CN115873074A - Active polypeptide OA-RD17 for promoting skin wound repair and application thereof - Google Patents

Abstract

The invention discloses an active polypeptide OA-RD17 for promoting skin wound repair and application thereof. The amino acid sequence of the polypeptide OA-RD17 is RDYCTPEDCDYDFSFPI. The application of the polypeptide OA-RD17 is the application in preparing medicines for promoting skin wound repair. The invention provides a novel polypeptide OA-RD17, and the polypeptide OA-RD17 has promotion effect on wound repair in vivo and in vitro and has excellent activity of promoting skin tissue regeneration. And OA-RD17 only has 17 amino acids, is easy to synthesize and low in cost, provides a new way for the development of skin wound repair medicines and provides a new direction for the development of skin wound treatment medicines.

Description

Active polypeptide OA-RD17 for promoting skin wound repair and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an active polypeptide OA-RD17 for promoting skin wound repair and application thereof.

Background

The skin is the largest organ of the human body and is not only the primary natural protective barrier of the body, but also plays a vital role in numerous physiological processes. Once the skin is damaged, the body initiates a cascade and fertilization-regulated wound repair process to restore the skin's barrier function and maintain homeostasis as soon as possible. However, the wound skin regeneration process is susceptible to self-disease and external environment, which may lead to chronic non-healing wounds or excessive healing of the wounds. Chronic non-healing wounds and excessive healing of wounds not only damage the normal physiological functions of the skin, but also have high clinical treatment cost, and the number of affected patients is increased year by year, thereby bringing huge psychological pressure and economic burden to the patients and heavy burden to the global health care system. Therefore, the quick and efficient regeneration and repair of the skin after being injured plays an important role in the restoration of normal physiological functions of organisms, the restoration of homeostasis and the reduction of psychological and economic burdens of patients and society.

In recent years, stem cell therapy, tissue engineering technology and keratinocyte transplantation provide a novel treatment strategy for wound repair, but the defects of high price, long treatment period and the like exist, and the wide popularization is difficult to realize. The medicine treatment still plays an important role in the treatment of skin wounds, and common clinical wound repair treatment medicines mainly comprise small molecular compounds, growth factors (rh-bFGF, EGF), animal and plant source medicines, low-viscosity dressings and the like. Although these drugs have good effects, they have many disadvantages such as difficult synthesis, high synthesis cost, harsh storage conditions, unstable active ingredients of the drugs and easy scar formation, and are difficult to satisfy the clinical requirements for drugs for treating skin wounds. Therefore, the discovery of novel efficient lead molecules for promoting skin tissue regeneration and the exploration of molecular mechanisms for promoting wound repair are important and necessary.

Compared with small molecular compound drugs, the polypeptide drugs have the characteristics of stronger activity, lower toxicity, higher safety, high development success rate, stronger targeting property and the like. In addition, the development of polypeptide drugs has a broad commercial market, and has gradually become a non-negligible "potential stock" in the field of drug development. The active peptide from the amphibian is expected to become one of important sources of novel regeneration-promoting drug lead molecules in the research and development field of the tissue regeneration-promoting drug, but the related reports are few, and the active peptide is still in the starting stage.

The present invention aims to provide a wound repair-promoting active peptide which exhibits a good activity of promoting the repair of skin wounds both in vivo and in vitro.

Disclosure of Invention

The first purpose of the invention is to provide an active polypeptide OA-RD17 for promoting skin wound repair, and the second purpose of the invention is to provide application of the active polypeptide OA-RD17.

The first object of the present invention is achieved by providing a polypeptide OA-RD17 having a skin wound repair promoting activity, wherein the amino acid sequence of the polypeptide is RDYCTPEDCDYDFSFPI, as shown in SEQ ID NO. 1.

The second aim of the invention is realized by the application of the active polypeptide OA-RD17 for promoting skin wound repair in the preparation of medicines for promoting skin wound repair.

The beneficial effects of the invention are as follows: the invention provides a novel polypeptide OA-RD17, and the polypeptide OA-RD17 has promotion effect on wound repair in vivo and in vitro and has excellent activity of promoting skin tissue regeneration. And OA-RD17 only has 17 amino acids, is easy to synthesize and low in cost, provides a new way for the development of skin wound repair medicines and provides a new direction for the development of skin wound treatment medicines.

Drawings

FIG. 1 is a primary structural diagram of the polypeptide OA-RD17 of the present invention;

FIG. 2 is a graph showing the HaCaT cell scratch repair-promoting activity of the polypeptide OA-RD17 of the present invention;

FIG. 3 is a graph showing the HaCaT cell proliferation-promoting activity of the polypeptide OA-RD17 of the present invention;

FIG. 4 is a graph showing the HaCaT cell migration-promoting activity of the polypeptide OA-RD17 of the present invention;

FIG. 5 is a diagram showing the activity of the polypeptide OA-RD17 in promoting the repair of the mouse full-cortical wound in an animal model, wherein A is a diagram showing the change in the repair of the mouse full-cortical wound, B is a diagram showing the repair of the mouse full-cortical wound, C is a diagram showing HE staining patterns at 4 th and 8 th days of the mouse full-cortical wound area skin tissue, D is a diagram showing the regeneration thickness of the mouse wound area skin tissue at 4 th and 8 th days of the wound, respectively, and E is a diagram showing the regeneration thickness of the mouse wound area skin tissue at 4 th and 8 th days of the wound, respectively.

Detailed Description

The invention is further described in detail below with reference to the drawings and examples, but the invention is not limited in any way, and any changes or modifications made based on the teachings of the invention fall within the scope of the invention.

The invention provides an active polypeptide OA-RD17 derived from rana obovata for promoting skin wound repair, wherein the amino acid sequence of the polypeptide OA-RD17 is RDYCTPEDCDYDFSFPI.

The invention also provides application of the polypeptide OA-RD17 in preparing a medicine for promoting skin wound repair.

The invention further provides a pharmaceutical composition comprising said polypeptide OA-RD17 as an active agent and a pharmaceutically effective carrier.

EXAMPLE 1 screening of polypeptide OA-RD17 having skin wound repair promoting activity

The first step is as follows: the sample is from skin of Rana yunnanensis. Adult Rana grahami (from Yunnan province of China) is bred in a 21 cm × 21 cm × 15 cm feeding jar for 7 days to adapt to the environment, water is periodically changed, and the adult Rana grahami is fed with Tenebrio molitor as feed. After the skin of the rana grahami is washed by deionized water, the skin of the rana grahami is killed quickly by smashing the brain marrow and is obtained.

The second step: construction of a rana grahami cDNA library: extracting RNA from the obtained skin tissue by using a total RNA extraction kit (TIANGEN, china), reversing RNA molecules into cDNA molecules after extraction is finished, and establishing a polypeptide cDNA library derived from the skin tissue of the rana grahami.

The third step: to screen for cDNA encoding mature OA-RD17, polymerase Chain Reaction (PCR) templates were derived from cDNA synthesized by SMART technology and 5'PCR primers (5' -CCAAA (G/C) ATGTTCACC (T/A) TGAAGAA-3 ') and 3' PCR primer (5 'ATTCAGGCCGAGCA TG-3') synthesized by BGI (China). The obtained PCR product was cloned into E.coli DH5a active cells and DNA sequencing was performed using an Applied Biosystems DNA sequencer (ABI 3730XL, foster, calif., USA), and as a result, as shown in FIG. 1, the nucleotide sequence of cDNA is shown in SEQ ID NO.2, the full length of cDNA is 365 bp (containing 365 bases) and encodes 64 amino acid residues, and the mature peptide sequence is "RDYCTPEDCDYDFSFPI" (base sequence underlined in FIG. 1). This peptide was a novel peptide and was named OA-RD17 by search.

And preparing the polypeptide OA-RD17 by adopting a conventional chemical synthesis or gene expression mode.

The polypeptide OA-RD17 of the present invention is synthesized by Wuhanbaiyixin biotechnology Limited.

Example 2 detection of in vitro skin wound repair-promoting Activity of polypeptide OA-RD17

1. In vitro cell scratch repair Activity detection

Keratinocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% antibiotics (100U/mL penicillin, 100U/mL streptomycin) at 37 ℃ and 5% CO ₂ . HaCaT was inoculated into 24-well plates (2X 10) ⁵ Cells/well) until a confluent monolayer was formed, and the wound was simulated by scratching the cell monolayer using a 200 μ L sterile pipette tip. After washing the cell debris with PBS, 500. Mu.L of PBS (Vehicle), rh-bFGF (1. Mu.g/mL) and OA-RD17 (10 n) were added to each well, respectivelyM) in a serum-free medium at 37 ℃ in 5% CO ₂ The culture was continued for 24 h under ambient conditions. Scratch healing was recorded using an inverted microscope (Zeiss, germany) taking pictures at 0 h,12 h,24 h, respectively.

The results are shown in FIGS. 2A-B: the polypeptide OA-RD17 can obviously promote the scratch repair of keratinocytes, and the scratch repair effect is close to 100% after the OA-RD17 is incubated for 24 hours.

2. In vitro cell proliferation Activity assay

Keratinocytes were seeded in 96-well plates (5X 10) ³ Perwell), after the cells adhered to the wall, different concentrations of polypeptide OA-RD17 (100 pM, 1 nM, 10 nM, 100 nM, 1. Mu.M) were added, and after continuous culture for 24 h, changes in cell proliferation were detected using MTS reagent.

The results are shown in FIG. 3: polypeptide OA-RD17 promotes keratinocyte proliferation in a concentration gradient-dependent manner, and OA-RD17 shows good cell proliferation promoting activity at a concentration of 1 nM.

3. In vitro cell migration Activity assay

The trans-well plate with filter membrane aperture of 8 μm is inserted into the 24-well plate of the lower chamber as the upper chamber, and then the keratinocyte and macrophage are respectively inserted into the 24-well plate at cell density of 1 × 10 ⁴ Perwell was inoculated into the upper chamber and OA-RD17 was added to a final concentration of 1 nM, followed by addition of medium containing 10% FBS to the lower chamber of a 24-well plate. 37. Incubate at C.for 24 h, wash with PBS, fix the cells with 4% paraformaldehyde and stain with 1 mg/mL crystal violet for 20 min. The upper chamber was washed with PBS and non-migrating cells were removed and photographed under an inverted microscope. After completion of the photographic recording, crystal violet was completely eluted using 33% acetic acid, and the cell mobility was calculated by detecting the absorbance value (OD value) at 570 nm with a microplate reader.

The results are shown in FIGS. 4A-B: the polypeptide OA-RD17 significantly promotes keratinocyte migration, and the number of the migrated cells reaches 2.34 times of that of a blank control.

In conclusion, OA-RD17 has good in vitro wound repair promoting activity.

Example 3 detection of polypeptide OA-RD17 in vivo skin wound repair-promoting Activity

The experimental method comprises the following steps: mice full skin wound experiment

1. Kunming mice were anesthetized with 1% sodium pentobarbital (60 mg/kg) and after removal of the back coat, two full-thickness wounds of 10 mm in diameter were created on both sides of the back using biopsy punches. Mice were randomized into four groups, blank (control, PBS), positive control (Positive, rh-bFGF,100 ng/ml), polypeptide (OA-RD 17,1 nM) and Scrambled peptide (1 nM). The drug is administered twice every day by 20 muL, and the change of the wound is recorded by taking pictures every other day after the operation till the 8 th day. The tissue of the wound on the back of the mouse was taken for histopathological analysis on the 4 th and 8 th days after the operation, respectively.

2. To assess the regeneration of wound tissue in mice after treatment in different groups, a Hematosin and Eosin (HE) staining assay was performed. Skin tissue was cut into tissue pieces 5 μm thick, sequentially deparaffinized, gradient hydrated and then HE stained. Wound tissue regeneration was recorded using an optical microscope at the same magnification, and changes in epidermal, neogenetic and epithelialization formation of skin of different pathological tissues were calculated using the software Image J, as shown in fig. 5, and the data were statistically analyzed in prism software.

As a result:

as shown in fig. 5A-B: the OA-RD17 can obviously promote the wound repair of the mouse back cortex, the OA-RD17 wound repair promoting activity after 8 days of treatment is similar to that of the positive control rh-bFGF, and the wound repair rate is close to 100%.

As shown in fig. 5C-E: the HE staining result shows that OA-RD17 obviously promotes the proliferation and migration of mouse wound epidermis to obviously accelerate re-epithelialization, and in addition, promotes the regeneration of granulation tissue in a wound area.

To summarize: the wound repair promoting peptide OA-RD17 provided by the invention has stronger activity of promoting skin tissue regeneration in vivo and in vitro, and has certain clinical application value.

Claims (3)

1. A polypeptide OA-RD17 with skin wound repair promoting activity, wherein the amino acid sequence of the polypeptide OA-RD17 is RDYCTPEDCDYDFSFPI.

2. Use of the polypeptide OA-RD17 of claim 1 for the preparation of a medicament for the treatment of skin wound repair.

3. A pharmaceutical composition comprising the polypeptide OA-RD17 of claim 1 as an active agent and a pharmaceutically effective carrier.

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