轻度浅表皮肤损伤容易自愈,而基底层的损伤则难以修复,常会伴有皮肤坏死、感染、增生性瘢痕等并发症,严重影响患者的生活质量,所以皮肤创面治疗和修复是亟待解决的临床问题。生长因子是一类能与细胞膜特异受体结合的活性蛋白多肽,广泛存在于生物体内,对机体的生长和发育具有调节作用。生长因子对人体的免疫、造血调控、肿瘤发生、炎症与感染、创伤愈合、血管形成、细胞分化、细胞凋亡、形态发生、胚胎形成等方面也有着重要的调控作用。近十余年来,已有包括表皮细胞生长因子(EGF)、成纤维细胞生长因子(FGF)、转化生长因子β(TGF-β)、血管内皮长因子(VEGF)以及血小板生长因子(PDGF)等应用于创面修复治疗,这些生长因子均有不同程度上促进急慢性创面愈合的作用,对其它疾病的治疗亦有积极的作用。The skin is the largest organ in the human body that is in direct contact with the external environment. It is often susceptible to a variety of physical, chemical and mechanical factors, resulting in varying degrees of damage. Mild superficial skin injuries are easy to heal on their own, while basal layer injuries are difficult to repair and are often accompanied by complications such as skin necrosis, infection, and hypertrophic scars, which seriously affect the patient's quality of life. Therefore, skin wound treatment and repair is a clinical problem that needs to be solved urgently. Growth factors are a class of active protein polypeptides that can bind to specific cell membrane receptors. They are widely present in organisms and have a regulatory effect on the growth and development of the body. Growth factors also have an important regulatory effect on human immunity, hematopoietic regulation, tumorigenesis, inflammation and infection, wound healing, angiogenesis, cell differentiation, apoptosis, morphogenesis, and embryogenesis. In the past decade, epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) have been used in wound repair treatment. These growth factors have the effect of promoting the healing of acute and chronic wounds to varying degrees, and also have a positive effect on the treatment of other diseases.

近年来,随着对创面愈合过程的深入研究,发现生长因子与修复细胞有密切关系,在创面修复中起关键作用。当细胞受到损伤因素刺激后,可释放多种生长因子,刺激同类细胞或同一胚层发育的细胞增生,参与损伤组织重建,促进修复过程。而创面愈合过程中主要涉及的生长因子包括成纤维细胞生长因子(FGF)、血小板源性生长因子(PDGF)、转化生长因子(TGF-α,TGF-β)、表皮细胞生长因子(EGF)、血管内皮细胞生长因子(VEGF)等,这些生长因子通过协同作用,在促进机体内多种类型组织细胞的分裂和增殖、基质合成和沉积、纤维组织和肉芽组织的形成,以及增加胶原合成能力与促进创伤后上皮再生、间质增生和新生血管形成等过程中发挥重要作用。In recent years, with the in-depth study of the wound healing process, it has been found that growth factors are closely related to repair cells and play a key role in wound repair. When cells are stimulated by damaging factors, they can release a variety of growth factors to stimulate the proliferation of similar cells or cells developed from the same germ layer, participate in the reconstruction of damaged tissues, and promote the repair process. The growth factors mainly involved in the wound healing process include fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF-α, TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), etc. These growth factors work synergistically to promote the division and proliferation of various types of tissue cells in the body, matrix synthesis and deposition, the formation of fibrous tissue and granulation tissue, and increase collagen synthesis capacity and promote post-traumatic epithelial regeneration, mesenchymal hyperplasia and neovascularization. Play an important role in the process.

生长因子在体内的短暂半衰期和在再生的特定阶段的需求是制约它们在临床广泛使用的瓶颈。因此,生物材料对生长因子的递送很重要,使用生物材料作为载体的主要优点是生长因子可以被包裹在生物材料中,避免它们在伤口环境中被降解。此外,还可以实现局部和持续的生长因子递送,确保组织再生所需的局部高浓度生长因子的供应,生物材料确保生长因子滞留在损伤部位,并提供宿主细胞可以在其上迁移的支架或结构支撑,以促进组织再生。例如,在治疗临界大小的骨缺损时,局部应用生长因子是较难实现的,而胶原海绵或磷酸三钙水泥等生物材料被用作骨再生中生长因子递送的载体。生物材料载体可用作不同的物理结构和形式,包括可注射水凝胶、支架、纳米颗粒和纳米纤维。此外,这些结构的组合也可用于生长因子的双重释放。这种灵活性较强的生物材料载体对生长因子的递送极具吸引力。The short half-life of growth factors in vivo and the need for specific stages of regeneration are bottlenecks that restrict their widespread clinical use. Therefore, biomaterials are important for the delivery of growth factors. The main advantage of using biomaterials as carriers is that growth factors can be encapsulated in biomaterials to avoid their degradation in the wound environment. In addition, local and sustained growth factor delivery can be achieved to ensure the supply of local high concentrations of growth factors required for tissue regeneration. Biomaterials ensure that growth factors are retained at the site of injury and provide a scaffold or structural support on which host cells can migrate to promote tissue regeneration. For example, when treating critical-sized bone defects, local application of growth factors is difficult to achieve, and biomaterials such as collagen sponges or tricalcium phosphate cement are used as carriers for growth factor delivery in bone regeneration. Biomaterial carriers can be used as different physical structures and forms, including injectable hydrogels, scaffolds, nanoparticles, and nanofibers. In addition, combinations of these structures can also be used for dual release of growth factors. This highly flexible biomaterial carrier is very attractive for the delivery of growth factors.

天然材料如蚕丝、胶原蛋白、明胶、壳聚糖、海藻酸盐、琼脂糖、透明质酸、纤维蛋白、弹性蛋白、淀粉、卡拉胶是常用的生长因子载体。这些材料可能具有一些固有的生物活性,可能有助于再生。这些材料通常溶于水,允许加载对生长因子的生物活性无害的温和制造条件。在大多数天然材料中,生长因子可浸润在材料支架中,或分散在材料支架中,通过扩散或支架的降解释放,对天然材料进行修饰则以实现生长因子控释。Natural materials such as silk, collagen, gelatin, chitosan, alginate, agarose, hyaluronic acid, fibrin, elastin, starch, and carrageenan are commonly used growth factor carriers. These materials may have some inherent biological activity that may aid regeneration. These materials are usually soluble in water, allowing for mild manufacturing conditions that are not detrimental to the biological activity of the growth factors. In most natural materials, the growth factors can be infiltrated into the material scaffold or dispersed in the material scaffold and released by diffusion or degradation of the scaffold. Modification of natural materials can achieve controlled release of growth factors.

藤壶Cp19k蛋白是藤壶胶复合物中的主要成分之一,其在藤壶粘附过程中主要起界面粘附的作用,然而,将藤壶Cp19k蛋白用于治疗难愈合的创面修复未见报道。Barnacle Cp19k protein is one of the main components of the barnacle glue complex, and it mainly plays a role in interfacial adhesion during the barnacle adhesion process. However, there are no reports on the use of barnacle Cp19k protein to treat difficult-to-heal wound repair.

发明内容Summary of the invention

为解决现有技术的缺陷,本发明提出藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病的制剂中的应用。具体而言,为了实现本发明的目的,本发明拟采用如下的技术方案:In order to solve the defects of the prior art, the present invention proposes the use of barnacle Cp19k protein in the preparation of a preparation for treating difficult-to-heal wound repair diseases. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

本发明一方面涉及藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病的制剂中的应用。One aspect of the present invention relates to the use of barnacle Cp19k protein in the preparation of a preparation for treating difficult-to-heal wound repair diseases.

在本发明的一个优选实施方式中,所述制剂为液体制剂。In a preferred embodiment of the present invention, the preparation is a liquid preparation.

在本发明的一个优选实施方式中,所述液体制剂的辅料为生理盐水。In a preferred embodiment of the present invention, the auxiliary material of the liquid preparation is physiological saline.

在本发明的一个优选实施方式中,所述制剂中还含有人成纤维生长因子2;优选地,不含有其它活性成分。In a preferred embodiment of the present invention, the preparation further contains human fibroblast growth factor 2; preferably, it does not contain other active ingredients.

在本发明的一个优选实施方式中,所述藤壶Cp19k蛋白和人成纤维生长因子2的比例为10:1-2;特别优选为10:1。In a preferred embodiment of the present invention, the ratio of the barnacle Cp19k protein to human fibroblast growth factor 2 is 10:1-2, and particularly preferably 10:1.

有益效果Beneficial Effects

本发明首次发现藤壶Cp19k蛋白在制备治疗难愈合的创面修复疾病中的应用,特别是与人成纤维生长因子2(FGF2)配合使用,不仅可以延长人成纤维生长因子2的半衰期,还可以进一步提高治疗难愈合的创面修复疾病的效果。The present invention is the first to discover the application of barnacle Cp19k protein in the preparation of treatment for difficult-to-heal wound repair diseases, especially when used in combination with human fibroblast growth factor 2 (FGF2), it can not only prolong the half-life of human fibroblast growth factor 2, but also further improve the effect of treating difficult-to-heal wound repair diseases.

具体实施方式DETAILED DESCRIPTION

为了进一步理解本发明,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅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。In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

实施例1Example 1

取1g藤壶Cp19k蛋白,分散在100g生理盐水中,4℃保存。Take 1 g of barnacle Cp19k protein, disperse it in 100 g of physiological saline, and store it at 4°C.

实施例2Example 2

取1g藤壶Cp19k蛋白和0.1g人成纤维生长因子2,分散在100g生理盐水中,4℃保存。Take 1 g of barnacle Cp19k protein and 0.1 g of human fibroblast growth factor 2, disperse them in 100 g of physiological saline, and store them at 4°C.

实施例3Example 3

取1g藤壶Cp19k蛋白和0.15g人成纤维生长因子2,分散在100g生理盐水中,4℃保存。Take 1 g of barnacle Cp19k protein and 0.15 g of human fibroblast growth factor 2, disperse them in 100 g of physiological saline, and store them at 4°C.

比较例1Comparative Example 1

取0.1g人成纤维生长因子2,分散在100g生理盐水中,4℃保存。Take 0.1 g of human fibroblast growth factor 2, disperse it in 100 g of physiological saline, and store it at 4°C.

实施例4:稳定性实验Example 4: Stability test

将制剂在室温下放置24小时,分别在0、12和24小时时采用人成纤维生长因子2(FGF2)Elisa试剂盒检测制剂中人成纤维生长因子2的相对含量,实验结果如下表1所示。The preparation was placed at room temperature for 24 hours, and the relative content of human fibroblast growth factor 2 (FGF2) in the preparation was detected using a human fibroblast growth factor 2 (FGF2) Elisa kit at 0, 12 and 24 hours, respectively. The experimental results are shown in Table 1 below.

表1:人成纤维生长因子2稳定性检测(以新制备的制剂含量为100%)Table 1: Stability test of human fibroblast growth factor 2 (the content of the newly prepared preparation is 100%)

制剂preparation 0小时0 hours 12小时12 hours 24小时24 hours 实施例2Example 2 100%100% 9595 9292 实施例3Example 3 100%100% 9494 9090 比较例1Comparative Example 1 100%100% 86%86% 75%75%

上述实验结果表明,制剂中的藤壶Cp19k蛋白能够提高人成纤维生长因子2的稳定性。The above experimental results show that the barnacle Cp19k protein in the preparation can improve the stability of human fibroblast growth factor 2.

实施例5:动物实验Example 5: Animal Experiment

兔耳增生性瘢痕模型建立:取健康成年家兔30只,雌雄各半,体重2.0±0.2kg。通过耳缘静脉注入1%戊巴比妥钠溶液(30mg/kg),75%酒精消毒,严格无菌操作,在兔耳腹侧中段沿长轴避开可见血管,采用打孔器制造4个直径为1cm的圆形全层皮肤缺损创面,创面深达软骨层,完整去除软骨膜,压迫止血后创面暴露,创面不进行任何处理,待自行愈合,在造创后16-18d,愈合区显现充血较明显、局部隆起、质地较硬的增生性瘢痕,则造模成功。Rabbit ear hypertrophic scar model establishment: 30 healthy adult rabbits, half male and half female, weighing 2.0±0.2kg were selected. 1% sodium pentobarbital solution (30mg/kg) was injected through the ear vein, and 75% alcohol was disinfected. Strict aseptic operation was performed. Four circular full-thickness skin defect wounds with a diameter of 1cm were made along the long axis of the middle ventral part of the rabbit ear, avoiding visible blood vessels. The wounds were deep into the cartilage layer, and the perichondrium was completely removed. After compression and hemostasis, the wounds were exposed. The wounds were not treated in any way and were allowed to heal on their own. At 16-18 days after the wound was made, the healing area showed obvious congestion, local protrusion, and hard hypertrophic scars, indicating a successful model.

将造模成功的兔子随机分为5组,分别为空白组、实施例1-3组、对比例1组,每组6只。其中,空白组不作任何处理,实施例1-3组、对比例1组分别喷洒本发明实施例1-3组、对比例1的制剂,每天涂抹3次,每次每处创面0.2ml,连续观察28天,收集各组瘢痕组织及其周边正常组织。The rabbits with successful modeling were randomly divided into 5 groups, namely, a blank group, Example 1-3 groups, and Comparative Example 1 group, with 6 rabbits in each group. Among them, the blank group was not treated in any way, while the Example 1-3 groups and Comparative Example 1 group were sprayed with the preparations of Example 1-3 groups and Comparative Example 1 of the present invention, respectively, 3 times a day, 0.2 ml per wound each time, and observed for 28 consecutive days, and the scar tissues and the surrounding normal tissues of each group were collected.

瘢痕增生指数:在兔耳瘢痕中央与周围组织取材,将切片行苏木精-伊红染色封片,于显微镜下拍照,测量并计算瘢痕增生指数。瘢痕增生指数=瘢痕最高点至耳软骨表面的垂直高度/周围正常皮肤厚度。Scar hyperplasia index: Samples were collected from the center and surrounding tissues of the rabbit ear scar, the sections were stained with hematoxylin-eosin, and the sections were sealed. The scar hyperplasia index was measured and calculated under a microscope. Scar hyperplasia index = vertical height from the highest point of the scar to the surface of the ear cartilage / thickness of the surrounding normal skin.

成纤维细胞数密度:经苏木精-伊红染色制成瘢痕组织切片,在400倍视野光镜下随机计数3个视野的成纤维细胞数量,求其平均值。Fibroblast density: Scar tissue sections were prepared by hematoxylin-eosin staining, and the number of fibroblasts in three fields of view were randomly counted under a 400-fold field of view light microscope to calculate the average value.

各组兔耳瘢痕增生指数、成纤维细胞数密度的测定结果见表2。The results of the measurement of scar hyperplasia index and fibroblast number density of rabbit ears in each group are shown in Table 2.

表2:瘢痕增生指数、成纤维细胞数密度结果Table 2: Scar hyperplasia index, fibroblast number density results

组别Group 瘢痕增生指数Scar hyperplasia index 成纤维细胞数密度(个/mm2)Fibroblast density (cells/mm 2 ) 空白组Blank group 3.18±0.183.18±0.18 6052±1836052±183 实施例1Example 1 1.53±0.211.53±0.21 3256±1923256±192 实施例2Example 2 1.32±0.281.32±0.28 2875±2032875±203 实施例3Example 3 1.41±0.191.41±0.19 2915±1592915±159 比较例1Comparative Example 1 2.05±0.252.05±0.25 4136±2434136±243

上述结果显示:与空白组和对比例1进行比较,本发明实施例1-3的瘢痕增生指数和成纤维细胞密度均具有统计学意义,说明藤壶Cp19k蛋白能够有效修复疤痕。特别是,本发明的实施例2和3,其相比于实施例1也具有统计学意义,说明本发明通过组合藤壶Cp19k蛋白和人成纤维生长因子2,可以相互协调,进一步协同增效地增强疤痕修复效果。The above results show that compared with the blank group and comparative example 1, the scar hyperplasia index and fibroblast density of Examples 1-3 of the present invention are statistically significant, indicating that the barnacle Cp19k protein can effectively repair scars. In particular, Examples 2 and 3 of the present invention are also statistically significant compared with Example 1, indicating that the present invention can coordinate with each other by combining barnacle Cp19k protein and human fibroblast growth factor 2 to further synergistically enhance the scar repair effect.

以上描述了本发明优选实施方式,然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims (7)

1. The application of barnacle Cp19k protein in preparing preparation for treating wound repair diseases difficult to heal.
2. The use according to claim 1, wherein the formulation is a liquid formulation.
3. The use according to claim 2, wherein the adjuvant of the liquid formulation is physiological saline.
4. Use according to any one of claims 1-3, wherein the formulation further comprises human fibroblast growth factor 2.
5. The use according to claim 4, wherein the formulation does not contain other active ingredients.
6. The use according to claim 4, wherein the weight ratio of barnacle Cp19k protein to human fibroblast growth factor 2 is 10:1-2.
7. The use according to claim 4, wherein the weight ratio of barnacle Cp19k protein to human fibroblast growth factor 2 is 10:1.
CN202410803289.4A 2024-06-20 2024-06-20 Application of barnacle Cp19k protein in the preparation of preparations for treating difficult-to-heal wound repair diseases Pending CN118557704A (en)

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