WO2021057279A1 - 一种凹耳蛙免疫调节肽及其应用 - Google Patents

一种凹耳蛙免疫调节肽及其应用 Download PDF

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WO2021057279A1
WO2021057279A1 PCT/CN2020/107720 CN2020107720W WO2021057279A1 WO 2021057279 A1 WO2021057279 A1 WO 2021057279A1 CN 2020107720 W CN2020107720 W CN 2020107720W WO 2021057279 A1 WO2021057279 A1 WO 2021057279A1
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macrophages
whp
immunomodulatory peptide
fibroblasts
skin
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French (fr)
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卫林
杨洋
徐薇
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the technical field of medical molecular biology, in particular to an immunomodulatory peptide of the frog ear and its application.
  • Wound healing is a general term for the pathological process caused by skin infection or physical trauma. It is a highly ordered biological process that requires various types of cells to restore tissue integrity. The wound healing process involves several different cell types, and the cell activity of any particular cell type may also change in different stages of repair. The complexity and coordination of the healing process are the main obstacles to the treatment method, so any therapeutic agent must be effectively sequenced to the appropriate stage. As a normal biological process of the human body, wound healing is achieved through four precise and highly programmed stages: hemostasis, inflammation, proliferation and remodeling. Wound healing is a dynamic process. The interruption, abnormality or prolongation of the process at each stage can lead to delayed or non-healing chronic wounds. Poor wound healing or wound rupture after surgery is a common symptom after surgery, causing a variety of poor prognosis, including wound infection and scar formation. Many postoperative infections form difficult-to-heal wounds, which bring great pain and psychological burden to patients.
  • the effective drugs that can promote wound healing are mainly growth factors, including epidermal growth factor (EGF), fibroblast growth factor-2 ( FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), keratinocyte factor-1 (KGF-1), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte Colony stimulating factor (G-CSF) and some other factors.
  • EGF epidermal growth factor
  • FGF-2 fibroblast growth factor-2
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived growth factor
  • KGF-1 keratinocyte factor-1
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte Colony stimulating factor
  • the skin of amphibians is an effective natural barrier and plays an important role in defense, respiration and water regulation. Due to the complex living environment of amphibians, their skin is often damaged by various organisms and non-biological species, such as predation, parasitic infection, microbial infection and some physical damage. However, it is surprising that the skin of amphibians still has good healing ability even without any post-injury care. This shows that amphibians have evolved excellent healing systems, and wound healing peptides are an important part of it.
  • amphibian-derived wound healing peptides such as the antibacterial peptides disclosed in CN201510000510.3 and CN201810366376.2
  • the mechanism of action of the healing peptides such as chemotaxis on neutrophils at the wound, and neutralization Whether granulocytes have direct chemotactic effect, whether they have direct chemotactic effect on macrophages, and the regulation of the interaction between effector cells, and other mechanisms are not in-depth enough.
  • Concave-eared frogs are mostly distributed in Huangshan Mountain, Anhui province, China. They hide in dark and humid cavities during the day, and often crawl on the shrubs, short grass branches or blades of grasses on the banks of mountain streams at night. Because its tympanic membrane is recessed and presents a slightly sloping external auditory canal, scientists have mostly focused on its ultrasound communication and behavioral studies using ultrasound communication. There have been no reports of biologically active peptides so far.
  • the purpose of the present invention is to provide an immunomodulatory peptide and its application.
  • the invention provides a new immunomodulatory peptide and its application.
  • the immunomodulatory peptide can be used as a novel wound healing polypeptide. template.
  • the first objective of the present invention is to disclose an immunomodulatory peptide (hereinafter named Ot-WHP), which includes the amino acid sequence shown in SEQ ID No. 1.
  • Ot-WHP immunomodulatory peptide
  • the above-mentioned Ot-WHP consists of 24 amino acids and has a molecular weight of 2666.19 Daltons.
  • the immunomodulatory peptide is derived from the concave-eared frog (Latin name Odorranatormota).
  • immunomodulatory peptides are derived from the skin of frog-eared frogs.
  • the second objective of the present invention is to disclose the application of immunomodulatory peptides in the preparation of medicines or skin care products for promoting skin wound healing.
  • drugs are used to promote the healing of human skin wounds.
  • the drug causes the migration of neutrophils and macrophages.
  • the drug induced mouse macrophages and skin wounds to produce chemokines and cytokines.
  • the drug activates the MAPKs, NF-excellentB and TGF- ⁇ /Smad signaling pathways of bone marrow-derived macrophages.
  • the drug promotes the proliferation of keratinocytes and fibroblasts.
  • the drug promotes the transformation of fibroblasts into fibroblasts and promotes the collagen production of fibroblasts.
  • the concentration of the immunomodulatory peptide in the drug is 20-100 ⁇ g/mL.
  • the present invention has at least the following advantages:
  • the present invention provides a new immunomodulatory peptide Ot-WHP, which has a small molecular weight and low production cost. It is applied to the preparation of drugs for promoting skin wound healing. Compared with growth factors, it has a broad application prospect and its effect The mechanism is clear, and it can be used as a new type of wound healing polypeptide template in the future.
  • the Ot-WHP provided by the invention can significantly promote wound healing in mice, and the Ot-WHP has a small molecular weight, a simple structure, and is easy for chemical synthesis and protein expression. Ot-WHP has a clear mechanism of action, and these advantages make it an excellent candidate peptide for a new generation of clinically promoting wound healing.
  • Figure 1 illustrates the activity peak measured at a wavelength of 280nm during the process of separating and preparing Ot-WHP from the skin secretion of the frog in Example 1;
  • Figure 2 is a photograph of skin wound healing of mice in different experimental groups in Example 3;
  • Figure 3 shows the test results of skin wound healing rate of mice in different experimental groups in Example 3;
  • Figure 4 shows the immunohistochemical and quantitative statistical results of neutrophils and macrophages of mice in different experimental groups in Example 4;
  • Figure 5 shows the results of the effect of Ot-WHP at different concentrations on the migration of neutrophils and macrophages in Example 5;
  • Figure 6 shows the test results of the effect of Ot-WHP at different concentrations on the production of chemokines and cytokines in mouse BMDMs cells and skin wounds in Example 6;
  • Figure 7 shows the test results of the activation of different signal pathways of different concentrations of Ot-WHP on mouse BMDMs cells in Example 7;
  • Figure 8 shows the test results of the effect of different concentrations of Ot-WHP on the proliferation of keratinocytes and fibroblasts in Example 8;
  • Figure 9 shows the test results of different concentrations of Ot-WHP promoting the conversion of fibroblasts into fibroblasts and collagen deposition in synergy with macrophages in Example 9.
  • Example 1 Separation and preparation of Ot-WHP from the skin secretion of frog-eared frog
  • Concave-eared frogs were collected from Huangshan, China, and stimulated with a little ether, a large amount of secretion will be seen on the back of the frog's body. Collect the secretion, centrifuge at 5000 ⁇ g for 10 minutes, collect the supernatant, and freeze-dry.
  • the lyophilized powder was dissolved in 10 mL of PBS (0.1M, pH 6.0), centrifuged at 5000 ⁇ g for 10 minutes, and the supernatant was collected and loaded onto Sephadex G-50 (Superfine, Amersham Biosciences, 2.6cm ⁇ 100cm)
  • PBS 0.1M, pH 6.0
  • the buffer of the filter chromatography is PBS (0.1M, pH 6.0)
  • one tube is collected every 10 minutes for every 3 mL, and about 200 tubes are collected.
  • Figure 1A is drawn according to the light absorption value of each tube.
  • the skin secretion of frog ear can be roughly divided into four absorption peaks.
  • the peaks are combined, and whether each peak has Promoting mouse skin wound healing activity, one of the peaks was found to have the function of promoting mouse skin wound healing (shown by the arrow in Figure 1A).
  • Example 1 The Ot-WHP purified in Example 1 was loaded onto the Shimadzu protein pulse liquid sequencer (PPSQ-31A; Shimadzu, Kyoto, Japan), and Edman degradation sequencing was performed according to the instrument instructions. After sequencing, the amino acid sequence was obtained. It is ATEWDLGPHGIRPLRPIRIRPLCG (SEQ ID No. 1).
  • Ot-WHP can be directly synthesized by chemical synthesis according to the known amino acid sequence of Ot-WHP.
  • mice Female, 6-8 weeks old, weighing 18-20g, purchased from Shanghai Slack Animal Co., Ltd.
  • the mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.1mL/20g), and the back hair was shaved with an electric razor and disinfected with iodophor.
  • a biopsy punch with a diameter of 8 mm was punched on the bare back. hole. After the operation, the mice were kept in separate cages until the end of the experiment.
  • mice were randomly divided into 6 groups, each with 5 mice, namely the negative control group (20 ⁇ L PBS), the natural Ot-WHP group (20 ⁇ L, 200 ⁇ g/mL), and the synthetic Ot-WHP group (20 ⁇ L, 200 ⁇ g/mL).
  • the negative control group (20 ⁇ L PBS
  • the natural Ot-WHP group (20 ⁇ L, 200 ⁇ g/mL
  • the synthetic Ot-WHP group (20 ⁇ L, 200 ⁇ g/mL
  • Disordered Ot-WHP group SEQ ID No. 2, 20 ⁇ L, 200 ⁇ g/mL
  • AH90 positive peptide control group (20 ⁇ L, 200 ⁇ g/mL
  • EGF growth factor control group 20 ⁇ L, 100 ⁇ g/mL
  • the corresponding drugs Drip it to the corresponding mouse wound once a day for 8 days.
  • Figure 2-3 shows the wound healing results of mice in different experimental groups taken at 0, 2, 4, 6, and 8 days after surgery; Figures 3A1-A4 are in turn. Test results of wound healing rate of mice in different experimental groups on 6 and 8 days.
  • Vehicle represents the negative control group; synthetic Ot-WHP represents the synthetic Ot-WHP group; NaturalOt-WHP represents the natural Ot-WHP group; Scrambled Ot-WHP represents the disordered Ot-WHP group.
  • the results show that both natural Ot-WHP and synthetic Ot-WHP can significantly promote the healing of mouse skin wounds, and the effect is equivalent to AH90 and EGF, while disordered Ot-WHP has no activity to promote wound repair.
  • Example 4 Ot-WHP recruits neutrophils and macrophages to migrate to the wound, accelerating the initial stage of inflammation in wound healing
  • mice were treated according to the method of Example 3.
  • the corresponding drugs in the negative control group and the natural Ot-WHP group were respectively dripped into the wounds of the corresponding mice at designated time points (0.5, 1, 2, 3 days) after the operation, and the mice were killed
  • the biopsy tissue containing the wound center was fixed with 10% formalin, then dehydrated by ethanol, washed with xylene, and finally embedded in paraffin.
  • the paraffin block was cut into 5 ⁇ m thin slices, after dewaxing, rehydration and antigen retrieval, immunohistochemical analysis was performed.
  • the sections were blocked with 5% BSA for non-specific sites, the primary antibody was 4°C overnight (anti-Ly6G for neutrophils, anti-F4/80 for macrophages), horseradish peroxidase and DBA is used to develop immunochromatography.
  • the infiltration of neutrophils and macrophages was performed by counting the number of cells stained with anti-Ly6G and anti-F4/80.
  • Figure 4A1-A4 are different groups of mice.
  • Figures 4C1-C4 are different groups of mice.
  • the immunohistochemical results of macrophages recruited on day 2 and day 3 are different groups of mice.
  • the statistical results of the number of neutrophils recruited on day 0.5 and day 1 is a different group of mice.
  • Example 5 Ot-WHP does not directly cause the migration of neutrophils and macrophages, but requires the coexistence of macrophages to cause the migration of neutrophils and macrophages
  • the prepared mouse neutrophils (neutrophils) or bone marrow-derived macrophages (BMDMs) were resuspended in 2% FBS
  • the cell density is 7 ⁇ 10 6 cells/mL
  • aspirate 500 ⁇ L of Ot-WHP (25, 50, 100 ⁇ g/mL, Ot-WHP dissolved in 2% FBS RPMI 1640 medium) or medium (control) was added to the lower chamber, and then cultured in a cell incubator at 37°C for 8 hours to collect the cells in the lower chamber.
  • Hemocytometer counts were used in the cell density of Ot-WHP (25, 50, 100 ⁇ g/mL, Ot-WHP dissolved in 2% FBS RPMI 1640 medium) or medium (control) was added to the lower chamber, and then cultured in a cell incubator at 37°C for 8 hours to collect the cells in the lower chamber. Hemo
  • mouse bone marrow-derived macrophages (BMDMs, 4 ⁇ 10 6 cells/mL, 500 ⁇ L) resuspended in 2% FBS in RPMI 1640 medium were plated in the lower chamber (24 Well plate), after adherence, the prepared mouse neutrophils (neutrophils, 7 ⁇ 10 6 cells/mL, 100 ⁇ L) or bone marrow-derived macrophages (BMDMs, 7 ⁇ 10 6 cells/mL, 100 ⁇ L) was added to a 3.0 ⁇ m pore size Transwell cell (upper chamber, 24-well plate), and then 500 ⁇ L of Ot-WHP (25, 50, 100 ⁇ g/mL, Ot-WHP was dissolved in 2% FBS in RPMI 1640 medium) ) Or culture medium (control) was added to the lower chamber, and then cultured in a 37°C cell incubator for 8 hours. The cells in the upper chamber were collected and counted with a hemocytometer. The cells that decreased in the upper chamber were the migrated cells.
  • BMDMs
  • Ot-WHP does not directly cause the migration of neutrophils (Figure 5A) and macrophages (Figure 5C) in the concentration range of 25-100 ⁇ g/mL, but macrophages coexist.
  • Ot-WHP was in the concentration range of 25-100 ⁇ g/mL, in a dose-dependent manner, it significantly caused the migration of neutrophils (Figure 5B) and macrophages (Figure 5D).
  • Ot-WHP can induce mouse macrophages and skin wounds to produce chemokines and cytokines
  • Ot-WHP can cause the migration of neutrophils and macrophages in the coexistence of macrophages
  • Base 2% FBS
  • Ot-WHP 25, 50, 100 ⁇ g/mL
  • PBS PBS
  • Ot-WHP can effectively induce the production of chemokines (CXCL1 and CCL2) and cytokines (TNF- ⁇ and TGF) in mouse skin wounds. - ⁇ 1).
  • Ot-WHP can activate the MAPKs, NF-excellentB, and TGF- ⁇ /Smad signaling pathways of mouse bone marrow-derived macrophages
  • BMDMs Mouse bone marrow-derived macrophages
  • MAPKs and NF- ⁇ B BMDMs were incubated with different concentrations of Ot-WHP (0, 25, 50, 100 ⁇ g/mL) for 30 min.
  • Ot-WHP 0, 25, 50, 100 ⁇ g/mL
  • TGF- ⁇ /Smad signaling pathway BMDMs were combined with Ot-WHP ( 0, 25, 50, 100 ⁇ g/mL) incubate for 24h or 100 ⁇ g/mL Ot-WHP with or without TGF- ⁇ 1 antibody.
  • Example 8 Ot-WHP does not directly promote the proliferation of keratinocytes and fibroblasts, but requires the coexistence of macrophages to promote the proliferation of keratinocytes and fibroblasts
  • Keratinocytes HaCat and mouse fibroblasts (5 ⁇ 10 3 cells/well, 100 ⁇ L/well) were inoculated into 96-well plates and incubated with different concentrations of Ot-WHP (25, 50, and 100 ⁇ g/mL) at the same time. Use PBS as a control. After incubating HaCat and Ot-WHP for 24 hours, and after incubating fibroblasts and Ot-WHP for 72 hours, add 10 ⁇ l of Cell Counting Kit-8 (CCK-8) solution to each well, and incubate at 37°C for 2-4 hours. Microplate reader Detect the light absorbance value at 450nm.
  • Ot-WHP 25, 50, and 100 ⁇ g/mL
  • HaCat was co-cultured with THP-1 derived macrophages
  • mouse fibroblasts were co-cultured with mouse macrophages BMDMs.
  • Corresponding macrophages (1 ⁇ 105 cells/well, 100 ⁇ L) and Ot-WHP (25, 50, 100 ⁇ g/mL) or Vehicle (PBS, control) of different concentrations were added.
  • Ot-WHP cannot directly promote the proliferation of keratinocytes (Figure 8A) and fibroblasts (Figure 8B); and only when macrophages coexist, can it promote keratinocytes (Figure 8C) And the proliferation of fibroblasts ( Figure 8D). It shows that Ot-WHP can promote the interaction between macrophages and keratinocytes/fibroblasts.
  • Example 9 Ot-WHP does not directly promote the conversion of fibroblasts to fibroblasts, nor does it directly promote the production of collagen by fibroblasts, but requires the coexistence of macrophages to promote fibroblasts to fibroblasts The transformation can promote the collagen production of fibroblasts
  • the method is the same as that in Example 8.
  • the fibroblasts treated in Example 8 were lysed with RIPA cell lysate, and the protein was extracted. Western blot was used to detect the expression of ⁇ -SMA. The higher the expression level of ⁇ -SMA, the fibroblasts are becoming The more fibroblasts are transformed); the supernatant of the fibroblasts treated in Example 8 is collected, and the production of collagen in the supernatant is quantitatively detected with an ELISA kit.

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Abstract

提供了一种免疫调节肽及其在制备促进皮肤伤口愈合药物或护肤品中的应用，该免疫调节肽包括SEQ ID No.1所示的氨基酸序列。还提供了该免疫调节肽促进伤口愈合的作用机制，可作为伤口愈合肽模板。

Description

一种凹耳蛙免疫调节肽及其应用 技术领域
本发明涉及医学分子生物学技术领域，尤其涉及一种凹耳蛙免疫调节肽及其应用。
背景技术
伤口愈合是皮肤感染或受到物理创伤后引起的病理过程的总称，需要各种不同类型细胞恢复组织完整性的一个高度有序的生物过程。创面愈合过程涉及几种不同的细胞类型，任何特定细胞类型的细胞活性在修复的不同阶段也可能变化。愈合过程的复杂性和协调性是治疗方法的主要障碍，因此任何治疗剂都必须有效地排序到合适的阶段。伤口愈合作为人体正常的生物过程，是通过四个精确和高度程序化的阶段实现的：止血，炎症，增殖和重塑。伤口愈合是一个动态过程，每个阶段过程中的中断，异常或延长可导致伤口愈合延迟或不愈合的慢性伤口。手术后伤口愈合不良或伤口破裂是外科手术后的常见症状，造成多种不良的预后，包括伤口感染、形成瘢痕等。许多术后感染形成难以愈合性伤口，给患者带来极大的痛苦和心理负担。
在过去的十几年里，各种新型的生物制剂疗法取得了一定的成效，目前可促进伤口愈合的有效药物主要是生长因子类，包括表皮生长因子(EGF)，成纤维生长因-2(FGF-2)，血管内皮生长因子(VEGF)，血小板来源的生长因子(PDGF)，角质形成细胞因子-1(KGF-1)，粒细胞巨噬细胞集落刺激因子(GM-CSF)，粒细胞集落刺激因子(G-CSF)和有一些其他的因子。虽然基于这些生长因子的治疗在临床上取得了一定的应用，但是这些生长因子的分子量大、生产成本很高，从而限制了他们在临床上的广泛应用。
两栖动物的皮肤是有效的天然屏障，在防御、呼吸和水分调节方面起重要作用。由于两栖动物生存环境复杂，其皮肤经常受到各种生物和非生物的损伤，比如捕食、寄生感染、微生物感染和一些物理损伤。然而令人惊奇的是，两栖动物的皮肤即使在没有任何伤后护理情况下仍然具有很好的愈合能力。这说明两栖动物已经进化出优秀的愈合系统，而伤口愈合肽是其重要的组成部分。虽然已有一些两栖动物来源的伤口愈合肽报道，如CN201510000510.3、CN201810366376.2所公开的抗菌肽，但愈合肽的作用机制，比如对伤口处中性粒细胞的趋化作用、对中性粒细胞是否具有直接趋化作用、对巨噬细胞是否具有直接趋化作用、对效应细胞之间互作的调节作用等机制的研究还不够深入。
凹耳蛙多分布于中国安徽省黄山，白天隐匿于阴暗潮湿的洞隙内，夜晚常匍匐在山溪两 岸的灌木、矮草枝或草丛的叶片上捕食。由于其鼓膜凹陷，呈一个略向前斜的外听道，科学家多将焦点集中到它的超声通讯以及利用超声通讯的行为学研究上，至今未有生物活性多肽的报道。
发明内容
为解决上述技术问题，本发明的目的是提供一种凹耳蛙免疫调节肽及其应用，本发明提供了一种新的免疫调节肽及其应用，可利用该免疫调节肽作为新型伤口愈合多肽模板。
本发明的第一个目的是公开一种免疫调节肽(以下命名为Ot-WHP)，其包括SEQ ID No.1所示的氨基酸序列。上述Ot-WHP)由24个氨基酸组成，分子量为2666.19道尔顿。
进一步地，免疫调节肽来源于凹耳蛙(拉丁文名为Odorranatormota)。
进一步地，免疫调节肽来源于凹耳蛙的皮肤。
本发明的第二个目的是公开免疫调节肽在制备促进皮肤伤口愈合药物或护肤品中的应用。
进一步地，药物用于促进人类的皮肤伤口愈合。
进一步地，在巨噬细胞存在下，所述药物引起中性粒和巨噬细胞的迁移。
进一步地，药物诱导小鼠巨噬细胞和皮肤伤口处产生趋化因子和细胞因子。
进一步地，药物激活骨髓来源的巨噬细胞的MAPKs、NF-кB和TGF-β/Smad信号通路。
进一步地，在巨噬细胞存在下，所述药物促进角质细胞和成纤维细胞的增殖。
进一步地，在巨噬细胞存在下，所述药物促进成纤维细胞向成纤维母细胞的转化并促进成纤维细胞的胶原产生。
进一步地，在巨噬细胞存在下，所述药物中免疫调节肽的浓度为20-100μg/mL。
借由上述方案，本发明至少具有以下优点：
本发明提供了一种新的免疫调节肽Ot-WHP，其分子量小，生产成本低，将其应用于制备促进皮肤伤口愈合药物中，相比于生长因子而言，应用前景广阔，且其作用机制明确，将来可作为新型的伤口愈合多肽模板。
本发明提供的Ot-WHP可以显著促进小鼠伤口愈合，而且Ot-WHP分子量小，结构简单，易于化学合成和蛋白表达。Ot-WHP作用机制明确，这些优点使其有望成为新一代临床促进伤口愈合的优良候选多肽。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1图示了实施例1中从凹耳蛙皮肤分泌液中分离制备Ot-WHP过程中在280nm波长下所测得的活性峰；
图2为实施例3中不同实验组小鼠的皮肤伤口愈合情况照片图；
图3为实施例3中不同实验组小鼠的皮肤伤口愈合率测试结果；
图4为实施例4中不同实验组小鼠的中性粒细胞和巨噬细胞的免疫组化和数量统计结果；
图5为实施例5中不同浓度的Ot-WHP对中性粒和巨噬细胞的迁移影响结果；
图6为实施例6中不同浓度的Ot-WHP对小鼠BMDMs细胞和皮肤伤口处的趋化因子和细胞因子产生情况的影响情况测试结果；
图7为实施例7中不同浓度的Ot-WHP对小鼠BMDMs细胞的不同信号通路的激活作用测试结果；
图8为实施例8中不同浓度的Ot-WHP对角质细胞和成纤维细胞的增值影响测试结果；
图9为实施例9中不同浓度的Ot-WHP促进与巨噬细胞协同作用的成纤维细胞向成纤维母细胞的转化以及胶原沉积情况测试结果。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：从凹耳蛙皮肤分泌液中分离制备Ot-WHP
凹耳蛙采集自中国黄山，并用少许乙醚刺激凹耳蛙，会看到蛙体背部有大量分泌液产生，收集分泌液，5000×g离心10分钟，收集上清，并冻干。冻干粉溶解于10mL的PBS中(0.1M，pH6.0)，并在5000×g离心10分钟，收集上清，上样到Sephadex G-50(Superfine，Amersham Biosciences，2.6cm×100cm)的葡聚糖凝胶层析柱中，进行过滤层析，过滤层析的缓冲为PBS(0.1M，pH6.0)，按照每3mL每10分钟收集一管，约收集200管。收集完毕后，检测各管的在280nm处的光吸收，根据各管的光吸收值，绘制出图1A。
根据图1A，凹耳蛙皮肤分泌液经Sephadex G-50葡聚糖凝胶过滤层析后，可以被粗分为四个吸收峰，合并各峰，并按照实施例3中检测各峰是否具有促进小鼠皮肤伤口愈合活性，其中发现一个峰具有促进小鼠皮肤伤口愈合的功能(图1A中箭头所示)。然后将图1A中的活性峰上样到C18(5μm particle size，
Figure PCTCN2020107720-appb-000001
pore size，250mm×4.6mm，Gemini，CA，USA) 反向高效液相色谱过滤层析，平衡相为加有0.1％三氟乙酸的水，用0-60％的乙腈梯度进行洗脱，洗脱时间为80分钟，检测280nm处的光吸收，光吸收特征如图1B所示，同上，进行促进皮肤伤口修复活性跟踪，图1B中的活性峰用箭头标出。接着，在和图1B同样的条件下，将图1B中的活性峰进行第二次反向高效液相色谱过滤层析，结果如图1C所示，活性峰用箭头标出，命名为Ot-WHP。
实施例2：Ot-WHP的氨基酸测序
将实施例1中纯化到的Ot-WHP上样到岛津蛋白脉冲液相测序仪(PPSQ-31A；Shimadzu，Kyoto，Japan)，按照仪器说明书，进行Edman降解测序，测序完毕后，获得氨基酸序列为ATAWDLGPHGIRPLRPIRIRPLCG(SEQ ID No.1)。
在实际应用过程中，可根据已知的Ot-WHP的氨基酸序列，采用化学合成法直接合成Ot-WHP。
实施例3：Ot-WHP可显著促进小鼠皮肤伤口愈合
实验动物BALB/c小鼠，雌性，6～8周龄，体质量18～20g，购于上海斯莱克动物有限公司。将小鼠腹腔注射2％的戊巴比妥钠(0.1mL/20g)使其麻醉后，用电动剃刀剃去背部毛发后碘伏消毒，在裸露的背部用直径8mm的活检打孔器打一个洞。术后小鼠单独分笼饲养直至实验结束。将小鼠随机分为6组，每组5只，分别为阴性对照组(20μL PBS)，天然Ot-WHP组(20μL，200μg/mL)，合成Ot-WHP组(20μL，200μg/mL)，乱序Ot-WHP组(SEQ ID No.2，20μL，200μg/mL)，AH90阳性多肽对照组(20μL，200μg/mL)，EGF生长因子对照组(20μL，100μg/mL)，将相应药物分别滴加到对应小鼠伤口处，每天一次，共8天。
结果如图2-3所示，其中图2为术后0，2，4，6，8天拍摄的不同实验组小鼠伤口愈合情况结果；图3A1-A4依次.为术后2，4，6，8天不同实验组小鼠伤口愈合率测试结果。图中，Vehicle代表阴性对照组；synthetic Ot-WHP代表合成Ot-WHP组；NaturalOt-WHP代表天然Ot-WHP组；Scrambled Ot-WHP代表乱序Ot-WHP组。结果表明，天然Ot-WHP和合成Ot-WHP都可以显著促进小鼠皮肤伤口愈合，效果与AH90和EGF相当，而乱序Ot-WHP则没有促进伤口修复的活性。
实施例4：Ot-WHP通过招募中性粒细胞及巨噬细胞迁移至伤口处，加速伤口愈合的炎症起始阶段
按照实施例3的方法处理小鼠，在阴性对照组和天然Ot-WHP组的相应药物分别滴加到对应小鼠伤口处术后指定时间点(0.5，1，2，3天)处死小鼠，取包含伤口中心的活检组织用10％的福尔马林固定，然后通过乙醇脱水，二甲苯清洗，最后石蜡包埋。将石蜡块切成5μm 的薄片，经过脱蜡，再水化和抗原修复后进行免疫组化分析。先将切片用5％的BSA封闭非特异位点，一抗4℃过夜(中性粒细胞的抗体anti-Ly6G，巨噬细胞的抗体anti-F4/80)，用辣根过氧化物酶和DBA来免疫显色。中性粒细胞和巨噬细胞的浸润通过统计免疫染色anti-Ly6G和anti-F4/80的细胞数。
结果如图4所示，图4A1-A4分别为不同组小鼠.术后第0.5天和1天招募的中性粒细胞的免疫组化结果，图4C1-C4分别为不同组小鼠.术后第2天和3天招募的巨噬细胞的免疫组化结果，图4B1-B2为不同组小鼠.术后第0.5天和1天招募的中性粒细胞的数量统计结果，图4D1-D2分别为不同组小鼠.术后第2天和3天招募的巨噬细胞的数量统计结果。结果表明，Ot-WHP可促进中性粒细胞及巨噬细胞迁移至伤口处。
实施例5：Ot-WHP不直接引起中性粒和巨噬细胞的迁移，而是需要巨噬细胞的共存才可以引起中性粒和巨噬细胞的迁移
为了明确Ot-WHP如何趋化中性粒细胞和巨噬细胞至伤口局部，将制备好的小鼠中性粒细胞(neutrophils)或骨髓来源的巨噬细胞(BMDMs)重悬于含有2％FBS的RPMI 1640培养基中，细胞密度为7×10 6cells/mL，吸取100μL加入到3.0μm孔径的Transwell小室中(上室，24孔板)，然后吸取500μL的Ot-WHP(25，50，100μg/mL，Ot-WHP溶解于2％FBS的RPMI 1640培养基中)或培养基(对照)加入到下室中，再在37℃细胞培养箱中培养8小时，收集下室中的细胞用血球计数板计数。
在共培养体系中，用2％FBS的RPMI 1640培养基重悬好的小鼠骨髓来源的巨噬细胞(BMDMs，4×10 6cells/mL，500μL)铺于共培养小室的下室(24孔板)，待贴壁后，将制备好的小鼠中性粒细胞(neutrophils，7×10 6cells/mL，100μL)或骨髓来源的巨噬细胞(BMDMs，7×10 6cells/mL，100μL)加入到3.0μm孔径的Transwell小室中(上室，24孔板)，然后吸取500μL的Ot-WHP(25，50，100μg/mL，Ot-WHP溶解于2％FBS的RPMI 1640培养基中)或培养基(对照)加入到下室中，再在37℃细胞培养箱中培养8小时，收集上室中的细胞用血球计数板计数，上室中减少的细胞即为迁移的细胞。
结果如图5所示，Ot-WHP在25-100μg/mL的浓度范围内，并不直接引起中性粒细胞(图5A)和巨噬细胞(图5C)的迁移，但有巨噬细胞共存时，Ot-WHP在25-100μg/mL的浓度范围内，以剂量依赖的方式，显著引起中性粒细胞(图5B)和巨噬细胞(图5D)的迁移。
实施例6：Ot-WHP可诱导小鼠巨噬细胞和皮肤伤口处产生趋化因子和细胞因子
为了明确为什么在巨噬细胞共存的情况下，Ot-WHP可引起中性粒细胞和巨噬细胞的迁移，将小鼠骨髓来源的巨噬细胞(BMDMs，5×10 5cells/well，DMEM培养基，2％FBS)铺于 24孔板中，加入Ot-WHP(25，50，100μg/mL)或等体积的PBS(Vehicle)，培养24小时候，收取上清，10000×g离心10分钟，测趋化因子和细胞因子。
结果如图6A1-A8所示，与Vehicle(PBS，对照)处理组相比，Ot-WHP以剂量依赖的方式，诱导小鼠BMDMs产生趋化因子(CXCL1，CXCL2，CXCL3和CCL2)和细胞因子(TNF-α，IL-1β，IL-6和TGF-β1)。
然后检测Ot-WHP是否可以诱导小鼠皮肤伤口处产生趋化因子和细胞因子。在指定时间点(第0，0.5，1，2，4和6天)取小鼠的伤口组织，用0.1M的PBS(含1mM的PMSF，1mL/g组织)，将伤口组织用玻璃匀浆器进行匀浆。匀浆完成后，12000×g、4℃离心30分钟，收集上清，测趋化因子(CXCL1和CCL2)和细胞因子(包括TNF-α和TGF-β1)。
结果如图6B1-B4所示，与Vehicle(PBS，对照)处理组相比，Ot-WHP可有效诱导小鼠皮肤伤口处产生趋化因子(CXCL1和CCL2)和细胞因子(TNF-α和TGF-β1)。
实施例7：Ot-WHP可激活小鼠骨髓来源的巨噬细胞的MAPKs，NF-кB，TGF-β/Smad信号通路
小鼠骨髓来源的巨噬细胞(BMDMs)接种至6孔板中无血清培养16h。对于MAPKs和NF-κB的检测，BMDMs与不同浓度的Ot-WHP(0，25，50，100μg/mL)孵育30min，对于TGF-β/Smad信号通路检测，BMDMs与不同浓度的Ot-WHP(0，25，50，100μg/mL)孵育24h或与100μg/mL Ot-WHP孵育时加或不加TGF-β1的抗体。
结果如图7所示，与Vehicle(PBS，对照)处理组相比，Ot-WHP以剂量依赖的方式激活小鼠BMDMs的MAPKs(ERK，JNK亚基，图7A，6C)、NF-κB(IκBα，p65亚基，图7B，6C)和Smad(Smad2，Smad3，图7D)信号通路，而且Ot-WHP对Smad信号通路的激活是依赖于Ot-WHP诱导的TGF-β的产生(图7E-G)。
实施例8：Ot-WHP不直接促进角质细胞和成纤维细胞增殖，而是需要巨噬细胞的共存才可以促进角质细胞和成纤维细胞的增殖
角质细胞HaCat与小鼠成纤维细胞(5×10 3cells/well，100μL/well)接种到96孔板中，与不同浓度的Ot-WHP(25，50，and 100μg/mL)共同孵育，同时用PBS作对照。HaCat与Ot-WHP孵育24小时后，成纤维细胞与Ot-WHP孵育72小时后，每孔加入细胞计数试剂盒-8(CCK-8)溶液10μl，37℃继续孵育2-4h，酶标仪检测450nm处的光吸光值。
对于共培养细胞，HaCat与THP-1来源的巨噬细胞共培养，小鼠成纤维细胞与小鼠巨噬细胞BMDMs共培养。先将HaCat(1×10 5cells/well)或小鼠成纤维细胞(1×10 5cells/well)提前一天接种到24孔板中(下室)，待贴壁后，然后在共培养小室(上室)中加入对应的巨噬细胞 (1×105cells/well，100μL)和不同浓度的Ot-WHP(25，50，100μg/mL)或Vehicle(PBS，对照)。HaCat与THP-1来源的巨噬细胞共培养24小时后，成纤维细胞与小鼠巨噬细胞BMDMs共培养72小时候后，弃掉共培养的上室，用CCK-8检测下室中的细胞活性。
结果如图8所示，Ot-WHP并不能直接促进角质细胞(图8A)和成纤维细胞(图8B)的增殖；而在巨噬细胞共存的条件下，才可以促进角质细胞(图8C)和成纤维细胞(图8D)的增殖。表明，Ot-WHP可以促进巨噬细胞与角质细胞/成纤维细胞之间的相互作用。
实施例9：Ot-WHP不直接促进成纤维细胞向成纤维母细胞的转化，不直接促进成纤维细胞的胶原产生，而是需要巨噬细胞的共存才可以促进成纤维细胞向成纤维母细胞的转化，才可以促进成纤维细胞的胶原产生
方法同实施例8，用RIPA细胞裂解液，裂解实施例8中处理的成纤维细胞，提取蛋白，用Western blot检测α-SMA的表达，α-SMA表达水平越高，代表成纤维细胞向成纤维母细胞转化的越多)；收集实施例8中处理的成纤维细胞的上清，用ELISA试剂盒定量检测上清中胶原的产生情况。
结果如图9所示，Ot-WHP对成纤维细胞α-SMA表达没有直接影响，Ot-WHP不直接促进成纤维细胞向成纤维母细胞的转化(图9A)，且Ot-WHP对成纤维细胞胶原生成没有直接影响，不直接促进成纤维细胞的胶原产生(图9B)；而是需要巨噬细胞的共存，才可以促进成纤维细胞α-SMA的表达，即促进成纤维细胞向成纤维母细胞的转化(图9C)，才可以促进成纤维细胞的胶原产生(图9D)。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2020107720-appb-000002

Claims (10)

  1. 一种免疫调节肽，其特征在于，其包括SEQ ID No.1所示的氨基酸序列。
  2. 根据权利要求1所述的免疫调节肽，其特征在于：所述免疫调节肽来源于凹耳蛙。
  3. 根据权利要求1所述的免疫调节肽，其特征在于：所述免疫调节肽来源于凹耳蛙的皮肤。
  4. 权利要求1-3中任一项所述的免疫调节肽在制备促进皮肤伤口愈合药物或护肤品中的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述药物用于促进人类的皮肤伤口愈合。
  6. 根据权利要求4所述的应用，其特征在于：在巨噬细胞存在下，所述药物引起中性粒和巨噬细胞的迁移。
  7. 根据权利要求4所述的应用，其特征在于：所述药物诱导小鼠巨噬细胞和皮肤伤口处产生趋化因子和细胞因子。
  8. 根据权利要求4所述的应用，其特征在于：所述药物激活骨髓来源的巨噬细胞的MAPKs、NF-кB和TGF-β/Smad信号通路。
  9. 根据权利要求4所述的应用，其特征在于：在巨噬细胞存在下，所述药物促进角质细胞和成纤维细胞的增殖。
  10. 根据权利要求4所述的应用，其特征在于：在巨噬细胞存在下，所述药物中免疫调节肽的浓度为20-100μg/mL。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101333258A (zh) * 2008-07-29 2008-12-31 辽宁大学 一种具有抗菌及促伤口愈合功能的融合多肽
CN102657845A (zh) * 2012-05-31 2012-09-12 中国科学院昆明动物研究所 无指盘臭蛙促皮肤修复多肽ah90和cw49的应用
US20180271938A1 (en) * 2011-09-12 2018-09-27 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
CN110577588A (zh) * 2019-09-29 2019-12-17 苏州大学 一种凹耳蛙免疫调节肽及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2332582T3 (da) * 2002-06-19 2014-01-20 Apeiron Biologics Ag ACE2-aktivering til behandling af hjerte-, lunge-og nyresygdomme samt forhøjet blodtryk
WO2006076736A2 (en) * 2005-01-14 2006-07-20 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods for modulating angiogenesis, lymphangiogenesis, and apoptosis with apelin compositions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101333258A (zh) * 2008-07-29 2008-12-31 辽宁大学 一种具有抗菌及促伤口愈合功能的融合多肽
US20180271938A1 (en) * 2011-09-12 2018-09-27 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
CN102657845A (zh) * 2012-05-31 2012-09-12 中国科学院昆明动物研究所 无指盘臭蛙促皮肤修复多肽ah90和cw49的应用
CN110577588A (zh) * 2019-09-29 2019-12-17 苏州大学 一种凹耳蛙免疫调节肽及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 16 October 2019 (2019-10-16), ANONYMOUS: "WHP precursor [Odorrana tormota]", XP055795729, retrieved from Genbank Database accession no. QEG59350 *
HE XIAOQIN, YANG YANG, MU LIXIAN, ZHOU YANDONG, CHEN YUE, WU JING, WANG YIPENG, YANG HAILONG, LI MIN, XU WEI, WEI LIN: "A Frog-Derived Immunomodulatory Peptide Promotes Cutaneous Wound Healing by Regulating Cellular Response", FRONTIERS IN IMMUNOLOGY, vol. 10, 17 October 2019 (2019-10-17), pages 1 - 20, XP055795714, ISSN: 1664-3224, DOI: 10.3389/fimmu.2019.02421 *
MIAO HUI-MIN,XIAO XIANG-HONG,SUN TING-TING,QI JUN,CHAI LONG-HUI,ZHANG JING-YU,GUO NA: "Study on Effect of Antimicrobial Peptides Dybowskin-1ST from Skin of Rana dybowskii on Wound Healing Skin of Mice", CHINESE JOURNAL OF EXPERIMENTAL TRADITIONAL MEDICAL FORMULAE, vol. 17, no. 23, 5 December 2011 (2011-12-05), pages 125 - 128, XP055795725, ISSN: 1005-9903, DOI: 10.13422/j.cnki.syfjx.2011.23.059 *

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