CN115850381A - Synthetic polypeptide and application thereof in promoting skin healing - Google Patents
Synthetic polypeptide and application thereof in promoting skin healing Download PDFInfo
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- CN115850381A CN115850381A CN202211600394.5A CN202211600394A CN115850381A CN 115850381 A CN115850381 A CN 115850381A CN 202211600394 A CN202211600394 A CN 202211600394A CN 115850381 A CN115850381 A CN 115850381A
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Abstract
The invention provides a synthetic polypeptide and application thereof in promoting skin healing, wherein the amino acid sequence of the provided synthetic polypeptide is SEQ ID NO. 1. The present invention also provides polypeptides derived from the above-described synthetic polypeptides. The synthetic polypeptide has obvious activity of promoting wound healing, can be used for accelerating wound healing of body surface skin and reducing scar generation. Therefore, the synthetic polypeptide has excellent activity of promoting wound healing, can be used as a production raw material or dressing of medicines, postoperative medical instruments, health care products, cosmetics and the like with the function of repairing skin, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of polypeptide and synthesis thereof, and particularly relates to a synthetic polypeptide and application thereof in promoting skin healing.
Background
The skin is used as the first protective barrier of the body and is very easy to be injured. If the skin wound is not properly treated, adverse effects such as skin wound infection or scar hyperplasia can be easily caused. Therefore, how to effectively promote skin wound repair is a great challenge for researchers. Wound healing is a complex biological process that is a key factor in restoring the integrity of the skin barrier.
The bioactive peptides play an indispensable role in maintaining the physiological activities and stabilizing the body. Abundant functions of bioactive peptides are the current popular research direction, wherein the functions of bioactive peptides extracted from animals and plants by means of separation are concerned, the bioactive peptides not only play a positive role in healing skin wounds, but also have good biological functions, and meanwhile, the bioactive peptides have the characteristics of trace, safety, high efficiency and the like, and are widely applied to the fields of medicine health and the like. However, at present, there are some problems in the related research on the effect of bioactive peptides in healing skin wounds, such as the preparation of bioactive peptides mostly uses the traditional enzymolysis method, the obtained products are mostly mixtures, and the analysis research on the bioactive peptide functional molecules is not sufficient; the molecular weight of the active peptide is small, and effective separation and purification means also become restrictive factors for research and industrial production of the active peptide. Regarding the bioactive peptide, the effect on the healing of skin wound should be considered, and the problem of skin irritation of the obtained product should also be considered.
Disclosure of Invention
The invention aims to provide a synthetic polypeptide and application thereof in promoting skin healing, and the synthetic polypeptide has good treatment effect on wound healing, good safety and obvious curative effect.
The invention firstly provides a synthetic polypeptide, which is characterized in that the amino acid sequence of the synthetic polypeptide is as follows:
Gly-Pro-Ser-Gly-Gly-Gly-Tyr-Asp-Phe-Gly-Leu-Pro-Gly-Ala-Pro-Gly-Phe-Val-Gly(GPSGGGYDFGLPGAPGFVG)(SEQ ID NO:1)；
the present invention also provides a polypeptide derived from the above-described synthetic polypeptide, which comprises:
1) 1, polypeptide obtained by substituting, deleting and adding one or more amino groups on the polypeptide of SEQ ID NO;
2) Polypeptide obtained by truncating the polypeptide of SEQ ID NO. 1;
3) A polypeptide obtained by connecting the polypeptides in 1) or 2) in series;
specifically, the amino acid sequence of the derivative polypeptide is as follows:
Gly-Leu-Pro-Gly-Val-Pro-Gly-Phe-Val-Gly(GLPGVPGFVG)(SEQ ID NO:2)；
Gly-Pro-Ser-Gly-Gly-Gly-Tyr-Asp-Phe-Gly-Leu-Pro-Gly(GPSGGGYDFGLPG)(SEQ ID NO:3)；
Gly-Leu-Pro-Gly-Val-Pro-Gly-Phe(GLPGAPGF)(SEQ ID NO:4)。
in yet another aspect, the present invention provides a conjugated polypeptide, which is a coupling product of the above synthetic polypeptide and a functional molecule;
the functional molecule is a molecule capable of enhancing the efficacy of the synthetic polypeptide, such as a polypeptide or polysaccharide;
the invention also provides an application of the synthetic polypeptide in preparing a medicament for promoting wound healing, a medical device after skin repair surgery, a product after skin repair surgery, a skin repair health product or a skin repair cosmetic.
The invention also provides a product with the function of promoting wound healing or skin repair, which comprises any one or more of the synthetic polypeptides or the derived polypeptides.
The synthetic polypeptide has obvious activity of promoting wound healing, can be used for accelerating wound healing of body surface skin and reducing scar generation. Therefore, the synthetic polypeptide has excellent activity of promoting wound healing, can be used as a production raw material or dressing of medicines, postoperative medical instruments, health care products, cosmetics and the like with the function of repairing skin, and has wide application prospect.
Drawings
FIG. 1: photographic picture of the effect of the polypeptide on the healing of mouse wound;
FIG. 2: graph of the effect of the polypeptide of sequence SEQ ID No. 1 on the proliferation of mouse embryonic fibroblasts NIH 3T3 cells, P <0.05 and P <0.01, compared to the blank;
FIG. 3: a photo picture of the effect of the polypeptide with the sequence SEQ ID NO. 1 on the migration of mouse embryo fibroblast NIH 3T3 cells, wherein the left picture is a blank control; the right figure is the polypeptide of SEQ ID NO. 1.
Detailed Description
The polypeptide mixture which is prepared by enzymolysis of pilose antler and has the effect of promoting skin healing is subjected to separation and purification of pilose antler polypeptide by adopting reversed phase liquid chromatography, is purified by adopting an Eclipse XDB-C18 chromatographic column and is sequenced to obtain the polypeptide with the effect of promoting skin healing, and the amino acid sequence of the polypeptide is GPSGGGYDFGLPGAPGFVG (SEQ ID NO: 1).
The present invention also provides polypeptides derived from the synthetic polypeptide of SEQ ID NO. 1 comprising:
1) 1, polypeptide obtained by substituting, deleting and adding one or more amino groups on the polypeptide of SEQ ID NO;
2) Polypeptide obtained by truncating the polypeptide of SEQ ID NO. 1;
3) A polypeptide obtained by connecting the polypeptides in 1) or 2) in series;
wherein 1) is a derivative polypeptide having high homology with SEQ ID NO. 1, and also has skin healing promoting effect;
2) Is obtained by truncating the polypeptide of SEQ ID NO. 1, and also has the effect of promoting skin healing; for example, the amino acid sequence is GLPGVPGFVG (SEQ ID NO: 2), the amino acid sequence is GPSGGGYDFGLPG (SEQ ID NO: 3), and the amino acid sequence is GLPGAPGF (SEQ ID NO: 4).
3) Is a conjugated polypeptide obtained by connecting the polypeptide of SEQ ID NO. 1 or 1) and optionally two or three of the polypeptides in 2) in series,
the conjugated polypeptide can also be a product obtained by coupling the polypeptide with a functional molecule.
The functional molecule is a molecule that enhances the efficacy of the synthetic polypeptide, such as a 3' plus polypeptide or polysaccharide.
The invention also provides an application of the synthetic polypeptide in preparing a medicament for promoting wound healing, a medical device after skin repair surgery, a product after skin repair surgery, a skin repair health product or a skin repair cosmetic.
The invention also provides a product with the function of promoting wound healing or skin repair, which comprises any one or more of the synthetic polypeptides or the derived polypeptides.
The polypeptide provided by the invention can be synthesized by conventional synthetic methods, such as liquid phase stepwise synthesis, solid phase synthesis and biological synthesis, and as a preferred embodiment of the polypeptide provided by the invention, the polypeptide is synthesized by a solid phase polypeptide synthesis process. In order to ensure the biological safety, the purity of the polypeptide is more than or equal to 95 percent. The product can be purified by HPLC.
The polypeptide and derivative products provided by the invention can be used for preparing medicines for treating skin defects and skin ulcers difficult to heal.
Further, the skin defect and the refractory skin ulcer disease can be large-area skin defect and refractory skin ulcer caused by various acute and chronic injuries such as inflammation, ulcer, severe burn, trauma, tumor after operation and congenital malformation.
The present invention will be described in detail with reference to examples.
Example 1: synthetic polypeptides
The polypeptide with the amino acid sequence of SEQ ID NO 1-4 is synthesized respectively, and the specific steps are as follows:
swelling resin: the resin was placed in a 150mL reaction column and 50mL DCM was added and soaked for 2h, the resin was washed with DMF, drained and repeated four times to complete the resin swelling. Deprotection: the first amino acid (with protection) at the Fmoc-C terminal, DCM and DIEA were weighed into a reactor, and the reactor was placed in a shaker at 30 ℃ for 2h. Blocking with methanol solution (methanol: DIEA: DCM = 1) for half an hour, then washing four times with DMF and draining. The Fmoc protecting group was removed by adding a 20% piperidine solution to the reactor. After deprotection was washed four times with DMF and then drained. And detecting a small amount of resin by an indetrione method, wherein the resin has color, which indicates that the deprotection is successful.
Amino acid coupling reaction: the Fmoc-C terminal second amino acid (with protection), HOBT and DIC were weighed into a reactor, and the reactor was placed in a shaker at 30 ℃ for 1h. And (3) detecting a small amount of resin by using an indantrione method, and if the resin has a color, indicating that condensation is incomplete, and continuing to react. If the resin is colorless, the reaction is complete; after the reaction was complete, the resin was washed four times with DMF and then drained. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF = 1) to the reactor and shaking on a decolourising shaker for 20 min. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed. And detecting a small amount of resin by an indetrione method, wherein the resin has color, which indicates that the deprotection is successful. Amino acids are connected in sequence according to the steps.
Cleavage and isolation of synthetic peptide: the polypeptide protecting group is completely cleaved off with a cleavage reagent and cleaved from the resin for purification. Separating the target peptide fragment from impurities by HPLC, and freeze-drying the target peptide fragment to obtain a product, wherein the purity of the composition is more than 97% as verified by mass spectrometry.
Example 2: influence of polypeptide on mouse wound healing speed and wound healing quality
Intravenous anesthesia was performed with 1mL/kg of 3% sodium pentobarbital. After preparation of the skin on the back of the mouse, the punch was adjusted to a diameter of 5cm and the tip was stained with methylene chloride. Surgical marks were printed on the dorsal cephalic 1/3 of the mice, respectively, centered on the spinal column. Sterilizing the skin of the operation area with strong iodine, and spreading sterile towel. The operation completely excises all skin and superficial fascia tissues of the designed wound surface along the blot in a uniform size under the sterile condition until reaching the deep fascia surface to stop bleeding completely. The self-made sterile drug guide tube with the sieve pores at the front end is buried under the sterile gauze on the wound surface on the back of the mouse, so that the drug delivery is convenient. The blank was given physiological saline and the other groups of mice were given the corresponding synthetic polypeptides (8 mg/mL), respectively. The incision is bandaged with sterile gauze. And (5) observing the healing condition of the wound surface, measuring the longitudinal diameter and the transverse diameter of the non-healed wound by using a vernier caliper on the 10 th day, and calculating the healing area. Area healed = total area of wound- (major diameter × minor diameter × 0.785).
The results show that the external application drug can obviously accelerate the healing of skin wounds. The sizes of wound surfaces of the polypeptides of SEQ ID NO. 1-4 are obviously lower than that of a blank control after 14 days of administration, and pathological histological observation shows that after 9 days, the area of a defect focus of a rat skin injury in the control group is larger, some epithelial tissues are completely deleted, some epithelial tissues are covered by partial epithelial tissues, but the epidermal layer is thinner and the continuity is poorer. After administration of the polypeptide, epidermal repair was more complete and epidermal thickness was thicker (table 1).
Table 1: comparative table for wound and wound healing speed
P <0.05, P <0.01 compared to blank.
Therefore, the histomorphosis result shows that the polypeptides of SEQ ID NO. 1-4 can accelerate the healing of skin wound.
Example 3: effect of Polypeptides on proliferation of mouse embryonic fibroblasts NIH 3T3 cells
NIH 3T3 cell concentration was diluted to 1X 10 4 cell/mL, pipetting 180. Mu.L of the cell suspension, plating into a 96-well plate, standing at 37 ℃ and 5% CO 2 Culturing in constant temperature dressing box for 24h. After the cells were completely attached to the surface, the wells were filled with 20. Mu.L of 10% FBS-DMEM culture containing the polypeptide of SEQ ID NO:1 in an amount of 200. Mu.g/mL, 100. Mu.g/mL, 10. Mu.g/mL, 1. Mu.g/mL, respectively. The blank control group was 10% FBS-DMEM medium, and each group had 6 duplicate wells. Standing at 37 deg.C, 5% CO 2 The culture was continued in the constant temperature dressing chamber for 48h. After completion of the culture, 20. Mu.L of 0.5mg/mL MTT solution was added to each well, and the concentration of CO was 5% at 37 ℃% 2 Incubating for 4 hr, discarding culture solution, adding dimethyl sulfoxide (DMSO) 150 μ L, shaking for 10min, and measuring absorbance at 570nm with enzyme readerAnd (4) luminosity. Calculating the proliferation rate of the cells: cell proliferation rate = [ A = 570nm (average of experimental groups) -A 570nm (blank control group)]/A 570nm (blank control group).
The result shows that the polypeptide of SEQ ID NO. 1 can obviously promote cell proliferation when acting on mouse embryo fibroblast NIH 3T3 24h at the concentration of 1-200 mug/mL (figure 2).
Example 4: effect of Polypeptides on migration of NIH 3T3 cells
Mouse embryo fibroblasts NIH 3T3 cells were cultured in DMEM medium containing 10% FBS, 1% penicillin-streptomycin diabody, serum-free medium or the polypeptide of SEQ ID NO:1 (10. Mu.g/mL) was added to each well, and the mixture was placed in an incubator for further 24 hours. Culturing the cells to be tested to logarithmic phase, digesting the cells, washing with PBS and serum-free medium for 1 time, suspending the cells with the serum-free medium, counting, and adjusting the concentration to 2 × 10 5 Per mL; 500. Mu.L of a medium containing 10% FBS was placed in the lower Transwell chamber, and 100 to 200. Mu.L of a cell suspension was placed in the upper chamber, followed by incubation for 24 hours; after the culture is finished, wiping off the non-migrated cells in the upper chamber; the lower surface of the membrane was immersed in a 4% paraformaldehyde solution, fixed for 20min, stained with crystal violet for 20min, cells were observed under a microscope for photographing, and the number of cells migrating to the lower surface of the PET membrane was calculated (Table 2).
Table 2: migration cell counting meter
P <0.05, P <0.01 compared to blank.
The results show that: compared with a blank control, 10 mu g/mL of the polypeptide of SEQ ID NO. 1 can remarkably promote the migration of NIH 3T3 cells.
Claims (10)
1. A synthetic polypeptide, wherein the amino acid of the synthetic polypeptide is SEQ ID NO. 1.
2. A derivative polypeptide comprising:
1) A polypeptide obtained by substituting, deleting, or adding one or more amino groups to the polypeptide of claim 1;
2) A polypeptide obtained by truncating the polypeptide of claim 1;
3) A polypeptide obtained by connecting the polypeptides in 1) or 2) in series.
3. The derivative polypeptide of claim 2, wherein the amino acid sequence of the derivative polypeptide is SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4.
4. A conjugated polypeptide, wherein said coupling polypeptide is a conjugate of the polypeptide of claim 1 and a functional molecule.
5. The conjugated polypeptide of claim 4, wherein the functional molecule is a polypeptide or a polysaccharide.
6. A tandem polypeptide, wherein said tandem polypeptide is constructed by tandem construction of not less than two polypeptides of claim 1, or a derivative polypeptide of claim 2.
7. Use of the polypeptide of claim 1 for the preparation of a preparation for promoting wound healing and skin repair.
8. The use according to claim 7, wherein the product is a pharmaceutical, nutraceutical or cosmetic product.
9. An article of manufacture for wound healing or skin repair comprising the polypeptide of claim 1.
10. The preparation of claim 7, further comprising a polypeptide of any one of claims 2-6.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211600394.5A CN115850381A (en) | 2022-12-12 | 2022-12-12 | Synthetic polypeptide and application thereof in promoting skin healing |
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| CN202211600394.5A CN115850381A (en) | 2022-12-12 | 2022-12-12 | Synthetic polypeptide and application thereof in promoting skin healing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117510592A (en) * | 2023-11-11 | 2024-02-06 | 永创恒新生物医学科技(北京)有限公司 | Bioactive peptide and application thereof in preparing skin wound repair cosmetics |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117510592A (en) * | 2023-11-11 | 2024-02-06 | 永创恒新生物医学科技(北京)有限公司 | Bioactive peptide and application thereof in preparing skin wound repair cosmetics |
| CN117510592B (en) * | 2023-11-11 | 2024-05-31 | 杭州达维先医药科技有限公司 | Bioactive peptide and application thereof in preparing skin wound repair cosmetics |
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