CN117298257A

CN117298257A - Use of human fibronectin or functional fragments thereof, screening method and kit

Info

Publication number: CN117298257A
Application number: CN202310869351.5A
Authority: CN
Inventors: 邵奇妙; 蔡佳宏; 赵娜
Original assignee: Hangzhou Enhe Biotechnology Co ltd
Current assignee: Hangzhou Enhe Biotechnology Co ltd
Priority date: 2023-07-14
Filing date: 2023-07-14
Publication date: 2023-12-29

Abstract

The invention belongs to the field of biological medicine and skin care, and relates to application, a screening method and a kit of human fibronectin or functional fragments thereof. In particular, the invention relates to the use of human fibronectin or a functional fragment of human fibronectin in the preparation of: a medicament for increasing the level of a protein or the expression level of a gene encoding the same, wherein the protein is selected from one or more of AQP1, AQP3, DSC3 and EPPK1; or a medicament for moisturizing skin, promoting wound healing of skin, or promoting skin repair by increasing the level of a protein or the expression level of a gene encoding the same, wherein the protein is selected from one or more of AQP1, AQP3, DSC3, and EPPK1. The invention also relates to a method for screening the functional fragments of human fibronectin and a kit.

Description

Use of human fibronectin or functional fragments thereof, screening method and kit

Technical Field

The invention belongs to the field of biological medicine and skin care, and relates to application, a screening method and a kit of human fibronectin or functional fragments thereof.

Background

Human Fibronectin (FN) is a macromolecular glycoprotein with a molecular weight of about 250kDa, which exists as a dimer. The entire molecule consists of multiple repetitive structural modules, including 12 FN type I repeats, 2 FN type II repeats, 15 constitutive expression, 2 alternating sheared FN type III repeats, and a non-homologous variable (V) or type III junction fragment (III CS) region. Wherein, FN I mainly binds to fibrin, heparin and collagen, FN II only participates in collagen binding, and FN III participates in binding with cells. Because fibronectin has specific functional and binding regions, it can bind to different types of cells, cytokines, and extracellular matrices. For example, fibronectin recognizes and binds to integrin heterodimers α5β1 and αvβ3 via arginine-glycine-aspartic acid sequences (Arg-Gly-Asp, RGD) on fibronectin 10Fn III and 9Fn III, thereby affecting cell adhesion, migration, and the like.

Human fibronectin is widely present in human tissues and tissue fluids, participates in migration, adhesion, proliferation, hemostasis, tissue repair and embryo development of cells, has the function of growth factors, can promote cell proliferation, induces epidermal cells to pass through granulation tissues, and promotes the reconstruction and normal keratinization process of the subcuticular basement membrane. Fibronectin is also involved in numerous pathological processes in the body. Fibronectin has a variety of adhesion functions, such as intercellular adhesion. Fibronectin is also important in wound healing. Soluble fibronectin deposited on damaged collagen and fibrin enhances platelet adhesion, phagocytic and fibroblast migration and cell proliferation [1]. Maxwell B.Johnson et al demonstrated that fibronectin could reduce the response to radiation in the mouse skin by a mouse wound model experiment. Furthermore, topically applied human fibronectin gels may significantly promote healing of irradiated skin wounds [2]. Fibronectin has a wide application value in cosmetics and after-treatment repair.

The aquaporin AQP is a transmembrane protein existing on a cell membrane, and can enable water molecules or glycerol molecules to rapidly pass through the cell membrane to help the cell to adjust the osmotic pressure, wherein AQP1 mainly plays a role in reabsorption of water in the kidney, and AQP3 is distributed on skin keratinocytes more to play a role in reabsorption of water and glycerol. AQP3 has been shown to be distributed in the human epidermis, mainly in the basal and acanthous layers, and less in the granular and stratum corneum [3]. AQP3 deficient mice reduce skin elasticity and slow the progress of barrier repair and wound healing [4]. There is also evidence that changes in AQP3 expression are directly related to skin moisturization and AQP3 deficient mice present dry skin symptoms [4,5]. As an action target of skin moisturization, AQP3 has been used as a screening target in the development of cosmetic raw materials, as in patent document 101453979B, describing the acceleration of aquaporin by a glyceroglycoside [6]. There are few reports of AQP1 on skin cells, AQP1 expression on skin fibroblasts is associated with fibrosis and edema of the skin, presumably AQP1 is associated with inflammatory regulation [7], but no reports of AQP1 expression on skin keratinocytes are associated.

More and more studies have demonstrated that the integrity of the skin barrier structure is closely related not only to the ceramide and phospholipid content of the epidermis layer, the integument and keratin, etc., but also to intercellular adhesion, such as Desmocollin (DSC) distributed on desmosomes between cells. DSC and Desmoglein (DSG) are the main two classes of transmembrane proteins on the hemidesmosome. Wherein DSC3 is critical for the integrity of the skin, spindle et al 2009 found that DSC3 mediated cell adhesion is important for the adhesion of keratinocytes, and on pemphigus patients' skin DSC3 is compromised [8]. Chen et al,2008 found that the absence of DSC3 resulted in impaired adhesion between cells, leading to epidermal blistering and telogen loss [9].

The periplaneta protein (EPPK 1) is a member of the Plakin family, belongs to the cell scaffold binding protein, and was first found to be an autoantigen extracted from the serum of patients with epidermoid bullae [10]. Ishikawa et al (2010) found that EPPK1 can promote keratinocyte keratin bundling in differentiation during wound healing, possibly by strengthening the keratin network to resist mechanical damage [11].

Currently, there is still a need to develop means for modulating AQP1, AQP3, DSC3 and/or EPPK1, as well as means for screening for functional fragments of human fibronectin.

Disclosure of Invention

The inventors have conducted intensive studies and creative efforts to obtain functional fragments of human fibronectin, which have the effects of effectively promoting skin repair, promoting cell adhesion, anti-inflammatory and/or promoting wound healing. Furthermore, the inventor also detects the expression of the AQP1 gene in the keratinocytes of the skin for the first time, the FN functional fragment is proved to be capable of promoting the expression of the AQP1 and AQP3 genes in the keratinocytes for the first time, and the FN functional fragment is proved to be capable of promoting the expression of DSC3 and EPPK1 for the first time, so that the adhesion of the epidermal cells is increased, and the skin barrier function is enhanced. The following invention is thus provided:

one aspect of the invention relates to the use of human fibronectin or a functional fragment of human fibronectin in the preparation of a medicament comprising:

a medicament for increasing the level of a protein or the expression level of a gene encoding the same, wherein the protein is selected from one or more of AQP1, AQP3, DSC3 and EPPK1;

or alternatively

A medicament for moisturizing skin, promoting wound healing in skin, or promoting skin repair by increasing the level of a protein or the expression level of a gene encoding the same, wherein the protein is selected from one or more of AQP1, AQP3, DSC3, and EPPK1.

In some embodiments of the invention, the use, wherein the amino acid sequence of the functional fragment of human fibronectin is as shown in SEQ ID NO. 1 and/or SEQ ID NO. 3.

In some embodiments of the invention, the use wherein the protein comprises AQP1 and one or more selected from AQP3, DSC3 and EPPK1.

In some embodiments of the invention, the use, wherein the protein is derived from human skin tissue or human skin cells such as human keratinocytes or human fibroblasts.

In some embodiments of the invention, the coding gene refers to a gene encoding a protein selected from one or more of AQP1, AQP3, DSC3, and EPPK1.

Another aspect of the invention relates to a method of screening for functional fragments of human fibronectin comprising the steps of:

(1) Adding a suitable concentration of a candidate human fibronectin fragment to human keratinocytes or human fibroblasts and culturing under suitable conditions for a period of time;

(2) Detecting the protein level or the expression level of the coding gene thereof, and if the protein level or the expression level is up-regulated, the human fibronectin fragment is a human fibronectin functional fragment;

wherein the protein is selected from one or more of AQP1, AQP3, DSC3 and EPPK1.

In some embodiments of the invention, the method of screening for a functional fragment of human fibronectin, wherein human keratinocytes or human fibroblasts without candidate human fibronectin fragments are provided as a blank.

In some embodiments of the invention, the method of screening for a functional fragment of human fibronectin, wherein human keratinocytes or human fibroblasts added with the polypeptide fragment of SEQ ID NO. 1 or SEQ ID NO. 3 are provided as positive controls.

In some embodiments of the invention, the method of screening for functional fragments of human fibronectin, wherein the concentration of the polypeptide fragment is greater than 3ppm, 3ppm to 50ppm, 3ppm to 30ppm, 3ppm to 20ppm, 3ppm to 15ppm, 3ppm to 6ppm, 6ppm to 15ppm, 3ppm, 6ppm, or 15ppm.

In some embodiments of the invention, the method of screening for functional fragments of human fibronectin, wherein the protein comprises AQP1 and one or more selected from the group consisting of AQP3, DSC3 and EPPK1.

In some embodiments of the invention, the method of screening for functional fragments of human fibronectin is a preliminary screening method; preferably, the method further comprises the step of further screening by a scoring assay, a cell adhesion assay, and/or an inflammation inhibition assay of macrophages.

Yet another aspect of the invention relates to a kit for screening for functional fragments of human fibronectin comprising:

a human keratinocyte or a human fibroblast, a cell culture medium of a human keratinocyte or a human fibroblast, and an agent for detecting the level of a protein or the expression level of a gene encoding the same, wherein the protein is selected from one or more of AQP1, AQP3, DSC3 and EPPK1.

In some embodiments of the invention, the kit wherein the agent that detects the level of a protein or the level of expression of a gene encoding the protein is an antibody to the protein or a PCR primer for the gene encoding the protein.

In some embodiments of the invention, the kit further comprises a polypeptide fragment as set forth in SEQ ID NO. 1 and/or SEQ ID NO. 3.

In some embodiments of the invention, the "one or more selected from AQP1, AQP3, DSC3, and EPPK 1" includes, but is not limited to, the following proteins or protein combinations:

AQP1；

AQP3；

DSC3；

EPPK1；

AQP1 and AQP3;

AQP1 and DSC3;

AQP1 and EPPK1;

AQP3 and DSC3;

AQP3 and EPPK1;

DSC3 and EPPK1;

AQP1, AQP3 and DSC3;

AQP1, AQP3 and EPPK1;

AQP1, DSC3 and EPPK1;

AQP3, DSC3 and EPPK1; or alternatively

AQP1, AQP3, DSC3 and EPPK1.

In the present invention, the term "functional fragment" refers to a truncated fragment of human fibronectin having all or part of the biological or pharmacological function of human fibronectin. The biological or pharmacological functions include, but are not limited to: promoting skin repair, promoting cell adhesion, anti-inflammatory and/or promoting wound healing, etc.

Advantageous effects of the invention

The invention proves that FN or FN functional fragments are likely to achieve the effects of improving skin barrier and increasing cell adhesion by regulating the gene expression of AQP1, AQP3, DSC3 and EPPK1, thereby providing a new idea for the application of the product in medicine or skin care.

Drawings

Fig. 1: 0417. PCR validation map after 0418 transformation of yeast GS 115. From left to right are markers, 0417 and 0418.

Fig. 2A: PCR validation map after 0419 transformation of yeast GS 115. 0419 and marker are in order from left to right.

Fig. 2B:0420 PCR validation panels after transformation of yeast GS 115. 0420 and marker are arranged from left to right.

Fig. 3A: western Blot validation of 0417 yeast fermentation broth. 0417 and marker are arranged from left to right.

Fig. 3B: western Blot validation of 0418 yeast fermentation broth. From left to right are markers and 0418.

Fig. 4: 0419. western Blot validation of 0420 yeast fermentation broth. 0419 and 0420 are in order from left to right.

Fig. 5: CCK experiments with 0417 keratinocytes, relative cellular activity at different concentrations; 0417 was shown to be safe for cells. NT represents no treatment.

Fig. 6: CCK experiments with 0418 keratinocytes, relative cellular activity at different concentrations; 0418 was shown to be safe for cells. NT represents no treatment.

Fig. 7: CCK experiments with 0419 keratinocytes, relative cellular activity at different concentrations; 0419 was shown to be safe for cells. NT represents no treatment.

Fig. 8:0420 CCK experiments with keratinocytes, relative cellular activity at different concentrations; 0420 was shown to be safe for cells. NT represents no treatment.

Fig. 9: 0417. 0420 has significant promotion effect on wound healing at 15ppm concentration. 0417 performed better at the same concentration. NT represents no treatment.

Fig. 10:0418 had a significant promoting effect on wound healing at 15ppm and 0419 had no promoting effect on wound healing at 15ppm concentration. NT represents no treatment.

Fig. 11:0417 had a significant cell adhesion promoting effect at a concentration of 10 ppm. 0420 had no effect on promoting cell adhesion at both concentrations. NT represents no treatment.

Fig. 12:0418 had a significant effect on promoting cell adhesion at a concentration of 10 ppm. 0419 had no effect on promoting cell adhesion at both concentrations. NT represents no treatment.

Fig. 13:0417 and 0418 both significantly inhibited IL-6 expression and had a concentration dependence. Wherein 0417 is better than 0418.NT represents no treatment.

Fig. 14:0417 and 0418 both have significant effects of inhibiting TNF- α expression and have concentration dependence. Wherein 0418 is slightly better than 0417 in effect. NT represents no treatment.

Fig. 15:0417 samples have promotion effect on the gene expression of AQP1 and AQP3 under different concentrations, and can obviously promote the expression of AQP1 and AQP3 under the conditions of 6ppm and 15ppm, and have concentration dependence. NT represents no treatment.

Fig. 16:0417 samples had a promoting effect on EPPK1 gene expression at different concentrations, and had a significant promoting effect at a concentration of 3ppm, reaching a maximum at 6ppm and a slight decrease in 15ppm. NT represents no treatment.

Fig. 17: semi-quantitative analysis of RHE immunohistochemical assay results. The results show that 0417 can increase the expression level of DSC3 by 230% compared to the control. NT represents no treatment.

The partial sequence related to the invention is as follows:

0417 amino acid sequence

PHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSD VPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQE FTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPA(SEQ ID NO:1)

0417 base sequence

GAATTCCCTCATTCTAGAAATTCTATCACTTTGACTAACTTGACTCCTGGTACTGAATATGTTGTTTCTATTGTTGCTTTGAACGGTAGAGAAGAATCTCCATTGTTGATTGGTCAACAATCTACTGTTTCTGATGTTCCAAGAGATTTGGAAGTTGTTGCTGCTACTCCAACTTCTTTGTTGATTTCTTGGGATGCTCCTGCTGTTACTGTTAGATATTATAGAATTACCTACGGTGAAACTGGTGGTAACTCTCCTGTTCAAGAGTTTACTGTTCCTGGTTCTAAATCTACTGCTACTATTTCTGGTTTGAAACCTGGTGTTGATTATACTATTACTGTTTACGCTGTTACTGGTAGAGGTGACTCTCCTGCTCATCATCATCATCACCATTAAGCGGCCGC(SEQ ID NO:2)

0418 amino acid sequence

GNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWCGTTQN YDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGH MMRCTCVGNGRGEWTCIAYS(SEQ ID NO:3)

0418 base sequence

GAATTCGGTAATTCTAACGGTGCTTTGTGTCATTTCCCTTTTCTTTACAATAACCATAACTACACTGACTGTACTTCTGAAGGTAGAAGAGATAATATGAAATGGTGTGGTACTACTCAAAACTACGATGCTGATCAAAAATTTGGTTTTTGTCCAATGGCTGCTCATGAAGAAATTTGTACTACTAATGAAGGTGTTATGTACAGAATTGGTGACCAATGGGATAAACAACATGATATGGGTCATATGATGAGATGTACTTGTGTTGGTAACGGTAGAGGTGAATGGACTTGTATTGCTTACTCTCATCATCATCATCACCATTAAGCGGCCGC(SEQ ID NO:4)

0419 amino acid sequence

PVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIK YEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQK SEPLIGRKKTVQKTPFVTH(SEQ ID NO:5)

0419 base sequence

GAATTCCCTGTTGTTATTGATGCTTCTACTGCTATTGATGCTCCATCTAATTTGAGATTCTTGGCTACTACTCCAAACTCTTTGTTGGTTTCTTGGCAACCTCCTAGAGCTAGAATTACTGGTTACATTATTAAGTACGAGAAGCCTGGTTCTCCTCCTAGAGAAGTTGTTCCTAGACCAAGACCAGGTGTTACTGAAGCTACTATTACTGGTTTGGAACCAGGTACTGAATACACTATCTATGTTATTGCTTTGAAGAACAACCAAAAGTCTGAACCATTGATTGGTAGAAAAAAGACTGTTCAAAAGACTCCTTTTGTTACTCATCATCATCATCATCACCATTAAGCGGCCGC(SEQ ID NO:6)

0420 amino acid sequence

PIAEKCFDHAAGTSYVVGETWEKPYQGWMMVDCTCLGEG SGRITCTSRNRCNDQDTRTSYRIGDTWSKKDNRGNLLQCICTGN GRGEWKCERHTSVQTTSSGSGPF(SEQ ID NO:7)

0420 base sequence

GAATTCCCAATTGCTGAAAAATGTTTCGATCATGCTGCTGGTACTTCTTATGTTGTTGGTGAAACTTGGGAAAAACCATACCAAGGTTGGATGATGGTTGATTGTACTTGTTTGGGTGAAGGTTCTGGTAGAATTACTTGTACTTCTAGAAACAGATGTAACGATCAAGATACTAGAACTTCTTATAGAATCGGTGACACTTGGTCTAAAAAGGATAATAGAGGTAACTTGTTGCAATGTATTTGTACTGGTAACGGTAGAGGTGAATGGAAGTGTGAAAGACATACTTCTGTTCAAACTACTTCTTCTGGTTCTGGTCCTTTCCATCATCATCATCACCATTAAGCGGCCGC(SEQ ID NO:8)

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Preparation example 1: design and preparation of truncated fragments of human fibronectin

1. Sequence design of truncated segment of human fibronectin

The truncated fragments of 4 human fibronectin are designed and preliminarily screened, and are respectively named 0417, 0418, 0419 and 0420, and the amino acid sequences of the truncated fragments are respectively shown as SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7; the corresponding coding nucleic acid sequences are shown as SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8 respectively.

2. Construction and amplification of recombinant vectors

The whole gene is synthesized into SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8, double enzyme digestion is carried out on a carrier template, T4 ligase is connected with a target gene sequence and a carrier recovered by gel cutting after double enzyme digestion, a pichia pastoris Top10 competent cell TOP10 strain is transformed, a monoclonal re-plasmid is extracted for amplification, and sequencing proves that the Top10 plasmid with a target gene is obtained.

3. Construction of Pichia pastoris recombinant strains

1% inoculum size TOP10 bacteria harboring the desired gene were inoculated into LLB liquid medium containing zeocin resistance at 37℃overnight at 250 rpm. The bacterial liquid precipitate was collected by centrifugation the next day and plasmid extraction was performed using plasmid extraction kit (Ai Baisen).

10. Mu.g of the extracted plasmid was added to SacI (thermo), and the mixture was digested at 37℃for 1 hour. The enzyme digestion product was recovered using a gel recovery kit (Ai Baisen). 2-5. Mu.g of linearized plasmid was added to 100. Mu.l of GS115 competent cells (Ai Baisen), transferred to a 2mm electric cuvette, and immediately after electric shock (parameters: voltage 2000V, capacitance 25. Mu.F, resistance 200. OMEGA., electric cuvette 2 mm) using an electric transducer, 1M of iceberg pear alcohol was added. The bacterial liquid in the electric rotating cup is evenly mixed and transferred into a centrifuge tube, kept stand for 1h at 30 ℃, 1mL of YPDS (YPDS composition: peptone 20g, yeast powder 10g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 11.8g and glycerin 10mL are added, water is added to complement to 1 liter, and after high-temperature sterilization, cooling solidification preservation) is carried out, and then shaking is carried out at 30 ℃ for 1h at 250 rpm. The supernatant was centrifuged to remove 100. Mu.l, and the remaining bacterial liquid was spread on YPDS-zeocin dishes and incubated at 30℃for 5 days. For the following PCR validation.

The grown monoclonal was picked up to 30. Mu.l of 0.25% SDS and boiled in water for 10min. After centrifugation, 5. Mu.L of the supernatant was added to 45. Mu.L of 25% glycerol and PCR was performed as a template.

Primer:

α-Factor：5'-TACTATTGCCAGCATTGCTGC-3'(SEQ ID NO:9)；

3-AOX：5'-GCAAATGGCATTCTGACATCC-3'(SEQ ID NO:10)。

the results of the PCR verification are shown in FIG. 1, FIG. 2A and FIG. 2B. The results showed that positive clones were obtained.

4. Fermentation

The monoclonal was added to 50mL of BMGY medium and incubated overnight at 28℃at 250rpm, the next day the pellet was collected by centrifugation and transferred to 100mL of BMMY medium, OD=1.0, 28℃at 250rpm for 96h, 1% methanol was added every 24 h.

5. Protein purification

And (3) taking the cultured thalli, centrifuging the bacterial liquid in a low-temperature centrifuge, and reserving the supernatant.

The supernatant obtained above was filtered through a 0.45 μm filter. 4ml of His filler was added to the filtered bacterial solution, incubated for 2h, and then protein purification was performed by means of an affinity column.

The method comprises the following steps:

1) Flowing the sample through the His column at a rate of 1 ml/min;

2) Equilibrate the column with Buffer a (50mM Tris PH8.0 150mM NaCl);

3) Elution was performed with 20mM, 300mM, 500mM imidazole.

4) The eluted samples were subjected to SDS-PAGE gel to analyze the presence of the target protein.

The results are shown in fig. 3A, 3B and 4. The results show that the proteins of interest 0417, 0418, 0419 and 0420 were produced.

Experimental example 1: cell safety test (CCK)

Cell culture: human primary keratinocyte [ ]Cell Technology) at 5% CO ₂ The cells were incubated at 37℃with complete medium containing 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% penicillin/streptomyin.

Human primary keratinocyte activity assay: human primary keratinocytes at 5% CO ₂ The cells were grown to 80-90% confluence in a culture medium expansion culture at 37℃using keratinocyte cells, digested with pancreatin and seeded in 96-well plates at 10000 cells/well. After cells were cultured in 96-well plates for 48h, sample-treated cells were prepared at different concentrations (n=3). After 48h, the cells were washed once with DPBS, incubated for 1h with CCK-8 reagent according to the instructions and OD was read at 450nm using an ELISA reader.

And calculating the average OD value ratio of the sample group and the control group to obtain the relative cell activity value, and judging the cytotoxicity effect of the sample. Relative% cell activity = (sample group OD value/control group OD value) x100%.

It is generally considered that the relative cell activity is 80% or more safe.

The results are shown in fig. 5 to 8.

FIG. 5 shows the effect of different concentrations of 0417 samples on keratinocyte viability. The results showed that 1-35ppm of the sample had no significant damage to cell viability, demonstrating safety to cells in this concentration range.

FIG. 6 shows the effect of different concentrations of 0418 samples on keratinocyte viability. The results showed that 1-17ppm of the sample had no significant damage to cell viability.

FIG. 7 shows the effect of 0419 samples on the activity of keratinocytes. The results showed that the samples were safe concentrations without any damage to cell activity in the range of 1-42 ppm.

FIG. 8 shows the effect of 0420 samples on keratinocyte activity. The results showed that the samples were safe concentrations without damaging cells in the range of 1-34 ppm.

Experimental example 2: scratch test

At 37 ℃,95% air humidity and 5% co ₂ Culturing primary human keratinocyte under the conditionCell Technology), cells were grown to 80-90% confluence, digested with pancreatin and plated into 12-well plates.

When cells were rapidly pooled in a 12-well plate, mechanical wounds were made to the grown monolayer cells with tips of micropipettes, then washed three times with PBS, and cell debris was washed away.

Immediately thereafter, 100ng/ml IGF-1 (insulin-like growth factor, positive control), 15ppm of the sample was treated and incubated for 24 hours.

Photographs were taken immediately after the wound was made, and further photographed after incubation for 24 hours.

The captured pictures were quantitatively analyzed with ImgaeJ software and the data from the different treatment groups were analyzed.

The following formula is adopted:

wound healing rate% = (area _0h Area-area _24h ) Area/area _0h x100％

Relative wound healing rate = (wound healing rate) _{Sample of} Wound healing Rate _Control )x100％。

The results are shown in fig. 9 to 10.

Figure 9 shows the efficacy of 0417 and 0420 samples on wound healing at 15ppm. The results show that the 0417 sample has more excellent effect on wound healing than the 0420 sample, and both have remarkable promotion effect on wound healing.

FIG. 10 shows the efficacy of 0418 and 0419 samples at 15ppm concentration on wound healing. The results show that 0418 has a significant promoting effect on wound healing, whereas 0419 has no significant promoting effect on wound healing.

Experimental example 3: cell adhesion experiments

Primary fibroblasts were seeded using extracellular matrix coated 96-well plates, 25,000 cells per well. The cells were incubated at 37℃with 95% humidity and 5% CO ₂ Is cultured under the condition of (2). After 18 hours of incubation, the cells were rinsed twice with PBS, tested for cell activity using the CCK-8 kit according to the test method required by the kit, and finally the absorbance was read at a wavelength of 450 nm.

The value of absorbance is proportional to the number of living cells adhering to the surface of the culture well. The cell adhesion can be calculated by the following formula:

relative cell adhesion% = (OD 450 sample/OD 450 control) X100%.

The results are shown in fig. 11 to 12.

FIGS. 11 and 12 show the promotion of cell adhesion by 0417 and 0420 samples, 0418 and 0419 samples, respectively, at different concentrations. The results showed that only 10ppm of samples 0417 and 0418 had significant promotion of cell adhesion, and none of the other samples had significant promotion.

Experimental example 4: macrophage inhibition assay

Culturing Raw 264.7 mouse macrophages in DMEM containing 10% FBSCell Technology), maintaining the temperature at 37 ℃,5% CO ₂ . When cells grew to 90% confluence, they were grown according to 8X10 ⁵ cells/mL concentration were seeded into 96-well plates and after 8 hours, samples of different concentrations were treated for 24 hours.

The cells were divided into different groups and each treatment was repeated three times: control (medium only), LPS (lipopolysaccharide) treated (LPS 1. Mu.g/mL), positive control (LPS+30. Mu.M dexamethasone) and treated (LPS+ samples) were used to analyze TNF-. Alpha.and IL-6 by ELISA.

The results are shown in fig. 13 to 14.

FIG. 13 shows the effect of different concentrations of 0417 and 0418 samples on the expression of inflammatory factor IL-6 by LPS. The results show that 0417 and 0418 samples at both concentrations showed significant inhibition of inflammatory factor IL-6.

FIG. 14 shows the effect of different concentrations of 0417 and 0418 samples on the expression of inflammatory factor TNF- α by LPS. The results show that 0417 and 0418 samples at both concentrations showed significant inhibition of inflammatory factor TNF- α.

Experimental example 5: gene analysis

Cell culture: normal human keratinocyte cell line (NHEKs)Cell Technology)

Culturing the keratinocyte cells in EpiLife (TM) medium (MEPI 500CA, thermo Fisher Scientific Inc., waltham, mass., USA) containing 60. Mu.M calcium at 37℃with a relative humidity of 95%,5% CO ₂ Is a culture vessel (Thermo Fisher 160 i). When cells were 80-90% confluent, transfer from the flask of T75 into a 24-well plate, 50,000 cells per well. Cells were treated with 0417 samples at different concentrations for 24 hours, rinsed once with cold PBS, and total RNA extracted with the RNeasy Mini Kit (Qiagen). Extracting total RNA and reverse transcription into cDNA, detecting gene expression by quantitative real RT-PCR (qRT-PCR). The primers used are shown in table 1 below.

TABLE 1

PCR conditions: pre-denaturation, 95 ℃ for 3min; then the mixture is circulated for 40 to 45 times at 95 ℃,5s and 60 ℃ for 30 to 40 s.

Statistical analysis:

the data are shown as mean ± standard error, t-test statistical method versus no treatment control. And (5) performing bidirectional contrast analysis.

The results are shown in fig. 15 and 16.

FIG. 15 shows that 0417 samples have a promoting effect on the gene expression of AQP1 and AQP3 at different concentrations, and that AQP1 and AQP3 can be significantly promoted at 6ppm and 15ppm, and that the concentration dependence is present, and that the higher the concentration of added 0417 samples, the higher the gene expression of AQP1 and AQP 3.

FIG. 16 shows that 0417 samples have a promoting effect on EPPK1 gene expression at various concentrations, already at a concentration of 3ppm, reaching a maximum at 6ppm and a slight decrease in 15ppm.

The results show that:

(1) 0417 sample was able to significantly promote the expression of AQP1 and it was the first time AQP1 expression was detected in skin keratinocytes.

(2) The inventors have discovered for the first time that fibronectin or a functional fragment thereof has an accelerating effect on AQP3 expression. The 0417 sample in this experiment is capable of significantly promoting the expression of AQP3, and considering that AQP3 has been widely validated and used as a key factor associated with skin moisturization, the present invention demonstrates the important role of the 0417 sample in skin moisturization.

(3) The 0417 sample can remarkably promote the expression of EPPK1, and based on the fact that EPPK1 plays an important role in coping with mechanical damage of skin, it is speculated that the 0417 sample can promote the expression of EPPK1 to enable the skin to have a rapid post-damage repair effect.

Experimental example 6: immunization groupChemical analysis

The 3D recombinant human epidermis model RHE is a 3D skin model constructed with human keratinocytes, can be used as a simulation of human skin, is often used as an in vitro model between cellular experiments and clinical experiments in cosmetic raw material efficacy evaluation, and generally has a further reference significance than cellular experiments.

On day 0, human keratinocytes were seeded on a 3D recombinant human epidermal model RHE (Ai Ji organism) and cultured with conventional medium. The culture was continued on day 1 by changing to medium containing 15ppm 0417 samples. On days 4-14, RHE were cultured on the gas-liquid interface. On day 14 RHE samples were fixed with 4% formalin fixation. The RHE samples were then embedded with paraffin. The embedded samples were cut into 5 μm flakes, then subjected to dewaxing and reabsorption processes, and the samples were stained with HE kit (Solarbio). Subsequent immunohistochemical staining was performed according to standard procedures as indicated by the antibody company's kit. Briefly, samples were first treated with a heat-mediated antigen (pH 6) containing sodium citrate buffer for 20 minutes. The samples were then treated with DSC3 antibody (Abcam) overnight (4 ℃). The next day was treated with a secondary antibody (Thermo) with alexaflor green fluorescence. Finally, photographs were taken with a Leica microscope. Semi-quantitative analysis was performed with ImageJ software based on the green fluorescence intensity of the pictures.

The results are shown in FIG. 17.

FIG. 17 shows the semi-quantitative analysis of the results of the RHE immunohistochemical experiments described above. The results show that 0417 can increase the expression level of DSC3 by 230% compared to the control.

The result shows that the 0417 sample has a remarkable promoting effect on the protein expression of DSC3, wherein DSC3 is an important transmembrane protein on semidesmosomes among keratinocytes on epidermis, the expression of the important transmembrane protein is directly related to the integrity of an epidermis barrier, and the experiment proves that the 0417 sample can promote the integrity of the skin barrier structure, increase the moisture retention of the skin and better cope with external stimulus.

Reference is made to:

[1] li Xiulan, shi Yijian, liu Jing. Fibronectin and its role in wound repair [ J ]. J.J.of Chinese, western and medical science, journal of surgery, 1998,4 (1): 60-62.

[2]Maxwell B.Johnson,Brandon Pang,et al.Topical Fibronectin Improves Wound Healing of Irradiated Skin[J].Scientific Reports,2017,7(Suppl 4):502-506.

[3]Sougrat R,Morand M,Gondran C,Barr′e P,Gobin R,Bont′eF,Dumas M,Verbavatz JM(2002)Functional expression of AQP3 in human skin epidermis and reconstructed epidermis.J Invest Dermatol 118(4):678-685.

[4]Hara M,Ma T,Verkman AS(2002)Selectively reduced glycerol in skin of aquaporin-3-defificient mice may account for impaired skin hydration,elasticity,and barrier recovery.J Biol Chem277(48):46616-46621.

[5]Ma T,Hara M,Sougrat R,Verbavatz JM,Verkman AS(2002)Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3.J Biol Chem 277(19):17147-17153.

[6] CN 101453979B cosmetic preparations containing aquaporin stimulators and uses thereof.

[7]Yamashita T,Asano Y,Saigusa R,Taniguchi T,Nakamura K,Miura S,Toyama T,Takahashi T,Ichimura Y,Hirabayashi M,Yoshizaki A,Miyagaki T,Sugaya M,Sato S(2018)Increased expression of aquaporin-1in dermal fibroblasts and dermal microvascular endothelial cells possibly contributes to skin fibrosis and edema in patients with systemic sclerosis.J Dermatol Sci.93(1),24-32.

[8]Spindler V，Heupel W-M，Efthymiadis A,Schmidt E,Eming R,Rankl C,Hinterdorfer P,Muller T,Drenchhahn A,and Waschke J,Desmocollin 3-mediated binding is crucial for keratinocyte cohersion and is impaired in pemphigus,J Biol Chem,284(44)30556-30564.

[9]Chen J,Den Z,Koch PJ,2008Loss of desmocollin 3in mice leads to epidermal blistering.J Cell Sci,2008,121(17)2844-2849.

[10]Fujiwara S，Shinkai H，Takayasu S，Owaribe K,Tsukita S,Kageshita T,A case of subepidermal blister disease associated with autoantibody against 450kD protein[J].J Dermatol，1992，19(10):610-613.

[11]Ishikawa K,Sumiyoshi H,Matsuo N,Takeo N,Goto M,Okamoto O,Tatsukawa S,Kitamura H,Fujikura Y,Yoshioka H,Fujiwara S,Epiplakin accelerates the lateral organization of keratin filaments during wound healing,J Dermatol Sci 2010(60):95-104.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of human fibronectin or a functional fragment of human fibronectin in the preparation of a medicament comprising:

or alternatively

2. The use according to claim 1, wherein the amino acid sequence of the functional fragment of human fibronectin is shown as SEQ ID NO. 1 or SEQ ID NO. 3.

3. The use according to any one of claims 1 to 2, wherein the protein comprises AQP1 and one or more selected from AQP3, DSC3 and EPPK1.

4. Use according to any one of claims 1 to 2, wherein the protein is derived from human skin tissue or human skin cells such as human keratinocytes or human fibroblasts.

5. A method of screening for functional fragments of human fibronectin comprising the steps of:

wherein the protein is selected from one or more of AQP1, AQP3, DSC3 and EPPK1.

6. The method for screening a functional fragment of human fibronectin according to claim 5, wherein human keratinocytes or human fibroblasts without candidate human fibronectin fragments are set as a blank.

7. The method for screening a functional fragment of human fibronectin according to any one of claims 5 to 6, wherein human keratinocytes or human fibroblasts added with the polypeptide fragments of SEQ ID NO. 1 and/or SEQ ID NO. 3 are provided as a positive control.

8. The method of screening for a functional fragment of human fibronectin according to claim 7, wherein the concentration of the polypeptide fragment is more than 3ppm, 3ppm to 50ppm, 3ppm to 30ppm, 3ppm to 20ppm, 3ppm to 15ppm or 6ppm.

9. The method of screening for a functional fragment of human fibronectin according to any of claims 5 to 9, wherein said protein comprises AQP1 and one or more selected from AQP3, DSC3 and EPPK1.

10. A kit for screening for functional fragments of human fibronectin comprising:

11. The kit of claim 10, wherein the reagent for detecting the level of a protein or the expression level of a gene encoding the protein is an antibody to the protein or a PCR primer for the gene encoding the protein.

12. The kit of any one of claims 10 to 11, further comprising the polypeptide fragment set forth in SEQ ID No. 1 and/or SEQ ID No. 3.