CN113520899B

CN113520899B - Recombinant collagen product for skin photodamage repair

Info

Publication number: CN113520899B
Application number: CN202110321963.1A
Authority: CN
Inventors: 肖建喜; 付彩虹
Original assignee: Collagen Wuhan Biotechnology Co ltd
Current assignee: Collagen Wuhan Biotechnology Co ltd
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2022-05-17
Anticipated expiration: 2041-03-25
Also published as: CN113520899A

Abstract

The invention belongs to the field of dermatology, and particularly relates to a recombinant collagen product for repairing skin ultraviolet injury. The invention provides a recombinant collagen, which has good repairing effect on ultraviolet-damaged skin, good adhesion effect on fibroblasts, no stimulation to skin and good safety, and can promote the proliferation of skin fibroblasts; meanwhile, the recombinant collagen can remarkably improve the inflammatory symptoms after skin injury, keep the moisture of the epidermal layer, induce the growth of epidermal cells of the skin, help to repair collagen fibers of the dermal layer and repair the skin with light injury, and can be used for preparing cosmetics or medical beauty products.

Description

Recombinant collagen product for skin photodamage repair

Technical Field

The invention belongs to the field of dermatology, and particularly relates to a recombinant collagen product for repairing skin photodamage.

Background

The skin is the largest organ of the human body, the total area of the skin can reach 1.5 to 2 square meters on average, and the total weight of the skin accounts for 5 to 15 percent of the total weight of the human body. The skin has important barrier function, can protect internal tissues and organs from being invaded by external viruses and physical and chemical injuries, and has the functions of perspiration, cold and heat feeling, pressure, body temperature regulation and the like. In daily life, the skin is inevitably irradiated by sunlight, and ultraviolet rays in the sunlight cause photodamage to the skin, including phototoxic reaction, photosensitivity, photoaging, and carcinogenesis. Most of ultraviolet rays in the sunlight are UVA with the wavelength of 320-400nm, and the UVA can permeate into the epidermis to generate the photooxidation of melanin, so that the skin is quickly changed into tan; UVB in the sun with the wavelength of 280-320nm can cause solar erythema, immunosuppression and carcinogenesis. Excessive ultraviolet radiation can cause not only acute effects such as erythema, inflammation, papules, blisters, flaking, pigmentation, etc. but also chronic effects such as skin aging, DNA abnormalities, malignant melanoma, etc.

Collagen is the major component of the dermis layer of the skin and is present in amounts up to 75% of the dry weight of the skin. Collagen in skin has a characteristic triple helix structure that self-assembles to form a periodic network of fibers that provide tensile strength and elasticity to the skin. Studies have shown that uv radiation affects the metabolism of collagen in the dermis. Collagen fiber bundles have certain elasticity, and ultraviolet irradiation may cause collagen fiber denaturation, thereby causing the reduction of skin elasticity. Collagen is used as key protein in skin, has the functions of good biocompatibility, moisturizing, nourishing skin and the like, and is widely applied to skin care products.

At present, collagen in the market mainly comes from tissues of animals such as fish, pigs, cows and the like, the risk of virus transmission may exist, and animal collagen has the defects of poor water solubility, complex processing process, easy inactivation of collagen in the preparation process and the like because the animal collagen has different sources and is difficult to control the quality. The recombinant collagen prepared by the genetic engineering technology completely eliminates the hidden danger of animal viruses, and has the advantages of easy quality control and uniform molecular weight.

The invention provides a recombinant collagen, which has good repairing effect on ultraviolet-damaged skin, good adhesion effect on fibroblasts, no stimulation to skin and good safety, and can promote the proliferation of skin fibroblasts; meanwhile, the recombinant collagen can remarkably improve the inflammatory symptoms after skin injury, keep the moisture of the epidermal layer, induce the growth of epidermal cells of the skin, help to repair collagen fibers of the dermal layer and repair the skin with light injury, and can be used for preparing cosmetics or medical beauty products.

Disclosure of Invention

The invention aims to provide the application of recombinant collagen in skin photodamage repair. The method specifically comprises the following steps:

in a first aspect, the invention provides the use of a recombinant collagen for the manufacture of a product for repairing photodamage to skin.

Preferably, the amino acid sequence of the recombinant collagen is as follows:

GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP。

preferably, the photodamage is ultraviolet damage.

Preferably, the product for repairing photodamage of skin comprises drugs, cosmetics, medical and cosmetic products, foods.

Preferably, the cosmetic comprises stock solution, lotion, essence cream, toner, conditioning water, cream, mask, makeup cream, eye cream, spray, sunscreen cream, foundation, body lotion.

Preferably, the medical cosmetic product comprises a stock solution, a dressing, a cold pack, a gel, a sponge, a film, a hydro-acupuncture, an implant, a milk, a cream, a paste.

In a second aspect, the present invention provides a use of recombinant collagen in the manufacture of a product for preventing photodamage to skin.

Preferably, the amino acid sequence of the recombinant collagen is as follows:

preferably, the photodamage is ultraviolet damage.

Preferably, the product for preventing photodamage to skin comprises a pharmaceutical, cosmetic, cosmeceutical, food product.

The invention has the beneficial effects that:

(1) the recombinant collagen provided by the invention has no cytotoxicity, good safety, no stimulation to skin, good adhesion to fibroblasts and capability of promoting the proliferation of skin fibroblasts;

(2) the recombinant collagen provided by the invention has a remarkable repairing effect on ultraviolet-damaged skin, can improve the inflammation symptom of the skin after being exposed to the sun, keeps the moisture of the epidermal layer, induces the epidermal cell growth of the skin, and helps to repair the collagen fiber of the dermal layer, and the repairing effect of the skin after being exposed to the sun is better than that of the existing collagen sold on the market;

(3) the recombinant collagen provided by the invention has good biocompatibility, no virus hidden danger, good water solubility, suitability for large-scale production and easy addition, and can be used for cosmetics or medical beauty products to help repair photodamaged skin.

Drawings

FIG. 1 circular dichroism spectroscopy of recombinant collagen prepared in example 1 of the present invention;

FIG. 2 shows the cytotoxicity of recombinant collagen prepared in example 1 of the present invention;

FIG. 3 shows the cell proliferation effect of the recombinant collagen prepared in example 1 of the present invention;

FIG. 4 shows the cell adhesion effect of the recombinant collagen prepared in example 1 of the present invention;

FIG. 5 shows skin irritation of New Zealand rabbits by the recombinant collagen prepared in example 1 of the present invention;

FIG. 6 HE staining results of mouse skin tissues of blank group, model group and experimental group;

FIG. 7 HE staining results of skin tissues of mice of different control groups;

FIG. 8 Masson staining results of mouse skin tissues of blank group, model group and experimental group;

FIG. 9 Masson staining results of skin tissues of mice in different control groups.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited to the following.

EXAMPLE 1 preparation of recombinant collagen

1. Determining the amino acid sequence comprising recombinant collagen as shown below:

MHHHHHHADEQEEKAKVRTELIQELAQGLGGFEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP。

2. construction of recombinant collagen expression strains

Synthesizing a nucleotide sequence encoding the amino acid sequence described in the above 1, constructing an expression plasmid containing the nucleotide sequence, and confirming the successful synthesis of the plasmid by DNA sequencing; transforming the plasmid into an escherichia coli BL21-DE3 strain, adding glycerol into the successfully transformed positive clone strain, and storing the strain in a refrigerator at-80 ℃ for later use;

3. preparation and purification of recombinant collagen

Taking a trace amount of frozen recombinant collagen expression strain, carrying out IPTG induction culture, treating by using trypsin, carrying out chromatography purification to obtain recombinant collagen, and freeze-drying and storing, wherein the amino acid sequence of the recombinant collagen is as follows:

example 2 detection of recombinant collagen

1. Circular dichroism detection

The recombinant collagen prepared in example 1 was dissolved in PBS buffer (pH 7.0), sodium acetate buffer (pH3.0) and ethylene glycol, respectively, to a final concentration of 1 mg/mL. And (3) performing spectrum scanning on the recombinant collagen solution by using a circular dichroscope, wherein the scanning wavelength is 210-260nm, the scanning temperature is 4 ℃, the wavelength stepping is 0.5nm, the average time is 0.5 second, and the CD spectra are average values of three times of scanning.

As shown in fig. 1, the recombinant collagen produced in example 1 of the present invention has a positive peak at about 220nm, and is a recombinant collagen having a triple helix structure, which corresponds to the triple helix structure of collagen.

2. Cytotoxicity test

HeLa cells grown completely adherent were digested with 0.25% trypsin and prepared to a cell density of 1X 10 using complete medium (1% FBS, 2% Penicilin-Streptomyces, DMEM medium)⁵Cell suspension per mL. 100 μ L of cell suspension was transferred and inoculated into 96-well culture plates and placed at 37 ℃ with 5% CO₂Culturing in an incubator with saturated humidity for 24 h. The complete culture solution was aspirated, and recombinant collagen solutions (0.001, 0.005, 0.01, 0.05, 0.1mg/ml) prepared in example 1 at different concentrations diluted in high-glucose DMEM medium were added to the experimental group, with 4 multiple wells per concentration; the control group is prepared by adding equivalent amount of high-glucose DMEM medium without recombinant collagen into cells, and the blank group is prepared by adding equivalent amount of DMEM medium without cells, and continuing to maintain at 37 deg.C and 5% CO₂Culturing in an incubator with saturated humidity for 24 h. To each group, 10. mu.L of CCK-8 reagent was added, incubated in a cell incubator for 1-4h, and the absorbance value (OD value) of each well was measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay. Cell viability was calculated from the absorbance mean of each group according to the following formula:

the experimental results are shown in fig. 2, and after the recombinant collagen prepared in the embodiment 1 of the present invention is added at different concentrations, the cell survival rates are all above 100%, and the recombinant collagen has no cytotoxicity and good safety.

3. Cell proliferation assay

Human skin fibroblast (HFF-1) grown to 70-80% of the culture dish area is digested with 0.25% pancreatin, and the whole culture solution is used to prepare the cell with density of 1 × 10⁵Cell suspension per mL. 100. mu.L of the cell suspension was inoculated into a 96-well plate and placed at 37 ℃ in 5% CO₂Culturing in an incubator with saturated humidity. After 24h of cell culture, the complete culture medium was aspirated. The experimental group was added with 0.1mg/ml of the recombinant collagen solution prepared in example 1 diluted in high-glucose DMEM medium (4 multiple wells were set); the control group is prepared by adding equivalent amount of high-glucose DMEM medium without recombinant collagen into cells, and the blank group is prepared by adding equivalent amount of DMEM medium without cells, and continuing to maintain at 37 deg.C and 5% CO₂Culturing in an incubator with saturated humidity for 24h, 48h, 72h and 96h respectively. At intervals, 10. mu.L of CCK-8 reagent was added to each group, incubated in a cell incubator for 1-4h, and the absorbance (OD) of each well was measured at a wavelength of 450nm using an enzyme linked immunosorbent assay. The relative proliferation rate (RGR) of the cells was calculated from the absorbance mean of each group according to the following formula:

as shown in fig. 3, the relative proliferation rates of the cells cultured up to 24h, 48h, 72h and 96h in the experimental group added with the recombinant collagen prepared in example 1 of the present invention are all greater than 100% and tend to increase with time, compared with the blank control group (100%), which indicates that the recombinant collagen prepared in example 1 of the present invention can promote the proliferation of human skin fibroblasts.

4. Cell adhesion experiment

The recombinant collagen prepared in example 1 of the present invention was diluted to 0.1mg/mL with PBS, 24-well cell culture plates without TC treatment were selected, and 500. mu.L of each well of the well plate was treated with 1% heat-denatured BSA and 0.1mg/mL of the recombinant collagen prepared in example 1, and incubated in a refrigerator at 4 ℃ for 24 hours. Mouse fibroblast (L929) growing to 70-80% of the culture dish area is prepared into a cell density of 3 × 10 by using a high-sugar DMEM culture medium⁵Cell suspension per mL; sucking out the liquid in the 24-well plate, and adding 500 mu L of mouse fibroblast suspension into each well; after 5h, the effect of cell adhesion was observed by means of an inverted microscope.

The experimental result is shown in fig. 4, the fibroblasts growing in Bovine Serum Albumin (BSA) are in a round and non-spread state, while most of the fibroblasts growing in the recombinant collagen are in a spread and adhered state, which indicates that the recombinant collagen prepared in example 1 of the present invention has good cell adhesion performance.

5. Skin irritation test

(1) Procedure of experiment

The back hairs on both sides of the spine of the New Zealand white rabbit are shaved off, the area is about 10cm multiplied by 15cm, and the hair removal cream is applied for 1 to 2 minutes and then washed off. After the hair removal, four areas of the same area are divided on the back and marked with a marker pen. After 24 hours of depilation, a nonwoven fabric (2.5 cm. times.2.5 cm) impregnated with the recombinant collagen solution (1mg/ml) prepared in example 1 was applied flat to two areas of the back skin of each rabbit, and the other two areas were used as controls and fixed with a band-aid from which the drug portion was removed. After 4h, the band-aid was cut off, the nonwoven fabric was removed, and the test site was gently scrubbed with warm water. After removing the nonwoven fabric for 1 hour, the contact area of the four experimental and control areas on the back of each rabbit was observed for the formation of erythema or eschar. Scoring was done according to GB/T16886.10-2005 and erythema and eschar formation were recorded: no erythema, 0 point; very slight erythema (barely visible), 1 point; clear erythema, 2 points; moderate erythema, score 3; severe erythema (purplish red) to eschar formation, 4 points. Edema formation: no edema, score 0; very mild edema (barely visible), 1 point; clear edema (swelling, no excess of regional margin), 2 points; moderate edema (swelling of about 1mm), score 3; severe edema (swelling over 1mm and beyond the contact zone) was scored 4. The sum of the two scores is the total score. The product was evaluated for non-irritation according to GB/T16886.10-2005 evaluation criteria for irritation. In addition, erythema and edema at the contact site at each time point of 24h, 48h and 72h after nonwoven fabric removal were observed and recorded.

(2) Results of the experiment

Skin irritation response skin appearance map: observing four experimental and control areas on the back of each white rabbit after 1h, 24h, 48h and 72h of the non-woven fabric is removed, observing whether erythema or eschar is formed on the contact part, grading and recording according to a grading standard, wherein the result is shown in figure 5, the experimental part of the rabbit of each time point experimental group has no erythema or eschar formation and is similar to the skin state of the control part, and the score is recorded as 0 according to the grading standard; there was no edema, similar to the skin condition of the control site, and the score was 0 according to the score criteria.

Skin irritation response score: according to table 1, the dressing containing the recombinant collagen prepared in this example 1 was divided into 0 points on average for the dorsal skin irritation score of 4 new zealand rabbits; a score of 0, graded according to the intensity of skin irritation, indicates no abnormal change, i.e. no irritation, on the skin contact area.

TABLE 1 skin irritation score Table

The experiments show that the recombinant collagen prepared in the embodiment 1 is of a triple helical structure, has no cytotoxicity and irritation, and is good in safety; but also can promote the proliferation of human skin fibroblasts and has good cell adhesion performance.

Example 2 ultraviolet injury skin repair experiment

(1) Procedure of experiment

The back hairs on both sides of the spine of the New Zealand white rabbit are shaved off, the area is about 10cm multiplied by 15cm, and the hair removal cream is applied for 1 to 2 minutes and then washed off. After the hair removal, four areas of the same area are divided on the back and marked with a marker pen. After 24 hours of depilation, nonwoven fabrics (2.5 cm. times.2.5 cm) impregnated with the recombinant collagen solution (1mg/ml) prepared in example 1 were applied flat to two areas of the back skin of each rabbit, the other two areas served as controls, and were fixed with a band-aid having the drug portion removed. After 4h, the band-aid was cut off, the nonwoven fabric was removed, and the test site was gently scrubbed with warm water. After removing the nonwoven fabric for 1 hour, the contact area of the four experimental and control areas on the back of each rabbit was observed for the formation of erythema or eschar. Scoring was performed according to GB/T16886.10-2005 and erythema and eschar formation were recorded: no erythema, 0 point; very slight erythema (barely visible), 1 point; clear erythema, 2 points; moderate erythema, score 3; severe erythema (purplish red) to eschar formation, 4 points. Edema formation: no edema, score 0; very mild edema (barely visible), 1 point; clear edema (swelling, no excess of regional margin), 2 points; moderate edema (swelling of about 1mm), score 3; severe edema (swelling over 1mm and beyond the contact zone) was scored 4. The sum of the two scores is the total score. The product was evaluated for non-irritation according to GB/T16886.10-2005 evaluation criteria for irritation. In addition, erythema and edema at the contact site at each time point of 24h, 48h and 72h after nonwoven fabric removal were observed and recorded.

(2) Results of the experiment

Skin irritation response score: according to table 2, the dressing containing the recombinant collagen prepared in this example 1 was divided into 0 points on average for the dorsal skin irritation score of 4 new zealand rabbits; a score of 0, graded according to the intensity of skin irritation, indicates no abnormal change, i.e. no irritation, on the skin contact area.

TABLE 2 skin irritation score Table

Example 2 ultraviolet injury skin repair experiment

1. Procedure of experiment

30 healthy adult mice of SPF grade, female, with a body weight of 27 + -2 g were selected. The mice were shaved on their backs with a hair clipper and depilated with depilatory cream in an area of approximately 2 x 4 cm. Mice were randomized into 6 groups: blank group, model group, experimental group (recombinant collagen solution prepared in example 1, concentration of 1.5mg/ml), control group 1 (commercial fish collagen solution, concentration of 1.5mg/ml), control group 2 (commercial pig collagen solution, concentration of 1.5mg/ml), control group 3 (commercial bovine collagen solution, collagen concentration of 1.5 mg/ml). Wherein the blank group was not treated except for depilatory; the model set does not undergo any repair process.

Establishing an ultraviolet injury model of the hairless mouse: irradiating model group and experimental group mice by UVA (wavelength: 320-440nm) and UVB (280-320nm) ultraviolet lamp tubes with continuous wavelengths, quantifying irradiation dose by an ultraviolet radiometer, wherein the total irradiation amount of UVA and UVB is 100mJ/cm²Can cause acute skin inflammation of the skin on the back of the mouse.

And (3) treatment: after 1h of irradiation, the damaged skin of the mice in the corresponding experimental group and the control group was smeared with the recombinant collagen solution, the commercially available fish collagen solution, the commercially available pig collagen solution and the commercially available bovine collagen solution prepared in example 1 of the present invention, respectively, at a dose of 4 ml/day. On

treatment days

2 and 4, mice are dislocated and killed, back skin is taken, and skin tissues are immediately frozen; a portion of the harvested skin tissue was fixed with 10% neutral formaldehyde for 48 h. The fixed skin tissue is taken, soaked in wax, dehydrated, transparent and embedded, and then paraffin section is carried out, wherein the section thickness is 3.5 mu m.

In order to observe the difference in the histology of the skin of the mice before and after the treatment, HE staining was performed on the skin sections of the mice on the 2 nd and 4 th days of the treatment to observe the morphology of the nucleus and epidermis, and Masson staining was performed on the skin sections to observe the morphological change of the collagen fiber.

HE staining: dewaxing and dehydrating the paraffin sections, then carrying out HE (high intensity intrinsic) staining, carrying out water immersion, staining with hematoxylin for 5-8min, and washing with running water for 1 min; differentiating the differentiation liquid for 1-3s, and washing for 1 min; returning the blue liquid to blue for 30-60 s; soaking in 95% ethanol for alcoholization for 1 min; dip-dyeing with alcohol-soluble eosin for 5-8 s; soaking in 95% ethanol I, II for 2-3s each; soaking in 100% ethanol I, II, and dehydrating for 1 min; drying by using an electric hair drier; the xylene is transparent for 5-15 min; sealing the neutral gum, drying at room temperature, and taking a picture.

Masson staining: dewaxing and dehydrating the paraffin section, performing Masson trichrome staining, staining for 5-10min by Weigert hematoxylin, and washing; differentiating the differentiation liquid for 5-15s, and washing with water; returning blue liquid to blue for 1-5min, and washing with water; dyeing ponceau fuchsin for 5-10 min; weak acid washing for 1 min; washing with phosphomolybdic acid solution for 1-2 min; weak acid washing for 1 min; dyeing for 2min by aniline blue; quickly dehydrating by 95% ethanol; absolute ethyl alcohol I, II and III are respectively 5-10 s; xylene I, II and III are respectively 1-2 min; and (5) sealing by using neutral gum.

2. Results of the experiment

2.1 HE staining of mouse skin

The blank mice were not irradiated with ultraviolet light and treated except for depilating, and as can be seen from fig. 6, on day 2 after depilating, the lower layer of the epidermal layer of the skin of the blank mice was observed to undulate in a wavy manner, wherein the epidermis grew downward to form a skin process; the skin of the back of the mouse is thin, the granular layer and the transparent layer are not obvious, and the basal layer and the spinous layer can be seen; the dermis is mainly composed of a papillary layer and a reticular layer, no obvious boundary exists between the papillary layer and the reticular layer, the fibers are compact and ordered, the shape of the cell nucleus is good, inflammatory cells are not generated, and the shape of the accessory is good. On day 4, the skin appendages of the mice in the blank group extended from the epidermis to the dermis, the fibers of the dermis were continuous and orderly arranged, the morphology of the cell nucleus was good, there were no inflammatory cells, and the morphology of subcutaneous fat and reticular fibers was good. As can be seen, the skin of the blank group of mice was entirely healthy on

days

2 and 4.

The mice of the model group after ultraviolet irradiation were not treated, and as can be seen from fig. 6, HE staining results of the skin on day 2 after ultraviolet irradiation showed that the skin of the mice had damaged the whole epidermis, edema of the local basal layer, and loss of the local structure; the dermal layer vessels dilated and congested, the reticular layer was loose and edematous, and a large amount of inflammatory cell infiltration was observed. On day 4, the epidermal structure of the skin of the mouse is more complete than that of the skin of the mouse on the next day, but the edema is thickened, the fibers of the dermal layer are obviously thickened and are more loose, the matrix among the fibers is increased, and a large amount of inflammatory cells are infiltrated. It can be seen that, in the absence of any treatment, on the fourth day after the uv light injury, the mice show a continuous increase in skin edema, a continuous increase in inflammatory cell infiltration, and an increase in the degree of injury.

Mice in the experimental group were treated with the recombinant collagen solution (1.5mg/ml, 4 ml/day) prepared in example 1 of the present invention by applying it 1h after the UV irradiation. As can be seen from fig. 6, after the experimental group was treated with the recombinant collagen solution prepared in example 1 of the present invention on day 2, the epidermal layer of the mouse was edematous and thickened, and had inflammatory cell infiltration; by day 4, the epidermal edema subsided, the dermal structure was intact, the collagen fibers were closely arranged, and the adnexa and subcutaneous fat morphology were good. As can be seen from the comparison of model groups, the recombinant collagen solution prepared in the embodiment 1 of the invention has a good effect on repairing the skin damaged by ultraviolet light, and the damaged skin of the mice recovers the fourth day after the damage, and the shape of the skin is similar to that of the skin of the healthy mice in the blank group.

Mice in control group 1 were treated with a commercially available fish collagen solution (1.5mg/ml, 4 ml/day) 1h after UV irradiation. As can be seen from FIG. 7, the epidermal layer of the mice is seriously damaged by the treatment of the 2 nd day, the dermal layer is loose and edema, inflammatory cell infiltration and blood vessel hyperemia and expansion are obvious. On day 4, the epidermis structure is complete but local basal layer is edematous, acanthosis is loosened, dermis layer is loose and edematous, fiber is thickened, stroma is increased, the number of accessory devices is obviously reduced, hair follicles are seriously damaged, and inflammatory cells are infiltrated. The results showed that the repair effect was not significant in control 1.

Mice in control group 2 were treated with a commercially available bovine collagen solution (1.5mg/ml, 4 ml/day) 1h after UV irradiation. As can be seen from fig. 7, by day 2, the mice had significant epidermal lesions, incomplete structures, partial exfoliation, edema of the dermis, thickening and breaking of the fibers, infiltration of inflammatory cells, damaged hair follicles (atrophy and disappearance of the fibrous sheath), vasodilation and congestion. On day 4, the epidermal structure of the skin of the mouse is complete but discontinuous compared with that on the next day, and the part of the skin is broken; edema of the dermis layer, thickening and breaking of fibers are more obvious, and blood vessels are dilated and congested; the number of accessories is significantly reduced. The results showed that the repair effect was not significant in control 2.

Mice in control group 3 were treated with a commercially available porcine collagen solution (1.5mg/ml, 4 ml/day) 1h after UV irradiation. As can be seen from fig. 7, by day 2 of treatment, the epidermal layer of the mice was significantly damaged, the structure was incomplete, and the local basal layer was damaged; loose edema of the dermis, infiltration of inflammatory cells, damaged hair follicles (atrophy or even disappearance of the fibrous sheath). On day 4, the epidermis is intact but partially defective, damaging the basal lamina, accompanied by acantholysis; the dermis is loose and edematous, the fiber is thickened, and the stroma is increased; the number of the accessory organs is obviously reduced, hair follicles are seriously damaged, and inflammatory cells infiltrate. The results showed that the repair effect was not significant in control group 3.

Therefore, the recombinant collagen prepared in the embodiment 1 of the invention has a remarkable repairing effect on the skin of the mouse with ultraviolet injury, and the repairing effect is superior to that of commercially available fish collagen, commercially available bovine collagen and commercially available pig collagen.

2.2 Masson staining of mouse skin

The blank group of mice was not exposed to uv irradiation and treatment except for hair removal. As can be seen from fig. 8, collagen fibers in the dermal papilla layer of the skin of the blank group of mice were fine and interwoven into a web on day 2; in addition, the hair-sebaceous gland unit, eccrine sweat gland and apocrine sweat gland are all surrounded by the thin reticular collagen, and the blood vessel is also surrounded by the thin layer of the thin collagen fiber; the collagen fibers of the reticular layer are the largest constituent of the dermis, and the collagen fibers therebetween form a coarse fiber bundle, which is slightly wavy in longitudinal section and extends in all directions in a horizontal plane, with fibroblasts interspersed among the collagen fibers. On day 4, the collagen fibers of the dermis layer of the skin of the mouse are compact and continuous, and the number and the shape of the accessories are good and are surrounded by the collagen fibers; the collagen fibers form relatively uniform and thick fiber bundles, which are slightly wavy in longitudinal section and extend in all directions in a horizontal plane, and fibroblasts are scattered among the collagen fibers. As can be seen, the collagen fibers and the appendages in the dermis layer of the skin of the blank mice on

days

2 and 4 were in good and healthy states.

Mice in the model group after uv irradiation were not treated. As shown in fig. 8, the collagen fibers in the dermis of the skin of the mice in the ultraviolet irradiation model group on day 2 were disorganized, and were significantly reduced, broken, crushed, thickened, twisted and gathered into a mass; collagen fibers in the dermis are coarser at day 4, and aggregate into larger collagen bundles, and blood vessels in the dermis dilate. It can be seen that the damage to collagen fibers in the skin of mice continued to worsen at the fourth day after the uv light damage without any treatment.

Mice in the experimental group were treated with the recombinant collagen solution (1.5mg/ml, 4 ml/day) prepared in example 1 of the present invention by applying it 1h after the UV irradiation. As shown in fig. 8, the collagen fibers in the dermis layer of the skin of the mouse are disorganized by the treatment day 2, and the collagen fibers are obviously reduced and are broken, crushed and curled; the collagen fibers of the dermis layer are obviously increased on the 4 th day, the arrangement is compact and orderly, the shape of the attachments is normal, the continuous collagen fibers are slightly wavy, and fibroblasts are distributed among the collagen fibers. Compared with the model group, the recombinant collagen solution has good repairing effect on collagen fibers of the dermis layer of the ultraviolet injury skin, and the collagen fibers are recovered to be in a health state similar to that of the blank group.

Mice in control group 1 were treated with a commercially available fish collagen solution (1.5mg/ml, 4 ml/day) 1h after UV irradiation. As can be seen in fig. 9, the collagen fibers in the dermis were significantly reduced, broken, kinked, aggregated, and dilated by blood vessel congestion by day 2; at the fourth day, the collagen fibers in the dermis layer are broken, aggregated into blocks, arranged loosely and infiltrated by inflammatory cells. The results showed that the repair effect was not significant in control 1.

Mice in control group 2 were treated with a commercially available bovine collagen solution (1.5mg/ml, 4 ml/day) 1h after UV irradiation. As can be seen in fig. 9, by day 2 of treatment, collagen fibers in the dermis layer were significantly reduced, broken, kinked, aggregated, and blood vessels were dilated and engorged with blood; at day 4, collagen fibers in the dermis layer were broken, aggregated into clumps, loose arrangement, blood vessel hyperemia and dilatation, and inflammatory cell infiltration. The results showed that the repair effect was not significant in control 2.

Mice in control group 3 were treated with a commercially available porcine collagen solution (1.5mg/ml, 4 ml/day) 1h after UV irradiation. As can be seen in fig. 9, collagen fibers in the dermis were significantly reduced, broken, distorted and aggregated, and inflammatory cell infiltration on day 2 of treatment; at the fourth day, the collagen fibers in the dermis are broken, aggregated and loosely arranged. The results showed that the repair effect was not significant in control group 3.

Therefore, the recombinant collagen provided by the invention has an obvious repairing effect on the skin of a mouse with ultraviolet injury, and the effect is superior to that of the collagen of commercially available fish, cattle and pig.

In conclusion, the recombinant collagen provided by the invention has a remarkable repairing effect on the skin of a mouse with ultraviolet injury, can promote the recovery of collagen fibers in the dermis of the skin with ultraviolet injury, and has an effect remarkably superior to that of various commercially available collagen products.

Claims

1. The application of the recombinant collagen in preparing products for repairing skin photodamage or preventing skin photodamage; the amino acid sequence of the recombinant collagen is shown as follows:

2. the use of claim 1, wherein the photodamage is ultraviolet damage.

3. Use according to claim 1, wherein the products for repairing photodamage to skin comprise cosmetics, cosmeceutical products, pharmaceuticals.

4. The use according to claim 3, wherein the cosmetic comprises a stock solution, an emulsion, an essence cream, a toner, a conditioning water, a cream, a mask, a makeup cream, an eye cream, a spray, a sunscreen cream, a foundation.

5. Use according to claim 3, wherein the medical cosmetic product comprises a stock solution, a dressing, a cold pack, a gel, a sponge, a film, a hydro-acupuncture, an implant, a milk, a cream, a paste.

6. Use according to claim 1, wherein the products for preventing photodamage to the skin comprise cosmetics, cosmeceutical products, pharmaceuticals.

7. The use according to claim 6, wherein the cosmetic comprises a stock solution, an emulsion, an essence cream, a toner, a conditioning water, a cream, a mask, a makeup cream, an eye cream, a spray, a sunscreen cream, a foundation.

8. Use according to claim 6, wherein the medical cosmetic product comprises a stock solution, a dressing, a cold pack, a gel, a sponge, a film, a hydro-acupuncture, an implant, a milk, a cream, a paste.