CN113929750B

CN113929750B - Polypeptide with skin barrier repairing function and application thereof

Info

Publication number: CN113929750B
Application number: CN202111391461.2A
Authority: CN
Inventors: 欧瑜; 杨明丽
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2023-04-11
Anticipated expiration: 2041-11-23
Also published as: CN113929750A

Abstract

The invention belongs to the field of polypeptides, and discloses a polypeptide with a function of repairing skin barrier and application thereof, wherein the peptide has an amino acid sequence shown in SEQ ID NO. 1. The peptide can obviously promote the proliferation, migration and differentiation of human keratinocytes in a certain concentration range, can obviously promote the recovery of damaged skin, relieve the abnormal thickening and abnormal differentiation of epidermis caused by skin damage, and has the function of promoting skin barrier repair.

Description

Polypeptide with skin barrier repairing function and application thereof

Technical Field

The invention belongs to polypeptides, and particularly relates to a polypeptide with a function of repairing skin barrier.

Background

The skin is the largest organ of the human body, covers the surface of the human body, is directly contacted with the external environment and has important barrier protection effect. The skin is structurally divided into epidermis, dermis, subcutaneous tissue, and various skin appendages. The epidermis is the outermost layer of the skin and has an important skin barrier function. The epidermis is mainly composed of keratinocytes and is divided into basal layer, spinous layer, granular layer and horny layer. Keratinocytes in the basal layer have the ability to proliferate, migrate and differentiate, and are epidermal "stem cells", which divide new keratinocytes, so that the divided cells migrate and differentiate upward to form a stratum spinosum and a stratum granulosum in turn, and finally reach the terminal differentiation stage to form a mature stratum corneum.

The stratum corneum is the final stage of keratinocyte differentiation, being a "brick wall structure". Keratinocytes differentiate to form organelle-free keratinocytes arranged to form a "brick wall structure," which refers to keratinocytes, "mortar," i.e., intercellular lipids, desmosomes connecting adjacent keratinocytes and tight junctions similar to "rebar". Intercellular lipids, desmosomes, tight junctions, etc., are all products of keratinocyte differentiation. The 'brick wall' and 'mortar' of epidermis make the epidermis form firm structure, restrict the water flow inside and outside the cell and among the cells, resist the harm such as external microorganism, chemical substance, ultraviolet ray, make the skin maintain important barrier function.

Normally, keratinocyte proliferation, migration, and differentiation are highly regulated, which enables the epidermis to develop to form a complete stratum corneum. The epidermis stratum corneum plays crucial effect in the skin barrier, and normal complete stratum corneum can effectively resist external environment's the infringement and the loss of internal moisture on the one hand, and on the other hand, the stratum corneum can perceive external condition and change, and after skin is impaired, can be with the deep transmission of signal to the skin, mobilize the organism and restore skin barrier function.

Once the skin barrier is damaged, the defense function of the skin is reduced, and the problems of increased percutaneous water loss, dry and sensitive skin, delayed wound healing, microorganism imbalance, reduced immune function, accelerated aging and the like are caused. Abnormalities in the structure and function of the skin barrier are manifestations of, and are important causes of, many skin disorders, such as atopic dermatitis, eczema, psoriasis, ichthyosis, and the like. In addition, these skin diseases are accompanied by the impairment of the skin barrier function, and if the skin barrier function is not repaired in time, the body is in a state of chronic inflammation, thereby aggravating the diseases and even inducing the occurrence of other diseases. Therefore, maintaining and repairing the skin barrier function is not only helpful for improving the skin condition, but also for preventing and treating skin diseases. The research and development of a new product with the function of repairing the skin barrier have important significance.

The polypeptide is used as an active ingredient and is more and more widely applied in the technical fields of biological medicines and cosmetics. The use of peptides as starting materials has several advantages: 1. the polypeptide participates in a plurality of physiological functions of the skin, and has wide application range; 2. the polypeptide has pertinence and selectivity, and the effect is better; 3. the safety is good, and the peptide has biocompatibility with human body and is not easy to be allergic; 4. the small molecular polypeptide is easy to synthesize, modify and optimize. Thus, the polypeptide has very important development value. A small molecular polypeptide containing 12 amino acids, namely SLDSTHTHAPWP, which can promote the proliferation, migration and differentiation of keratinocytes is screened, and further animal experiments prove that the small molecular polypeptide has the function of repairing skin barriers.

1. Summary of the invention

The purpose of the invention is as follows: the invention aims to disclose the application of a peptide in repairing skin barriers, and the peptide is used as an effective component in medicaments for treating skin barrier damage and cosmetics for repairing the skin barriers. The peptide can effectively promote the proliferation, migration and differentiation of keratinocytes, promote the recovery of damaged skin and has the functions of maintaining and repairing skin barriers.

The technical scheme is as follows: use of a polypeptide having the amino acid sequence sldsthiappp for repairing a skin barrier. The invention refers to a polypeptide with SLDSTHTHAPWP amino acid sequence, which is abbreviated as HCSP4. The amino acid sequence shown is as follows: ser-Leu-Asp-Ser-Thr-His-Thr-His-Ala-Pro-Trp-Pro.

A polypeptide, characterized by: the amino acid sequence is SEQ ID NO.1 and is written as HCSP4.

A composition characterized by: comprises the polypeptide and pharmaceutically or cosmetically acceptable auxiliary materials. The composition is characterized in that the pharmaceutically or cosmetically acceptable auxiliary materials comprise diluent filler, adhesive, wetting agent, absorption enhancer, lubricant or stabilizer to form a preparation.

The polypeptide or the composition is applied to preparing medicines or cosmetics for repairing skin barriers.

The peptide may be modified by methods conventional in the art.

Advantageous effects

1. The invention firstly discovers that the HCSP4 can promote the repair of the skin barrier and can be used as a medicine for preventing and treating diseases related to the damaged skin barrier function or a cosmetic for improving the skin barrier function through in vivo and in vitro experiments.

Specifically, in vitro experiments, HCSP4 promotes proliferation, migration, and differentiation of HaCaT cells (human immortalized keratinocytes). In animal experiments, the invention proves that HCSP4 can accelerate the recovery of the skin barrier by establishing a mouse skin barrier function damage model, can effectively relieve pathological symptoms after the skin barrier function is disturbed, and can prevent the abnormal thickening and abnormal differentiation of the epidermis after the skin barrier is damaged.

Description of the drawings:

FIG. 1 shows HCSP4 purity;

FIG. 2 shows molecular weight identification of HCSP 4;

FIG. 3 shows the effect of HCSP4 at different concentrations for 24h (A)) And 48h (B) effect on HaCaT cell proliferation (compared to control, ^* P<0.05， ^** P<0.01， ^*** P<0.001)

FIG. 4 is a graph of the effect of HCSP4 on Ki67 expression in HaCaT cells;

fig. 5 is a graph of the effect of HCSP4 on HaCaT cell migration, where a is a migration picture, B is a mobility line graph (compared to control, ^* P<0.05， ^** P<0.01， ^*** P<0.001)；

FIG. 6 shows the effect of HCSP4 on HaCaT cell differentiation marker gene expression (compared to control, ^* P<0.05， ^** P<0.01， ^*** P<0.001)；

FIG. 7 shows the repair of the damaged epidermal barrier of mice by HCSP 4;

FIG. 8 shows the effect of HCSP4 on the morphology of mouse epidermis;

figure 9 shows the effect of HCSP4 on the thickness of the mouse epidermis (compared to the control, ^### P<0.001; compared with the matrix group, ^*** P<0.001)；

FIG. 10 shows the effect of HCSP4 on the expression of genes involved in epidermal differentiation in mice, where A is Filaggrin, B is Involucrin, C is lorarin, D is Caspase-14, E is Keratin-1 (compared with the control group, ^### P<0.001; in comparison with the matrix group, ^* P<0.05， ^** P<0.01， ^*** P<0.001)。

Detailed Description

Example 1: synthesis and purity of HCSP4

The HCSP4 is synthesized by Shanghai bioengineering, inc., has a purity of 98.9% (FIG. 1) and a mass spectrometric identification molecular weight of 1348.8Da (FIG. 2).

Example 2: effect of HCSP4 on HaCaT cell proliferation, migration, differentiation

1. MTT method for detecting influence of HCSP4 on HaCaT cell proliferation

Culturing HaCaT cells was seeded in 96-well plates, and culturing was continued for 24h at 37 ℃ 5% CO2 in DMEM medium containing 10% newborn calf serum, after which the medium was carefully discarded with a pipette and replaced with serum-free DMEM medium containing different concentrations of HCSP4 (0. Mu.M, 1. Mu.M, 3. Mu.M, 10. Mu.M, 30. Mu.M, 100. Mu.M, 300. Mu.M) for 24h or 48h. After completion of the incubation, 10. Mu.L MTT solution was added to each well, the incubation was continued for 4 hours at 37 ℃ 5% CO2, the medium in the wells was carefully discarded, 150. Mu.L of dimethyl sulfoxide (DMSO) was added to each well and shaken for 10 minutes, the absorbance of each well was measured at a wavelength of 490nm and the cell viability was calculated.

As a result: after HCSP4 treated HaCaT cells for 24h, as shown in FIG. 3A, HCSP4 showed no significant change in HaCaT cell growth at concentrations of 1. Mu.M and 3. Mu.M, promoted HaCaT cell proliferation in a dose-dependent manner at 10-100. Mu.M, and at 300. Mu.M (P < 0.05) was inferior to that of the 100. Mu.M CSP4 treated group (P < 0.001). After HCSP4 treated HaCaT cells for 48h, as shown in FIG. 3B, there was no significant difference in the growth of HaCaT cells at a concentration of 1. Mu.M in HCSP4, and the proliferation of HaCaT cells was promoted in a dose-dependent manner within 3-100. Mu.M, but at 300. Mu.M, the effect was not as good as that of the 100. Mu.M HCSP4 treated group. After HCSP4 treatment on HaCaT cells for 24h and 48h, the cell survival rate is increased compared with that of a control group, and the increase trend is that the cell survival rate is increased firstly and then decreased secondly. Therefore, the experiment is carried out by selecting three concentrations of 10, 30 and 100 mu M to act on the HaCaT cells for 24h in the later period. The result shows that HCSP4 can obviously promote the proliferation of HaCaT cells within a certain concentration range.

2. Method for detecting influence of HCSP4 on Ki67 expression in HaCaT cells by adopting cell immunofluorescence method

Ki-67 is a marker of cell proliferation and reflects the level of cell proliferation. Taking HaCaT cells in good condition in logarithmic growth phase, inoculating into 96-well plate, placing at 37 deg.C, 5% ₂ The culture box is used for culturing for 24 hours. After the cell density reached about 50%, the cells were incubated for 24h in serum-free DMEM medium containing different concentrations of HCSP4 (10. Mu.M, 30. Mu.M, 100. Mu.M). The medium was discarded, and 100. Mu.L of 4% formaldehyde solution was added to each well, and the mixture was allowed to stand at room temperature for 20min to fix the cells. The cells were rinsed 3 times with 100. Mu.LPBS for 5min each time, and the fixative was washed away. Blocking was performed by adding 100. Mu.L of blocking solution, 5% of BSA +0.1% by volume, tritonX-100inPBS, and allowing to stand at room temperature for 1 hour. The blocking solution was discarded and 50. Mu.L of diluted Ki67 antibody (blocking solution diluted) was added overnight at 4 ℃. Primary antibody recovery, 100 μ LPBS rinsing 3 times, each timeThe next 5min, wash away the excess primary antibody. In the dark, 50. Mu.L of diluted fluorescent secondary antibody (diluted with blocking solution) was added, immediately placed in a box, protected from light, and allowed to stand at room temperature for 1 hour. Discard secondary antibody, not recycle, rinse 3 times with 100 μ LPBS for 5min each time, and wash off excess secondary antibody. 35 μ of LDAPI (diluted as described) was added and incubated for 10min in the dark, the specimens were nucleated, and after recovery of DAPI, washed 3 times with PBS. And (5) observing and photographing by a fluorescence microscope.

As a result: as shown in fig. 4, the fluorescence signal of Ki67 was increased in the HCSP4 group compared to the control group, indicating that the Ki67 protein expression level was increased. Thus, cellular immunofluorescence results further indicate that HCSP4 is capable of promoting HaCaT cell proliferation over a range of concentrations.

3. In-vitro scratch test is adopted to detect the influence of HCSP4 on HaCaT cell migration

And vertically drawing 3 lines at intervals of 1cm on the back of a 6-hole plate by using a marker pen. HaCaT cells were cultured at 1X 10 ⁵ cells/ml are inoculated on a 6-hole cell plate, when the cells grow to 80-90% fusion, the culture medium is sucked away, a 20-microliter sterilization gun head is used for being compared with a ruler, vertical scratches are formed, and a cross point is formed with a back line, so that fixed-point observation and photographing are facilitated. The scratched cells were removed by gentle washing 3 times with PBS. 2mL of serum-free medium containing different concentrations of HCSP4 (0, 10, 30, 100. Mu.M) was added for conventional culture. At

different time points

0, 12 and 24h, the area of the scratch injury is observed under an inverted microscope and photographed, and the healing rate of the area of the scratch injury is calculated by using software ImageJ. Mobility = (original area-area after scratch)/original area × 100%.

As a result: the effect of HCSP4 on HaCaT cell migration is shown in FIG. 5, and when the concentration of HCSP4 is 100. Mu.M, the cell migration rate is increased at different time points after scratching compared with the control group; wherein the mobility of the 100 mu MHCSP4 reaches 70.8 +/-8.6 percent compared with the mobility of a control group (57.9 +/-1.8 percent) at 24h after scratching, the mobility of the 100 mu MHCSP4 reaches 83.1 +/-5.4 percent compared with the mobility of the control group (70.1 +/-2.4 percent) at 48h after scratching, and the mobility is obviously different from that of the control group (P < 0.05). The results of HCSP4 at concentrations of 10 and 30 μ M showed that HCSP4 promoted the migration of HaCaT cells at a concentration of 100 μ M, although there was no significant difference in the migration rate of HaCaT cells compared to the control group.

4. The method for detecting the content of HCSP4 in Filaggrin, involucrin, loricrin, caspase-14, DNA by adopting a qPCR method,

Influence of mRNA expression level of cell differentiation marker gene such as Keratin 1.

Culturing HaCaT cells, inoculating to a 96-well plate, culturing at 37 ℃ 5% CO2 for 24 hours, removing the medium, adding a culture medium containing different concentrations of 0, 10, 30, 100. Mu. MHCSP4, 1mM CaCl ₂ The culture medium is incubated for 24 hours. After the incubation was completed, the culture solution was discarded and washed twice with PBS. Adding 1ml of LRNAzol into each well, repeatedly blowing to ensure that the cells are completely lysed, and standing at room temperature for 5min. To the above solution was added 0.2mL of chloroform, the tube cap was closed, shaken vigorously for 15s, and left at room temperature for 3min. Then, the mixture was centrifuged at 12000g for 15min at 4 ℃. The solution now divided into three layers, with RNA present in the upper aqueous phase. Aspirate 500. Mu.L of the upper aqueous phase and transfer to a new centrifuge tube. Adding equal volume of isopropanol into the solution, mixing by inversion, and standing at room temperature for 10min. Centrifuge at 12000g for 10min at 4 ℃ and discard the supernatant. The pellet was washed gently by adding 1mL of 75% ethanol to each tube, centrifuged at 12000g at 4 ℃ for 5min, and the supernatant was carefully discarded. After standing at room temperature for several minutes, air-dried. A certain amount of RNase-free water was added to each tube to dissolve RNA sufficiently. The RNA concentration was measured by NanoDrop and the integrity of the RNA was verified by agarose gel electrophoresis. Synthesizing DNA by reverse transcription according to an RT-PCR reverse transcription reaction system, and designing and synthesizing a primer. The mRNA expression levels of the differentiation genes Filaggrin, involucrin, loricrin, caspase-14 and keratin1 of the HaCaT cells are detected by a qPCR method. The method is prepared according to a qPCR reaction system, and qPCR is carried out on an ABIQuantStudio fluorescent quantitative PCR instrument. Using GAPDH as internal reference, normalizing Ct value of each target gene, and adopting 2 as gene expression difference calculation method ^-△△Ct The method comprises the following specific calculation method:

△Ct _sample ＝Ct _{target gene of sample to be tested} -Ct _{Reference gene of sample to be detected}

△Ct _control ＝Ct _{Control sample Gene of interest} -Ct _{Reference gene of control sample}

△△Ct＝△Ct _sample -△Ct _control

Folddifference＝2 ^-△△Ct

TABLE 1.QPCR primer sequences

As a result: as shown in FIG. 6, filaggrin, involucrin, loricrin, caspase-14 and keratin1 are all epidermal differentiation related genes. Calcium chloride promotes keratinocyte differentiation, thus 1mM CaCl ₂ The group was positive. Compared with the control group, the positive group significantly promotes the expression of Filaggrin, involucrin, loricrin, keratin1 and Caspase-14 (P)<0.05、P<0.01、P<0.001). HCSP4 promotes the expression of Filaggrin, involucrin, loricrin, keratin1 in a concentration-dependent manner in the range of 10-100. Mu.M. When the concentration of HCSP4 is 10 mu M, the expression of Filaggrin and Involucrin can be obviously promoted, and the obvious difference (P) between the expression and the expression of the Involucrin and the expression of the Filaggrin and the Involucrin and the expression of the Involucrin in the HCSP4 is obviously different from that in a control group (P)<0.01). When the concentration of HCSP4 is 30 and 100 mu M, the expression of Filaggrin, involucrin, loricrin and keratin1 can be obviously promoted, and the significant difference (P) is obtained when the concentration is different from that of a control group<0.05、P<0.01、P<0.001). As for the expression of Caspase-14, the 10 and 30 mu MHCSP4 groups have no significant difference compared with the control group, and 100 mu MHCSP4 can significantly promote the expression of Caspase-14 (P<0.05). The results show that HCSP4 can increase the expression of genes Filaggrin, involucrin, loricrin, caspase-14 and Keratin1 related to HaCaT cell differentiation under certain concentration.

Example 3: preparation examples

A composition with skin barrier repairing effect comprises the following raw materials: 5g of glycerol, 1g of triethanolamine, 4g of liquid paraffin, 2.5g of vaseline, 6g of stearic acid, 1.5g of glyceryl monostearate, 5.6g of urea, 0.03g of HCSP4 peptide and water to make up to 50g.

The preparation method comprises the following steps:

oil phase: adding liquid paraffin, heating to 80 deg.C, melting, sequentially adding vaseline, stearic acid, and glyceryl monostearate, heating to melt, and keeping at 80 deg.C.

Water phase: adding glycerol and triethanolamine into distilled water, heating to 80 deg.C, adding 0.1% methyl p-hydroxybenzoate, rapidly adding urea, stirring, heating to 80 deg.C, and keeping the temperature.

Emulsification: slowly adding the water phase into the oil phase, continuously stirring in the same direction, and continuously stirring. Cooling to below 40 deg.C, adding HCSP 4-containing water solution, stirring, and completely condensing.

Example 4: skin barrier repair effect of HCSP4 on mice with damaged skin barrier

And establishing a mouse skin barrier injury model by using a tape pasting method. ICR female mice (provided by the university of Yangzhou, comparative medicine center) with SPF grade, 4-6 weeks of age and weight of 15-22g were selected for the experiment. The experimental animals are bred in the experimental animal center of Chinese university of pharmacy at 22 + -2 deg.C and humidity of 50-60% under 12 hr illumination and black-night circulation. After 1 week of adaptive feeding, the animals were randomly divided into 5 groups, namely, a control group, a model group, a ground group, an HCSP4 low dose group (0.02%; HCSP4 peptide), and an HCSP4 high dose group (0.06%; HCSP4 peptide), respectively. After shaving the back hair of the mice with a shaver, the mice were depilated with a depilatory cream, and one day after skin preparation, the mice in the control group, model group, matrix group, HCSP4 low-dose group, and HCSP4 high-dose group were molded. The molding method is that after the mouse is anesthetized (4% chloral hydrate is injected into abdominal cavity), the mouse is fixed on a foam board, the skin on the back of the mouse is repeatedly stuck by a strong adhesive tape, and the sticking is stopped when the back of the mouse shows congestion. After molding, the same amount of the corresponding cream was applied to the back molding area of mice in the matrix group, the HCSP4 low dose group and the HCSP4 high dose group every day.

Skin tissues in a molding area on the back of a mouse on the 8 th day after molding are taken, cut into a proper size, adhered to tin foil paper (preventing the skin tissues from curling in a fixing solution), fixed in a 4% paraformaldehyde fixing solution, and then subjected to skin tissue slicing and HE staining. The HE section is used for observing the morphological change of skin tissues under a 20-fold microscope, and pictures are taken to measure the thickness of the epidermis. And (3) taking the skin tissue of the back molding area of the mouse on the 8 th day after molding, extracting RNA in the tissue, performing qPCR (quantitative polymerase chain reaction) experiment, and detecting the differentiation related marker gene.

As a result: as shown in FIG. 7, after modeling, the skin on the back of the mice was red and shiny, the red was due to the thick and congested capillaries, and the shiny was due to the exudation of exudate. The first day after molding, the wound surface began to scab, and the exudate covered the surface to form scabs. Compared with the model group, the wound on the back of the mice in the matrix group, the low dose group and the high dose group scab more quickly. On the fourth day after the administration, the scab on the back began to fall off, no significant falling off of the scab on the back was observed in the model group mice, the edge of the scab on the matrix group was fallen off, and the scab was significantly fallen off in both the low dose group and the high dose group. On the eighth day after administration, the scab had completely fallen off, and some areas were redder than the surrounding skin after the scab had fallen off in the model group, and some areas of the matrix group also appeared red, and the wound on the back of the low dose group and the high dose group had completely healed, and had uniform skin color, and no redness was seen. The above results indicate that HCSP4 is able to promote impaired skin repair after an impaired epidermal barrier.

HE sections were observed under a microscope as shown in fig. 8. The control mice had no abnormal skin tissue structure, regular arrangement and no inflammatory cell infiltration. Compared with the normal group, the horny layer of the model group is obviously thickened and has inflammatory cell infiltration; the stratum corneum of the blank matrix group was also abnormally thickened and infiltrated with inflammatory cells, but the condition was reduced compared to the model group. Compared with the model group and the stroma group, the HCSP4 group has different degrees of improvement on pathological changes, obviously thinned stratum corneum, reduced inflammatory cells and a structure tending to a normal group.

The skin thickness was measured by ImageJ software. The results are shown in fig. 9, where the skin thickness of the model group is significantly increased (P < 0.001) compared to the control group. Compared with the stroma group, the epidermal thickness of the HCSP4 low-dose group and the high-dose group was significantly reduced (P < 0.001), and the epidermal thickness tended to that of the control group. This result indicates that HCSP4 can alleviate the symptoms of epidermal hyperplasia after the skin barrier is damaged.

In order to explore the influence of HCSP4 on the differentiation of mouse epidermal keratinocytes, mRNA expression levels of epidermal differentiation related genes Filaggrin, involucrin, loricrin, caspase-14 and Keratin-1 in skin tissues were detected by a qPCR method. The results of qPCR are shown in FIG. 10, compared with the control group, the levels of mRNA expression of Filaggrin, loricrin, caspase-14 and Keratin-1 in the skin of the model group are obviously increased (P < 0.001), and the expression level of Involucrin is obviously reduced (P < 0.001), which indicates that the epidermis is abnormally differentiated after the back skin of the mouse is damaged by the adhesive tape. Compared with the stroma group, the skin of mice in the HCSP4 group all showed reduced expression levels of Filaggrin, loricrin, caspase-14 and Keratin-1 (P <0.05, P < -0.01, P < -0.001), and the expression level of Involucrin was significantly reduced (P < 0.01), and showed certain dose dependence. The results show that compared with the control group, the expression of the epidermal differentiation related gene of the mice in the model group is abnormally increased or reduced; the expression trend of the related genes of the HCSP4 group is opposite to that of the model group and the stroma group, and the expression level of the related genes of the HCSP4 group is close to that of the control group. This result demonstrates that HCSP4 significantly reduces the abnormal differentiation of the epidermis after the skin of mice is damaged.

The animal experiment results show that HCSP4 can not only accelerate the repair of the skin barrier, but also prevent the abnormal thickening and abnormal differentiation of the epidermis after the skin is damaged.

Sequence listing

<110> university of Chinese pharmacy

<120> polypeptide with function of repairing skin barrier and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> HCSP4(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Ser Leu Asp Ser Thr His Thr His Ala Pro Trp Pro

1 5 10

<210> 2

<211> 20

<212> DNA

<213> Filaggrin-Forward prime(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

tgaggcatac ccagaggact 20

<210> 3

<211> 20

<212> DNA

<213> Filaggrin-Reverse primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

ctgtatcgcg gtgagaggat 20

<210> 4

<211> 21

<212> DNA

<213> Involucrin-Forward primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

gccaggtcca agacattcaa c 21

<210> 5

<211> 24

<212> DNA

<213> Involucrin-Reverse primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

gggtggttat ttatgtttgg gtgg 24

<210> 6

<211> 20

<212> DNA

<213> Caspase-14-Forward primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

accgcacatc aggatctctc 20

<210> 7

<211> 20

<212> DNA

<213> Caspase-14-Reverse primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

ggcttagagg caagggtgag 20

<210> 8

<211> 24

<212> DNA

<213> Keratin-1-Forward primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

atttctgagc tgaatcgtgt gatc 24

<210> 9

<211> 21

<212> DNA

<213> Keratin-1-Reverse primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

cttggcatcc ttgagggcat t 21

<210> 10

<211> 21

<212> DNA

<213> GAPDH-1-forward primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 10

gaaggtcgga gtcaacggat t 21

<210> 11

<211> 20

<212> DNA

<213> GAPDH-1-reverse primer(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

tgacggtgcc atggaatttg 20

Claims

1. The application of a polypeptide or a composition formed by the polypeptide in preparing a medicine or a cosmetic for repairing skin barriers is characterized in that the amino acid sequence of the polypeptide is SEQ ID NO.1; the composition comprises polypeptide and pharmaceutically or cosmetically acceptable auxiliary materials, wherein the pharmaceutically or cosmetically acceptable auxiliary materials comprise diluents, binders, wetting agents, absorption enhancers, lubricants or stabilizers.