Cell-like growth factors based on ai-assisted design and their use in the treatment of skin diseases

Abstract

The present application relates to an AI-assisted design-based cell-like growth factor and its use in treating skin diseases. The amino acid sequence of the cell-like growth factor is shown as SEQ ID No. 1, which can be used to treat skin damage, skin aging or skin pigmentation.

Description

Technical Field

[0001] This invention relates to the field of functional genes, and in particular to cell growth factor-like substances and their use in the treatment of skin diseases. Background Technology

[0002] The skin is the largest organ in the human body, mainly composed of the epidermis, dermis, and subcutaneous tissue. The epidermis, the outermost layer of skin tissue, is composed of keratinocytes, melanocytes, Langerhans cells, Merkel cells, etc., and its main functions are barrier defense, resisting mechanical and chemical stimuli, and ultraviolet radiation. The dermis, composed of collagen, elastic fibers, matrix, blood vessels, nerves, hair follicles, sweat glands, etc., is the supporting structure and supply system of the skin. The subcutaneous tissue, mainly composed of adipose tissue, plays a role in energy storage, buffering, and heat preservation.

[0003] Despite the rapid advancements in modern medicine, some unresolved issues remain in the treatment of skin injuries, the prevention of pigmentation, and the treatment of wrinkles from both clinical and research perspectives. For example, in the area of ​​skin injuries, scar formation and wound healing in diabetic patients are the biggest challenges; regarding skin aging, there is currently no method that can comprehensively prevent dermal atrophy, collagen loss, and the aging of elastic fibers; and in the case of skin problems such as pigmentation, due to the complexity of the causes and the high recurrence rate, people have been seeking products that can quickly remove pigmentation, provide long-lasting safety, and avoid excessive irritation. Summary of the Invention

[0004] One aspect of the present invention provides a cell growth factor, the amino acid sequence of which is shown in SEQ ID No. 1.

[0005] The second invention provides a fusion protein comprising a small ubiquitin-like modifying factor and a cell growth factor as described in the first invention.

[0006] In one specific embodiment, the fusion protein further includes a visual reporter protein.

[0007] In one specific embodiment, the visual reporter protein is a fluorescent protein.

[0008] In one specific embodiment, the visual reporter protein is one of green fluorescent protein, red fluorescent protein, blue fluorescent protein, and cyan fluorescent protein. For example, the visual reporter protein can be green fluorescent protein with the amino acid sequence described in CN2021107794524 as shown in SEQ ID No. 23.

[0009] In one embodiment, the fusion protein further includes a peptide linker connecting the visual reporter protein and the small ubiquitin-like modifying factor. For example, the amino acid sequence of the peptide linker may be as shown in SEQ ID No. 25 as described in CN2021107794524.

[0010] In one specific embodiment, the cell growth factor is located at the nitrogen terminus or the carbon terminus.

[0011] The third invention provides a nucleic acid encoding a cell growth factor as described in the first invention or a fusion protein as described in any one of the second inventions.

[0012] In one specific embodiment, the sequence of the nucleic acid encoding the cell growth factor is shown in SEQ ID No. 2.

[0013] In one specific embodiment, the nucleotide sequence encoding the visual reporter protein may be as shown in SEQ ID No. 48 as described in CN2021107794524.

[0014] In one specific embodiment, the nucleotide sequence encoding the small ubiquitin-like modifying factor may be as shown in SEQ ID No. 49 as described in CN2021107794524.

[0015] In one specific embodiment, the nucleotide sequence encoding the polypeptide linker may be as shown in SEQ ID No. 50 as described in CN2021107794524.

[0016] The fourth invention provides the use of the cell growth factor as described in the first invention, the fusion protein as described in any one of the second invention, or the nucleic acid as described in the third invention in the preparation of a medicament for treating at least one of skin lesions, skin aging, or skin pigmentation. The lesions can be in humans or animals (e.g., cats, dogs, cattle, horses, sheep, pigs, chickens, ducks, geese, etc.), meaning that the cell growth factor of the present invention can be prepared into a medicament for human use or into a veterinary drug for animal use.

[0017] In one specific embodiment, the skin lesion is a skin lesion in a person with diabetes or other animals (e.g., mammals such as mice or rats).

[0018] In one specific embodiment, the skin aging can manifest as skin wrinkles, meaning that the cell growth factor, fusion protein, or nucleic acid of the present invention can be used for skin anti-wrinkle.

[0019] In one specific embodiment, the skin pigmentation includes at least one of melasma, sun spots, and age spots, meaning that the cell growth factor, fusion protein, or nucleic acid of the present invention can be used for skin anti-pigmentation, such as melasma, sun spots, or age spots.

[0020] The beneficial effects of this invention: The polypeptide designed in this invention possesses multiple beneficial biological functions of cell growth factors, including promoting skin wound healing, promoting skin cell proliferation, enhancing the synthesis of skin extracellular matrix, and inhibiting skin cell aging induced by external environment and harmful compounds, indicating that it is a cell growth factor-like substance. Based on the findings of this invention, the cell growth factor-like substance of this invention can be used to treat skin damage, skin pigmentation, or skin aging. Furthermore, when treating skin damage, the cell growth factor-like substance of this invention can leave no scars or leave only minor scars, and these minor scars gradually disappear over time. Attached Figure Description

[0021] Figure 1 The results show the SDS-PAGE electrophoresis analysis of strain pGS-GFLP / BL21(DE3) before and after induction. Lane M: protein molecular weight marker; Lane 1: bacterial suspension sample before pGS-GFLP / BL21(DE3) induction; Lane 2: bacterial suspension sample after pGS-GFLP / BL21(DE3) induction; the arrow points to the induced expression of the fusion protein GFP-SUMO-GFLP.

[0022] Figure 2 The results show the SDS-PAGE electrophoresis of strain pET-SUMOP / BL21(DE3) before and after induction. Lane M: protein molecular weight marker; Lane 1: bacterial suspension sample before pET-SUMOP / BL21(DE3) induction; Lane 2: bacterial suspension sample after pET-SUMOP / BL21(DE3) induction; the arrows indicate the induced SUMO protease.

[0023] Figure 3 The SDS-PAGE electrophoresis results of each sample during the expression and purification of cell growth factor-like molecules are shown. Lane M: protein molecular weight marker; Lane 1: pGS-GFLP / BL21(DE3); Lane 2: supernatant; Lane 3: SP flow-through buffer; Lane 4: elution buffer containing 0.3 mol / L sodium chloride; Lane 5: elution buffer containing 0.6 mol / L sodium chloride.

[0024] Figure 4 The assay showed that cytokines promote the proliferation of skin fibroblasts. ** indicates p < 0.01 compared with the negative control; *** indicates p < 0.001 compared with the negative control group.

[0025] Figure 5 This study demonstrates the detection of cell growth factor-like mechanisms promoting extracellular matrix synthesis in skin fibroblasts, specifically through transcriptional analysis of the genes for type I collagen, type III collagen, and elastin in cells. * indicates p < 0.05 compared to the negative control; ** indicates p < 0.01 compared to the negative control; *** indicates p < 0.001 compared to the negative control.

[0026] Figure 6 The image shows a photograph of skin lesions in a mouse model of type 2 diabetes treated with cell growth factor-like therapy.

[0027] Figure 7 This study demonstrates the detection of cell growth factor-like inhibitory effects on skin fibroblast senescence, specifically through staining analysis of senescence-associated β-galactosidase (SA-β-Gal).

[0028] Figure 8 The image shows photos of skin treated with cell growth factor-like growth factor (CGF) treatment for sunspots. The white arrows indicate the treatment sites. Detailed Implementation

[0029] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0030] Unless otherwise specified, the biochemical reagents, proteases, etc. used in the embodiments of the present invention can be purchased commercially.

[0031] Unless otherwise specified, the molecular biology manipulation methods used in the embodiments of the present invention are based on "Molecular Cloning: A Laboratory Manual" (Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0032] The Escherichia coli pET-21a expression vector (catalog number: 69740) and Escherichia coli BL21(DE3) (catalog number: 69450) were purchased from Sigma-Aldrich.

[0033] The DEAE anion exchange gel (DEAE Sepharose Fast Flow, catalog number: 17070905), nickel-nitrotriacetic acid affinity chromatography gel (NI-NTA) (catalog number: 17371203), SP cation exchange chromatography gel (SP Sepharose Fast Flow, catalog number: 17108703), and Sephadex G-25 filtration chromatography gel (molecular sieve, catalog number: 17003302) used for protein purification were purchased from Cytiva Biotechnology (Hangzhou) Co., Ltd.

[0034] Human dermal fibroblasts were purchased from Wuhan Pronosei Life Science Co., Ltd., product number: CP-H103.

[0035] The RNA extraction kit (catalog number: R0026) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0036] The Cell Senescence β-Galactosidase Staining Kit (Catalog No.: C0602) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0037] LB liquid medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 1000 ml distilled water, sterilized at 121°C for 20 minutes.

[0038] Basic phosphate buffer: 5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, pH=7.4.

[0039] Example 1: Artificially Designed Cell Growth Factor

[0040] Artificial design of cell growth factors: 1) Using the AI ​​(Artificial Intelligence) software RFDiffusion (version 1.1.0), a polypeptide backbone was designed, and then the direction of polypeptide generation and local structure were manually adjusted step by step to obtain the cell growth factor backbone sequence; 2) Using the AI ​​software ProteinMPNN (version 1.0.1), various amino acids were randomly filled based on the cell growth factor backbone sequence to obtain more than 2,000 first candidate sequences of cell growth factors; 3) Each first candidate sequence of cell growth factors was input into the AlphaFold 3 analysis platform for evaluation to screen out the second candidate sequences of cell growth factors; 4) Using the GROMACS (version 2020.6) software, molecular dynamics simulation analysis was performed on each second candidate sequence of cell growth factors to screen out the third candidate sequences of cell growth factors.

[0041] One of the third candidate sequences for a cell growth factor is shown in SEQ ID No. 1.

[0042] Example 2: Construction and expression of cell growth factor-like vectors

[0043] The coding sequence of the growth factor-like peptide (GFLP) shown in SEQ ID No. 1, i.e. the gene sequence of this type of growth factor shown in SEQ ID No. 2, was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0044] A cell growth factor-like gene fragment was cloned into the downstream of the GFP-SUMO nucleic acid encoded by the E. coli expression vector pET-GFP-SUMO (see Example 3 in CN2021107794524) using genetic engineering methods, forming a nucleic acid encoding GFP-SUMO-GFLP. This nucleic acid was then transformed into the E. coli host BL21(DE3). Positive recombinant bacteria were selected using ampicillin resistance screening and named pGS-GFLP / BL21(DE3), where the positive recombinant plasmid is pGS-GFLP. The fusion protein encoded by the GFP-SUMO-GFLP nucleic acid is a cell growth factor-like protein with GFP-SUMO fused to its N-terminus; this fusion protein is abbreviated as GFP-SUMO-GFLP.

[0045] The nucleic acid encoding the small ubiquitin-like modifying protease Ulp1 (SUMO protease) (see SEQ ID No. 52 in CN2021107794524) was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0046] Then, the Ulp1 gene was cloned into the E. coli expression plasmid vector pET-21a using genetic engineering methods, and transformed into the E. coli host BL21(DE3). Positive recombinant bacteria pET-SUMOP / BL21(DE3) were selected by ampicillin resistance screening, among which the positive recombinant plasmid was pET-SUMOP.

[0047] The pGS-GFLP / BL21(DE3) strain was inoculated into a test tube containing 5 mL of LB liquid medium containing ampicillin and activated by incubation at 37°C for 12 hours. The activated bacterial solution was then inoculated at 1% (v / v) into 50 mL of fresh LB liquid medium containing ampicillin and cultured at 37°C with shaking at 220 rpm until OD reached. 600=1.0 (approximately 2 to 3 hours), after sampling (pre-induction bacterial culture), add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol / L, and continue induction culture at 37°C for 4 hours to obtain the post-induction bacterial culture. Centrifuge the pre-induction and post-induction bacterial cultures at 12,000 rpm to collect the bacterial cells, and resuspend the cells in phosphate buffer (137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L sodium dihydrogen phosphate, 2 mmol / L potassium dihydrogen phosphate, 0.2% surfactant NP-40, pH=7.0) to obtain the pre-induction and post-induction bacterial suspensions. Perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on the pre- and post-induction bacterial suspensions. The results are shown in the figure. Figure 1 . Figure 1 The results showed that the fusion protein GFP-SUMO-GFLP was successfully expressed in Escherichia coli BL21(DE3).

[0048] The SUMO protease expression strain pET-SUMOP / BL21(DE3) was induced to express SUMO protease using the same method described above. The induced bacterial culture was obtained by centrifugation, and the cells were resuspended in phosphate buffer to obtain a bacterial suspension. The bacterial suspension was then analyzed by SDS-PAGE electrophoresis. The results are shown below. Figure 2 . Figure 2 The results showed that SUMO protease was successfully expressed in Escherichia coli BL21(DE3).

[0049] Example 3: Purification of Cell Growth Factor-like Forms

[0050] The pGS-GFLP / BL21(DE3) cryopreserved glycerol culture was inoculated at a ratio of 1:100 (volume / volume) into 3.7 L of fresh LB liquid medium containing 100 mg / L ampicillin and cultured at 37°C and 220 rpm for 4 hours with shaking. Subsequently, the culture was inoculated at a 10% inoculum volume into 200 L of LB liquid medium containing 5% (mass / volume) glucose and a final concentration of 100 μg / mL ampicillin and cultured at 37°C. When the glucose content in the medium decreased to 0.1% (mass / volume), fed-batch feeding was started. The feed medium was LB liquid medium containing 20% ​​(mass / volume) glucose and a final concentration of 100 μg / mL ampicillin. The fermentation parameters were set as follows: pH = 7.0-7.2; dissolved oxygen > 30%; stirring speed 250-650 rpm. When the wet bacterial weight reached 35 ± 1 g / L, IPTG was added to a final concentration of 1 mmol / L for induction. After 4 hours of induction, the bacterial cells were collected by centrifugation to obtain pGS-GFLP / BL21(DE3) cells.

[0051] pET-SUMOP / BL21(DE3) was fermented using the same method, and the cells were collected by centrifugation to obtain pET-SUMOP / BL21(DE3) cells.

[0052] Weigh 20 g of pGS-GFLP / BL21(DE3) bacterial cells and 1 g of pET-SUMOP / BL21(DE3) bacterial cells, mix them (the purpose is to utilize SUMO protease to enzymatically digest the fusion protein GFP-SUMO-GFLP to obtain a cell growth factor-like protein with the sequence shown in SEQ ID No. 1), and resuspend the bacterial cells in 200 mL of basal phosphate buffer to prepare a bacterial suspension. Place the bacterial suspension in an autoclave and homogenize it twice consecutively at 4°C and pressures of 800 and 1200 bar. Collect the lysate and place it in a sterile centrifuge tube, centrifuge at 12000 rpm for 30 minutes, and collect the supernatant.

[0053] The supernatant was loaded onto a DEAE anion exchange gel for chromatography, and the DEAE eluent was collected. The DEAE eluent was then loaded onto a nickel-nitrotriacetic acid (NiNTA) affinity gel equilibrated with imidazole-containing phosphate buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 10 mmol / L imidazole, pH 7.4), and the NI-NTA eluent was collected. The NI-NTA eluent was then loaded onto an SP cation exchange gel, and gradient elution was performed using elution buffers containing increasing concentrations of sodium chloride. The elution buffer formulations were as follows: a low-concentration sodium chloride elution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.3 mol / L sodium chloride, pH 7.4); a medium-concentration sodium chloride elution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 0.6 mol / L sodium chloride, pH 7.4); and a high-concentration sodium chloride elution buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, 1.0 mol / L sodium chloride, pH 7.4). pGS-GFLP / BL21(DE3) cells, supernatant, and elution buffers from each gradient were collected and analyzed by SDS-PAGE electrophoresis. The results showed that the cell growth factor-like compound was present in the elution buffer containing 0.6 mol / L sodium chloride.

[0054] The eluent containing cell growth factor was loaded onto a Sephadex G-25 filter chromatography gel. The eluent containing a medium concentration of sodium chloride was replaced with basal phosphate buffer to remove sodium chloride, resulting in a cell growth factor solution with a final concentration of 0.5 mg / mL. The solution was then stored at -80°C.

[0055] Example 4: Cell growth factor-like mechanisms promote the proliferation of skin fibroblasts and the synthesis of extracellular matrix proteins.

[0056] The ability to promote the proliferation of skin fibroblasts: Human dermal fibroblasts in the logarithmic growth phase were injected with 1×10⁻⁶ cells. 3Cells were seeded at a density of 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium / F-12 (DMEM / F-12, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 41420ES76) and cultured at 37°C with 5% CO2 for 24 hours. The original medium was discarded, and DMEM / F-12 medium containing 0.5% FBS was added, followed by 12 hours of culture. The medium was then discarded again, and DMEM / F-12 medium containing 0.5% FBS and a cell growth factor (CGF) solution were added to bring the final concentration of CGF in the medium to 100 ng / mL. Cells were cultured for 0, 24, 36, and 48 hours. Then, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide solution (MTT, Shanghai Beyotime Biotechnology Co., Ltd., catalog number: ST316) was added to each cell culture well. After incubation for 4 hours, 100 μL of dimethyl sulfoxide (DMSO, Shanghai Beyotime Biotechnology Co., Ltd., catalog number: ST038) was added to each well, and the mixture was thoroughly mixed with a shaker. The absorbance was measured at 450 nm using a microplate reader. Negative control group: The difference from the above treatment was that the cell growth factor at a final concentration of 100 ng / mL was replaced with basal phosphate buffer (5.1 mmol / L potassium dihydrogen phosphate, 14 mmol / L dipotassium hydrogen phosphate, pH=7.4), where the volume of basal phosphate buffer added was the same as the volume of cell growth factor solution added. Results are shown below. Figure 4 . Figure 4 The results showed that, compared with the negative control, the cell growth factor-like enzyme had a significant ability to promote the proliferation of skin fibroblasts.

[0057] Enhanced ability to promote the synthesis of extracellular matrix (ECM) in skin fibroblasts: Human dermal fibroblasts in logarithmic growth phase were injected with 1×10 3 Cells were seeded at a density of 10% fetal bovine serum (FBS) in DMEM / F-12 medium and cultured at 37°C with 5% CO2 for 24 hours. The original medium was discarded, and DMEM / F-12 medium containing 0.5% FBS was added, followed by 12 hours of culture. The medium was then discarded again, and DMEM / F-12 medium containing 0.5% FBS and a cell growth factor (CGF) solution were added to bring the final CGF concentration to 100 ng / mL. The cells were cultured for another 24 hours. Total RNA was then extracted from the cells using an RNA extraction kit, and the type I collagen gene (Collagen type I alpha 1 chain) was analyzed by quantitative real-time PCR (qRT-PCR). Col1a1Type III collagen gene (collagentype III alpha 1 chain, Col3a1 ) and elastin gene ( Elastin The transcriptional changes of elastin (Eeln) were analyzed, with cytoskeletal protein (β-Actin) as an internal control. The sequences of the upstream primer F-Col1a1 for amplifying the type I collagen gene fragment are shown in SEQ ID No. 3 and the downstream primer R-Col1a1 in SEQ ID No. 4; the sequences of the upstream primer F-Col3a1 for amplifying the type III collagen gene fragment are shown in SEQ ID No. 5 and the downstream primer R-Col3a1 in SEQ ID No. 6; the sequences of the upstream primer F-Eln for amplifying the elastin gene fragment are shown in SEQ ID No. 7 and the downstream primer R-Eln in SEQ ID No. 8; and the sequences of the upstream primer F-Actin for amplifying the cytoskeletal protein (β-Actin) are shown in SEQ ID No. 9 and the downstream primer R-Actin in SEQ ID No. 10. Negative control group: The difference from the above treatment was that the final concentration of cell growth factor (CGF) was replaced with basal phosphate buffer, and the volume of basal phosphate buffer added was the same as the volume of CGF solution added. Results are shown below. Figure 5 . Figure 5 The results showed that, compared with the negative control, the cell growth factor-like enzyme promoted a 1.95-fold upregulation of type I collagen gene transcription, a 1.4-fold upregulation of type III collagen gene transcription, and a 1.35-fold upregulation of elastin gene transcription in cells. This indicates that the cell growth factor-like enzyme has the ability to promote the synthesis of extracellular matrix by skin fibroblasts.

[0058] Based on the cell growth factor of the present invention, which promotes the proliferation of skin fibroblasts and the synthesis of extracellular matrix of skin fibroblasts, it can be used to treat skin damage, skin aging and skin pigmentation problems.

[0059] Example 5: Treatment of skin lesions in type 2 diabetic animals with cell growth factor-like inhibitors

[0060] Establishment of a type 2 diabetic mouse model: Three-month-old male adult mice were purchased and housed in an environment with a constant temperature of 24±2 degrees Celsius, a relative humidity of 50% to 70%, and a day-night ratio of 12:12. After being fed a high-fat, high-sugar diet (Shanghai Maokang Biotechnology Co., Ltd., product number: MS1607) for 30 consecutive days, streptozotocin (Shanghai Maokang Biotechnology Co., Ltd., product number: MS1601) was injected intraperitoneally at a dose of 40 mg / kg for 4 consecutive days, once a day. One week later, the blood glucose concentration of the animals after fasting for 10 hours was measured using a glucometer. A blood glucose concentration of 11.1 mmol / L or higher was considered a successful model establishment.

[0061] Establishing a type 2 diabetes mouse skin injury model: Type 2 diabetes mice were anesthetized with 0.5% sodium pentobarbital. Then, the hair on their backs was shaved with an electric razor, and an appropriate amount of depilatory cream was evenly applied to the shaved area. After waiting 3 to 5 minutes, excess depilatory cream was wiped away with a cotton ball, and the skin at the hair removal site was disinfected with povidone-iodine. Under aseptic conditions, a skin tissue punch was used to press the skin at the hair removal site to create two symmetrical, equal-sized, full-thickness skin wounds with a diameter of 8 mm.

[0062] Treatment of skin lesions in type 2 diabetic mouse models: The 0.5 mg / mL cell growth factor (CGF) solution prepared in Example 2 was diluted with basal phosphate buffer to a concentration of 100 μg / mL. For each mouse, two wounds were treated: one as the experimental group (left, labeled a), with 10 μL of the 100 μg / mL CGF solution applied daily; the other as the negative control (right, labeled b), with 10 μL of basal phosphate buffer applied daily. Wound healing was photographed and recorded every 24 hours for 10 consecutive days. See attached image. Figure 6 The experimental results showed that cell growth factor-like agents could rapidly promote wound healing. After 10 days of treatment, the wounds treated with cell growth factor-like agents healed significantly faster than the negative control.

[0063] Example 6: Cell growth factor-like inhibitors hydrogen peroxide-induced senescence of skin fibroblasts

[0064] Human dermal fibroblasts in logarithmic growth phase were seeded at a density of 1 × 10³ cells / well in DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and cultured at 37°C with 5% CO2 for 24 hours. The cells were then divided into three groups: experimental group, model group, and control group. In the experimental group, FBS, a cell growth factor (CGF) solution, and hydrogen peroxide were added to the DMEM / F-12 medium to achieve a final FBS concentration of 0.5%, a CGF concentration of 100 ng / mL, and a hydrogen peroxide concentration of 200 μmol / L. After culturing at 37°C for 2 hours, the medium was replaced with fresh DMEM / F-12 medium containing 10% FBS and cultured at 37°C with 5% CO2 for 24 hours. The model group differed from the experimental group in that the CGF solution was replaced with basal phosphate buffer, and the volume of basal phosphate buffer added was equal to the volume of the CGF solution added in the experimental group. The difference between the control and experimental groups was that the cell growth factor solution was replaced with basal phosphate buffer, and the volume of basal phosphate buffer added was the sum of the volumes of cell growth factor solution and hydrogen peroxide added in the experimental group. After culture, the activity of β-galactosidase in fibroblasts was detected using a cell senescence β-galactosidase staining kit. The results are shown in [Figure 1]. Figure 7 Hydrogen peroxide exposure can induce rapid cellular senescence and be accompanied by an increase in the activity of senescence-associated β-galactosidase (SA-β-Gal). Figure 7 The results showed that the number of SA-β-Gal positive cells treated with cell growth factor-like agents was significantly less than that in the model group, indicating that cell growth factor-like agents have the function of inhibiting skin fibroblast senescence induced by hydrogen peroxide.

[0065] Example 7: Subcutaneous injection of cell growth factor-like substances improves sunspots.

[0066] The 0.5 mg / mL cell growth factor solution prepared in Example 2 was diluted with basal phosphate buffer to a 50 μg / mL cell growth factor solution.

[0067] A 50 μg / mL solution of cell growth factor was injected into the epidermis and dermis of the face of six volunteers at the site of sunspots using a syringe. Injections were given once a week for four consecutive weeks. Photos were taken with the same parameters before injection (day 0) and after four injections (day 28). Photos of one subject before and after cell growth factor injection are shown below. Figure 8 The results showed that cell growth factor-like substances can effectively promote skin turnover in sunspot areas and reduce the area of ​​pigmentation in these areas.

Claims

1. A cell growth factor having the amino acid sequence shown in SEQ ID No.

1.

2. A fusion protein comprising a small ubiquitin-like modifying factor and a cell growth factor as described in claim 1, wherein the small ubiquitin-like modifying factor is a SUMO protease and the cell growth factor is located at the C-terminus.

3. The fusion protein according to claim 2, characterized in that, The fusion protein also includes a visual reporter protein.

4. The fusion protein according to claim 3, characterized in that, The visual reporter protein is a fluorescent protein.

5. The fusion protein according to claim 3, characterized in that, The visual reporter protein is one of green fluorescent protein, red fluorescent protein, blue fluorescent protein, and cyan fluorescent protein.

6. The fusion protein according to claim 3, characterized in that, The fusion protein also includes a peptide linker, which connects the visual reporter protein and the small ubiquitin-like modifier.

7. A nucleic acid encoding the cell growth factor as described in claim 1 or the fusion protein as described in any one of claims 2 to 6.

8. The nucleic acid according to claim 7, characterized in that, The sequence of the nucleic acid encoding the cell growth factor is shown in SEQ ID No.

2.

9. Use of the cell growth factor of claim 1, the fusion protein of any one of claims 2 to 6, or the nucleic acid of claim 7 or 8 in the preparation of a medicament for treating at least one of skin lesions, skin aging, or sunburn.

10. The use according to claim 9, characterized in that, The skin lesion is a skin lesion in a person with diabetes or other animal; and / or Skin aging manifests as wrinkles.

Images