CN115252763A - REG3A-DGTM photosensitive hydrogel composition and application thereof in inducing epidermis to regenerate and repair wound surface - Google Patents

Abstract

The invention discloses a photosensitive hydrogel composition for promoting wound surface rapid healing and application thereof, and a method for inducing large-area skin wound surface epidermis in-situ regeneration to promote wound surface rapid healing, wherein photosensitive hydrogel with excellent biocompatibility, pancreatic regeneration-derived protein REG3A and DGTM factor for inducing wound surface epidermis regeneration are combined to form REG3A-DGTM photosensitive hydrogel. After the combined photosensitive hydrogel is smeared on the surface of a large-area wound surface, the wound surface is quickly sealed (10-30 seconds) under the regulation and control of light, REG3A and DGTM factors are slowly released to the wound surface, REG3A prevents wound infection by inhibiting bacterial growth and controls the expression of inflammatory factors to improve the wound microenvironment, and DGTM induces mesenchymal stem cells to be reprogrammed into epidermal keratinocytes, so that the large-area wound surface can be regenerated and repaired in situ without debridement and surgical skin grafting.

Description

REG3A-DGTM photosensitive hydrogel composition and application thereof in inducing epidermis to regenerate and repair wound surface

Technical Field

The invention relates to the fields of biochemistry, molecular biology, immunology, material biology and dermatology, and relates to a photosensitive hydrogel composition for promoting rapid healing of a wound surface, and application and a preparation method thereof.

Background

The skin is the largest organ of the human body and mainly plays a role in protecting the body, perspiring, feeling cold and hot and bearing pressure. The skin of human and higher animals is composed of three layers, epidermis, dermis, and subcutaneous tissue. The skin has a barrier effect: on one hand, the loss of water, electrolyte and the like in the body is prevented; on the other hand, the utility model can prevent the invasion of harmful substances outside and protect the organism from ultraviolet rays, chemical substances, allergens and microorganisms outside, thus forming a natural barrier to protect the host. Meanwhile, the body hair in the skin can maintain the body temperature, and the sweat can expel toxin and maintain the internal environment balance of body fluid. Subcutaneous adipose tissue can maintain body temperature and secrete growth factors and cytokines [ 1 ].

Loss of integrity of most skin due to injury or disease can lead to severe disability. Extensive skin damage, if not repaired in a timely manner, can cause a variety of complications, which can be life threatening in the severe cases. For example, extensive damage to the skin can result in a large amount of fluid loss, which can lead to an imbalance in the human body's internal environment, leading to organ failure. In addition, loss of the skin barrier leads to the body being more susceptible to infection by external microbes, leading to sepsis [ 2 ]. Moreover, excessive inflammation at skin wounds can also lead to a decrease in the rate of skin wound repair, and scars are more likely to appear [ 3 ]. In addition to acute wounds, chronic skin wounds such as pressure ulcers and diabetic foot ulcers are also on the steady rise. According to statistics, the U.S. costs for skin treatment in 2013 reach $ 750 billion, while the proportion of patients who die due to burns or other causes that damage the large area skin barrier reaches 15% [ 4 ]. Therefore, it is important to find a way to seal wounds in time after the appearance of large skin wounds to control fluid loss and prevent wound infection and inflammation.

Wound healing is an orderly process of change. However, in the healing of large-area infectious skin defects, secondary retreatment, namely skin grafting or suturing, is often needed to cure the large-area infectious skin defects [ 5 ]. There are many new materials currently used in the treatment of wounds, among which hydrogels [ 6 ]. A photoinduced imine crosslinked hydrogel system is a novel photosensitive hydrogel system, wherein aldehyde groups are generated on hyaluronic acid HA-NB grafted with o-nitrobenzyl alcohol (NB) photoresponse groups under 395nm illumination, and then the aldehyde groups react with amino-containing materials (such as chitosan, gelatin, polylysine, protein, platelet-rich plasma PRP and the like) and amino groups on the surface of biological tissues to form imine bonds, so that chemical bond fixation of hydrogel and biological tissues at the action part is realized while the hydrogel is constructed in situ [ 7 ]. The method has excellent operability and gelling controllability of the photo-crosslinking hydrogel. In the process of wound repair, newly-born cells and tissues can be fully migrated and permeated into hydrogel, the function of a hydrogel scaffold material is better exerted, and the healing process of the wound is accelerated, so that the photoinduced imine cross-linked hydrogel has important clinical transformation value [ 8 ].

In addition, wound infection is a major challenge in the care of large wounds and is also the most common cause of death after trauma. Antibiotics are currently used primarily to inhibit microbial infections during anti-infection procedures. However, the use of antibiotics can lead to the development of resistant bacteria and fungi, thereby posing a significant challenge in the treatment of skin lesions [ 7 ]. The invention finds that the antibacterial protein islet regeneration source protein (Reg 3A) not only has the function of inhibiting bacterial growth [ 10 ], but also can promote cell proliferation [ 11 ] and inhibit wound inflammation [ 12 ]. However, it is not known whether REG3A (human)/Reg III γ (murine) mixed with hydrogel can play multiple roles of rapidly closing a wound, promoting wound healing, preventing wound infection, and controlling wound inflammation.

In addition to the primary treatments of hemostasis, wound closure, inflammation control, and infection prevention, large areas of skin burn wounds require a secondary treatment, namely skin grafting, to restore an intact skin barrier. The source of the skin graft is mainly skin of other parts of a human body or skin of other animals, such as pig skin and the like, and the skin graft is transplanted to a human body and easily causes the same problem as other types of organ transplantation, namely immunological rejection. Since patients with extensive burns and wounds often have a degree of immune dysfunction, the risk of infection increases and immunosuppressive therapy is undesirable. In 2018, kurita in japan reported a result of their study: after four transcription factors DGTM (DNP 63 alpha, GRHL2, TFAP-2 alpha and C-Myc) are transferred into the mouse mesenchymal stem cells, the mesenchymal stem cells can be successfully induced to be differentiated into epithelioid cells. The cells were identified to have morphology and characteristics similar to mouse keratinocytes and to be able to form 3D skin in vitro [ 13 ]. Meanwhile, the lentivirus supernatant with four transcription factors of DGTM is added into the wounds of mice [ 14 ], so that the skin ulcer surfaces of the mice can be quickly and effectively epithelialized. However, it is completely unknown whether the combined use of DGTM and REG3A/Reg III gamma, hydrogel can promote DGTM to induce the reprogramming of mesenchymal stem cells into keratinocytes in the wound by rapidly closing the wound to prevent the loss of body fluid, the infection of the wound and controlling the inflammation of the wound, so as to regenerate the epidermis in situ to repair the large-area burn wound in situ without surgical skin grafting. Therefore, the patent uses the combination of DGTM, REG3A and photosensitive hydrogel to achieve the purpose of rapidly repairing the large-area burn wound surface without debridement and skin grafting.

Disclosure of Invention

The invention relates to the fields of biochemistry, molecular biology, immunology, material biology and dermatology, and relates to a method for promoting in-situ regeneration and rapid healing of epidermis of large skin wound by combining photosensitive hydrogel, REG3A protein with bactericidal and anti-inflammatory effects, and four factors of DNP63A (D), GRHL2 (G), TFAP2A (T) and MYC (M) (also called DNP63 alpha, GRHL2, TFAP2 alpha and C-Myc).

Aiming at the problem that the large area of the ulcer wound surface is difficult to regenerate and repair in situ so as to endanger the life of a patient, the invention plans to utilize photosensitive hydrogel which has excellent biocompatibility and is easy to control to quickly seal the wound, and uses the combination of pancreatic regeneration source protein REG3A with the functions of diminishing inflammation, sterilizing and promoting the repair of the wound surface and four transcription factors DGTM (DNP 63 alpha, GRHL2, TFAP2 alpha and C-Myc) capable of inducing the regeneration of the wound surface skin to induce the in-situ regeneration of the epidermis, thereby realizing the purpose of quickly repairing the large area of the wound surface without debridement and surgical skin grafting.

The invention provides a REG 3A-photosensitive hydrogel, which comprises the photosensitive hydrogel and a REG3A protein.

The invention relates to a novel photoinduced imine crosslinking hydrogel system, in the hydrogel system, aldehyde group is generated by hyaluronic acid HA-NB grafted with an o-nitrobenzyl alcohol (NB) photoresponse group under 395nm illumination, and then the aldehyde group reacts with amino-containing materials (such as chitosan, gelatin, polylysine, protein, platelet-rich plasma PRP and the like) and amino on the surface of biological tissue to form imine bond, so that chemical bond fixation between hydrogel and biological tissue at the action site is realized while in-situ construction of hydrogel.

The invention also provides a preparation method of the REG 3A-photosensitive hydrogel, and the preparation method of the REG 3A-photosensitive hydrogel comprises the steps of mixing the REG3A protein and the photosensitive hydrogel, incubating the mixture on a wound surface, and irradiating the wound surface for 10-30s by using an ultraviolet lamp for solidification.

In the invention, the REG3A protein is a protein with biological activities of inhibiting bacterial growth and wound inflammation and promoting keratinocyte proliferation, the homologous protein of the REG3A protein in a mouse is RegIII gamma, the amino acid sequence is shown as SEQ ID NO.1 and 2, and the nucleotide sequence is shown as SEQ ID NO.3 and 4.

Specifically, the RegIII gamma amino acid sequence of the mouse is shown as (SEQ ID NO. 1); the amino acid sequence of human REG3A is shown as (SEQ ID NO. 2); the RegIII gamma nucleotide sequence of the mouse is shown as (SEQ ID NO. 3); the nucleotide sequence of human REG3A is shown in (SEQ ID NO. 4).

The invention provides a method for reprogramming mesenchymal stem cells at a wound into keratinocytes, which comprises the steps of transferring four transcription factors DGTM into the mesenchymal stem cells of a mouse by using lentivirus infection, and then inducing by using F-medium to obtain epithelioid cells. The cells were identified to have morphology and characteristics similar to mouse keratinocytes and to differentiate into stratified epidermis in vitro.

The present invention also provides a REG3A-DGTM photosensitive hydrogel composition comprising the REG 3A-photosensitive hydrogel as described above and DGTM factors for inducing regeneration of wound surface epidermis. Wherein, the DGTM factor refers to four transcription factors of DNP63 alpha, GRHL2, TFAP-2 alpha and C-Myc, the amino acid sequence of the DGTM factor is shown in SEQ ID NO.5, 6, 7 and 8, and the nucleotide sequence thereof is shown in SEQ ID NO.9, 10, 11 and 12.

Specifically, the amino acid sequence of DNP63 alpha is shown as (SEQ ID NO. 5);

the nucleotide sequence of DNP63 alpha is shown as (SEQ ID NO. 9);

the amino acid sequence of GRHL2 is shown as (SEQ ID NO. 6);

the nucleotide sequence of GRHL2 is shown as (SEQ ID NO. 10);

the amino acid sequence of TFAP2a is shown as (SEQ ID NO. 7);

the nucleotide sequence of TFAP2a is shown as (SEQ ID NO. 11);

the amino acid sequence of C-Myc is shown as (SEQ ID NO. 8);

the nucleotide sequence of C-Myc is shown as (SEQ ID NO. 12).

The DGTM factors (DNP 63 alpha, GRHL2, TFAP-2 alpha, C-Myc) induce reprogramming of mesenchymal cells into keratinocytes.

The invention also provides application of the REG3A-DGTM photosensitive hydrogel in preparing a medicament for inhibiting cytokine production in wounds.

Wherein the cytokines comprise TNF alpha and IL-6.

The present invention also provides a pharmaceutical composition or a reagent or a kit containing the REG 3A-photosensitive hydrogel, and/or DGTM factor, as described above, and/or the REG3A-DGTM photosensitive hydrogel composition, as described above.

The invention provides application of the REG 3A-photosensitive hydrogel, the REG3A-DGTM photosensitive hydrogel composition, the pharmaceutical composition or the reagent or the kit in preparation of drugs for treating large-area skin wounds, drugs for inhibiting generation of wound surface cytokines and drugs for promoting proliferation of keratinocytes.

The invention also provides methods for preparing the REG 3A-photosensitive hydrogel, the REG3A-DGTM photosensitive hydrogel composition, the pharmaceutical composition or the reagent or the kit.

The present invention also provides REG 3A-photosensitive hydrogel as described above, REG3A-DGTM photosensitive hydrogel composition as described above, pharmaceutical composition or reagent or kit as described above, in a method for promoting healing of large area skin wounds, administering REG 3A-photosensitive hydrogel and/or REG3A-DGTM photosensitive hydrogel composition to a subject individual.

The beneficial effects of the invention include: the invention provides a novel treatment method for in-situ repair of large-area skin burn wound surface by combining photosensitive hydrogel capable of rapidly closing the wound, REG3A protein capable of promoting keratinocyte proliferation, inhibiting bacterial growth and controlling wound inflammation and DGTM induction technology.

Specifically, the method comprises the following steps:

(1) Vector construction: uses lentivirus vector pLVX-IRES-zsGREEN vector which can be used for eukaryotic cell gene overexpression as construction vector, connects DNP63 alpha, GRHL2, TFAP2 alpha and C-Myc gene segments cloned from keratinocyte into the vector, and carries out sequencing on the constructed plasmid to determine the plasmid is correct and then is used for later use.

(2) And (3) slow virus packaging: and co-transferring the constructed lentivirus vector and lentivirus assembly vectors PSPax2 and PMD2.G into HEK293T cells by using PEI. Cell culture supernatants were collected 48 hours after transfection. The resulting lentivirus supernatant was filtered through a 0.45um filter for use.

(3) And (3) slow virus concentration: 5XPEG8000 concentrated solution is added into the packaged lentivirus supernatant, and the mixture is placed in a 4-degree refrigerator for standing overnight after being shaken uniformly. The next day, the supernatant was discarded after centrifugation at 4500rpm at 4 degrees for 30 minutes using a refrigerated centrifuge, and viral particles were resuspended at 1/100 of the stock volume to give a 100-fold concentrated viral suspension.

(4) DGTM lentivirus induces differentiation of mesenchymal stem cells into keratinocytes:

taking a pregnant mouse which is pregnant for 13.5 days, separating embryonic fibroblast of the pregnant mouse, and culturing to a P3 generation postspecies 24-well plate. The original medium was discarded, and four-factor lentiviral supernatants were added to each well of a 24-well plate in a ratio of dg: T: M = 1. The virus-containing medium was discarded the next day and replaced with fresh DMEM medium. Fluid was changed continuously until day 3 post viral infection. The culture solution was changed to a keratinocyte-inducing Medium F-Medium (3, [ 1 ], [ v/v ] F-12 ], [ ham ] -DMEM,5% FBS, 0.4. Mu.g/ml hydrocortisone, 5. Mu.g/ml insulin,8.4ng/ml cholera toxin,10ng/ml EGF, 24. Mu.g/ml adenin, 100U/ml penicilin, 100. Mu.g/ml streptomycin) on day D3, the solution was changed daily, morphological change of the cells was observed, and cellular RNA was collected to detect marker expression of the keratinocyte.

(5) DGTM-induced differentiation of keratinocytes in vitro into the epidermis: first, a dermal equivalent was constructed: a piece of human foreskin with the size of 2cmx2cm is taken, thoroughly cleaned, soaked in a 1M NaCl solution in an incubator at 37 ℃ overnight, the epidermis on the foreskin is removed the next day, the remaining part of the foreskin is soaked in DMEM containing 400U/Ml P/S for minutes, then soaked in DMEM containing 400U/Ml P/S for 30 minutes, and finally the dermis is soaked in DMEM containing 50U/Ml P/S for 6 days. After loading the dermal equivalent on an aluminum holder, a plastic ring (diameter 1 cm) was placed on top and the resulting epidermal progenitors were induced by resuspension in an F-medium containing Y27632, seeded at a cell density of 4X 10 in the ring⁵Per cm². Ca of the culture Medium in the first 6 days of culture²⁺The concentration was gradually increased to 1.8mM. On day 7, the volume of medium in the loop was reduced to the level of the epithelial cell sheet, allowing the epithelial cells to grow at the gas-liquid interface. On day 14, the epidermis formation was examined histologically.

(6) REG3A/RegIII gamma protein purification: streaking escherichia coli BL21 which is preserved at-80 ℃ and contains the over-expression REG3A plasmid on a TSB culture plate, selecting a single colony on the next day, inoculating the single colony in a TSB liquid culture medium for overnight culture, and transferring the overnight culture into a fresh TSB culture medium for continuous culture; when OD600 is 0.6-0.8, adding isopropyl thiogalactoside (IPTG) for induction expression, collecting induced thallus, and performing bacteria breaking and washing; and respectively taking samples of the supernatant before and after induction, the bacteria-breaking supernatant and the precipitate and the washed supernatant precipitate to carry out SDS-PAGE to detect the expression condition of the target protein. After the inclusion bodies are dissolved, renaturation and dialysis of protein are carried out, after filtration sterilization by a filter membrane, the inclusion bodies are put on a cation column (Capto SP ImpRes cation column), naCl solutions with different concentrations are used as elution buffer for elution, and the elution condition of the target protein is detected by collecting the eluate and carrying out SDS-PAGE. Collecting elution samples according to the display result, dialyzing, ultrafiltering and concentrating, collecting samples before and after ultrafiltration, carrying out SDS-PAGE to detect the expression condition of the target protein, and subpackaging the rest samples and freezing at-80 ℃ for later use.

(7) DGTM-REG3A photosensitive hydrogels promoted large area wound healing in mice: counting each virus particle to 10⁹The required volume of the virus suspension is obtained by dripping the virus suspension on the wound part of the mouse, mixing 100ug Reg III gamma with 100ul hydrogel and smearing the mixture on the wound part to seal the wound. After treatment, the dorsal wound of the mice was photographed daily until the wound was completely healed. The wound healing rate was counted using Excel treatment after analysis of the mouse back wound size using Image J, and the analysis results were plotted in GraphPadPrism 8.3.

Drawings

FIG. 1 shows that the optimal MOI of DNP63A, GRHL2, TFAP2A and C-Myc for lentivirus infection of MEF are 1.

Fig. 2 shows that the optimal combination ratio of DGTM lentivirus-induced MEFs to differentiate into keratinocytes is 1.

FIG. 3 shows that the induction of DGTM into MEF cells using lentivirus resulted in the appearance of cells with similar morphology to keratinocytes at different days.

FIG. 4 shows the successful overexpression of DGTM transcription factors after transfection of DGTM factors into MEF cells using lentiviruses.

FIG. 5 shows that the expression of the marker K8\ K18 of ectodermal cells and the marker protein K14\ CDH1 of keratinocytes increases with the induction time after transfection of DGTM factors into MEF cells using lentiviruses.

FIG. 6 shows that the keratinocytes induced by DGTM are able to differentiate into epidermal layers in the mouse dermis.

FIG. 7 shows the purification of Reg III γ.

FIG. 8 shows that Reg III γ inhibits the expression of poly (I: C) -induced inflammatory factors IL-6, TNF- α in keratinocytes induced by DGTM.

FIG. 9 shows that Reg III gamma promotes the increase in expression of a keratinocyte proliferation-associated gene PCNA induced by DGTM.

FIG. 10 shows the size of the wound area and the statistical rate of wound healing in mice treated with various combinations of photoactivated hydrogel + RegIIIγ + DGTM, showing that the combination of photoactivated hydrogel + RegIIIγ + DGTM significantly promotes wound healing in large areas.

FIG. 11 shows the reduction of the expression of the inflammatory factors Il-6, tnf-alpha in the mouse wound using various combinations of the photosensitive hydrogel + Reg III gamma + DGTM.

The specific implementation mode is as follows:

the present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1: optimal MOI detection for viral transfection

Taking pregnant mouse 13.5 days of pregnancy, separating embryo fibroblast of fetal mouse, culturing to obtain P3 generation postspecies 24-well plate with cell density of 4000-5000/cm². The next day, the 24-well plates were removed, the original medium of the cells was discarded, and the volume of the medium was adjusted to 1: 10. 1: 100. 1:200 cells to virus ratio, and the next day after overnight cell infection the virus-containing medium was discarded and replaced with fresh DMEM medium. Continuing to culture until 48At h, the change in expression of green fluorescence was observed under a fluorescence microscope.

The experimental results show that as shown in fig. 1, the optimal MOI of four genes including DNP63a, GRHL2, TFAP2a and C-Myc lentivirus infected MEF are 1.

Example 2: the optimal combination ratio of DGTM lentivirus to induce differentiation of MEFs into keratinocytes is 1.

Taking pregnant mice 13.5 days of pregnancy, separating embryonic fibroblasts of the pregnant mice, and culturing the embryonic fibroblasts until P3 generation offspring 24-well plates. The cell-original culture medium was discarded, and the following ratios of 1. The virus-containing medium was discarded the next day and replaced with fresh DMEM medium. Fluid was changed continuously until day 3 post viral infection. RNA samples were collected on day 4, and the expression of the keratinocyte marker gene Keratin14 and the fibroblast marker gene Fsp1 were detected by RT-PCR (FIG. 2). The experimental results show that different ratios of DGTM induced day 4, there was not much difference in Fsp1 expression in the cells, but the expression of K14 was significantly increased. Wherein the DGTM ratio is in the range of 1. This suggests that Tfap2a may inhibit keratinocyte proliferation, so we finally believe that DGTM is the most suitable combination for inducing differentiation of MEF into keratinocytes at 2.

Example 3: DGTM induces mouse embryo fibroblast to differentiate into keratinocyte

Taking pregnant mice 13.5 days of pregnancy, separating embryonic fibroblasts of the pregnant mice, and culturing the embryonic fibroblasts until P3 generation offspring 24-well plates. The original medium was discarded, and four-factor lentiviral supernatants were added to each well of a 24-well plate in a ratio of dg: T: M = 1. The virus-containing medium was discarded the next day and replaced with fresh DMEM medium. Fluid was changed continuously until day 3 post viral infection. The culture solution was changed to a keratinocyte-inducing Medium F-Medium (3, [ 1, [ v/v ] F-12 ], [ ham ] -DMEM,5% FBS, 0.4. Mu.g/ml hydrocortisone, 5. Mu.g/ml insulin,8.4ng/ml cholera toxin,10ng/ml EGF, 24. Mu.g/ml adenin, 100U/ml penicillin, 100. Mu.g/ml streptomycin) on day D3, the solution was changed every day, the morphological change of the cells was observed, and cellular RNA was collected to detect marker expression of keratinocytes.

The results of the experiments show that after 5 days of induction, cells with a morphology similar to that of keratinocytes were observed (FIG. 3) and that fluorescence emission from these cells could be detected, indicating that DGTM was successfully expressed in the cells. The present invention also detected that the expression of four genes, dnp63a, grhl2, tap 2a, C-Myc, were all significantly increased on day D3 by RT-PCR (FIG. 4).

Simultaneously, RNA from cells at D0, D3 and D7 days during induction was taken to detect the expression of ectodermal cell marker genes (K8, K18) and keratinocyte marker genes (K14, cdh 1) (FIG. 5). Real-time quantitative PCR detected a 4-fold increase in K14 expression and a 20-fold increase in Cdh1 expression at D7 (fig. 5). Secondly, the invention discovers that the cell expression K8 in D3 days is increased by 2 times, and the K18 is increased by 1.5 times. But the expression of K8 and K18 in the cells decreased again to the same level as in day D0 on day D7. These experimental results show that MEF cells eventually differentiate into keratinocytes under the action of DGTM.

Example 4: formation of epithelial tissue in vitro by keratinocytes obtained by DGTM induction

Keratinocytes induced by infection of fibroblasts with DGTM at a cell density of 4X 10⁵Per cm²The amount of (a) was inoculated into a plastic ring in the dermis of the mouse with the epidermis removed. After the cells are attached to the wall and the medium in the plastic ring is aspirated, 1.8mM Ca is applied to the outside of the plastic ring²⁺The medium (2) was subjected to induction culture at a gas-liquid interface for 14 days, and 14 days later, a tissue sample was taken, fixed with 4% PFA, dehydrated and cleared to prepare a paraffin section, and then subjected to HE staining, and as a result, as shown in FIG. 6, the skin inoculated with the induction keratinocytes was differentiated to form a stratified epidermis.

Example 5: purification of Reg iii γ protein:

streaking the bacteria preserved at-80 ℃ on a plate culture medium, selecting a single colony on the next day, inoculating the single colony in a liquid culture medium for overnight culture, and transferring the overnight culture to a fresh culture medium for continuous culture; when OD600 is 0.6-0.8, adding isopropyl thiogalactoside (IPTG) for induction expression, collecting induced thallus, and performing bacteria breaking and washing; samples of the supernatant before and after induction, the supernatant after disruption and the supernatant after precipitation and washing were taken to perform SDS-PAGE to detect the expression of the target protein (FIGS. 7A and B). After the inclusion bodies were dissolved, renaturation and dialysis of the protein were performed, and after the inclusion bodies were filtered and sterilized with a filter, the resultant was applied to a column (Capto SP ImpRes cation column), eluted with an elution buffer containing 20, 50, 100, 200, 300, 500mM and 1m nacl (dissolved in 20mM PB at ph 6.5), and the eluate was collected and subjected to SDS-PAGE to detect the elution of the target protein (fig. 7C). Collecting elution samples according to the display result, dialyzing, ultrafiltering and concentrating, collecting samples before and after ultrafiltration, carrying out SDS-PAGE to detect the expression condition of the target protein, and subpackaging the rest samples and freezing at-80 ℃ for later use.

Example 6: reg III gamma inhibits poly (I: C) -induced inflammatory factor expression in DGTM-induced keratinocytes.

Culturing the induced cell seed 24-well plate until the density is 80%, adding Reg III gamma with different concentrations to stimulate for 10 minutes, then adding poly (I: C) with 20ug/ml to stimulate the cell for 24 hours, adding Trizol to extract RNA, inverting to cDNA, and detecting the expression of inflammatory factors IL-6 and TNF-alpha by quantitative PCR.

As shown in FIG. 8, the poly (I: C) -induced inflammation increased with time, but Reg III γ inhibited the poly (I: C) -induced expression of Tnf- α and Il-6, wherein the Il-6 expression decreased by nearly 20% and the Tnf- α expression decreased by nearly 30%. These experimental results demonstrate that Reg iii γ is able to inhibit inflammatory factor expression in keratinocytes.

Example 7: reg III gamma promotes proliferation of keratinocyte induced by DGTM

Culturing the induced cell seed 24-well plate until the density is 80%, adding Reg III gamma for stimulating for 24 hours, then adding RIPA (Ribose nucleic acid) cracking protein for 30 minutes, scraping the cell by using a cell scraper, ultrasonically cracking for 30 minutes, then centrifuging at low temperature for 15 minutes, then sucking protein supernatant, and determining the protein concentration by using a BCA (burst amplification) method. After the concentration was adjusted to be uniform, expression of cell proliferation gene PCNA was detected by Western Blot. The experimental results show that, as shown in fig. nine, the expression of the cell proliferation gene PCNA of the induced skin cells increases more than two-fold with the increase of the concentration of Reg iii γ, indicating that Reg iii γ stimulation can promote the induced keratinocyte proliferation.

Example 8: different combinations of Reg III gamma + DGTM + photosensitive hydrogel for promoting healing of large-area wounds of mice

To verify that the REG3A-DGTM photosensitive hydrogel combination can play a role in promoting wound healing in mice, the present invention performed different treatments on the wounds of mice after making 1.5cm diameter wounds on the back of C57. Through counting the wound healing rate of the mice from D0 to D9 days, the invention finds that the wound healing speed of the mice coated with the Reg III gamma-photosensitive hydrogel mixture is the fastest after DGTM lentivirus is dripped, and the speed is obviously different from other groups. The healing rate is from slow to fast: control < Hydrogel < Hydrogel + DGTM < RegIIIgamma < Hydrogel + RegIIIgamma < RegIIIgamma-DGTM-Hydrogel. The results of these experiments demonstrate that the Reg III gamma-DGTM-hydrogel combination is able to greatly accelerate the rate of wound healing in mice (FIG. 10).

Example 9: DGTM-Reg III gamma photosensitive hydrogel for inhibiting expression of inflammatory factors at wounds of mice

In order to prove that Reg III gamma can play a role in inhibiting wound inflammation at the wound of a mouse, RNA samples of tissues at the wound of each group of mice on the 3 rd day and the 6 th day of wound healing are collected after the wound of the mouse with the Reg III gamma-DGTM-hydrogel group heals the fastest healing, and the expression of inflammatory factors Il-6 and Tnf-alpha is detected by real-time quantitative PCR (figure 11). Firstly, on the sixth day of wound healing, the present inventors found that the expression of inflammatory factors in the wounds of mice treated with Reg iii γ was significantly down-regulated: compared with DGTM-hydrogel group, the inflammatory factor expression of the wounds of mice in the Reg III gamma-DGTM-hydrogel group is regulated by nearly 3 times, and the inflammatory factor expression of mice injected with Reg III gamma through single skin is the lowest. Secondly, on the ninth day of wound healing, since the inflammatory phase of wound healing had elapsed at this time, the inflammatory factors of the Hydrogel-treated group were decreased as compared to that of day 6, but the expression of inflammatory factors of the Hydrogel + DGTM group remained high: the expression of the inflammatory factor Il-6 is increased 6 times than that of the control group, and the expression of Tnf-alpha is increased 25 times than that of the control group. The reason for this analysis may be that the antiviral response of the cells at the wound site is activated due to the addition of lentiviruses. In summary, the above experimental results demonstrate that Reg iii γ is able to inhibit inflammation at the wound of mice.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be determined by the appended claims.

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SEQUENCE LISTING

<110> university of east China

<120> REG3A-DGTM photosensitive hydrogel composition and application thereof in inducing epidermis regeneration and repairing wound surface

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 174

<212> PRT

<213> Artificial sequence

<400> 1

Met Leu Pro Arg Ile Thr Ile Thr Ile Met Ser Trp Met Leu Leu Ser

1 5 10 15

Cys Leu Met Leu Leu Ser Gln Val Gln Gly Glu Val Ala Lys Lys Asp

20 25 30

Ala Pro Ser Ser Arg Ser Ser Cys Pro Lys Gly Ser Arg Ala Tyr Gly

35 40 45

Ser Tyr Cys Tyr Ala Leu Phe Ser Val Ser Lys Asn Trp Tyr Asp Ala

50 55 60

Asp Met Ala Cys Gln Lys Arg Pro Ser Gly His Leu Val Ser Val Leu

65 70 75 80

Ser Gly Ala Glu Ala Ser Phe Leu Ser Ser Met Ile Lys Ser Ser Gly

85 90 95

Asn Ser Gly Gln Tyr Val Trp Ile Gly Leu His Asp Pro Thr Leu Gly

100 105 110

Tyr Glu Pro Asn Arg Gly Gly Trp Glu Trp Ser Asn Ala Asp Val Met

115 120 125

Asn Tyr Ile Asn Trp Glu Thr Asn Pro Ser Ser Ser Ser Gly Asn His

130 135 140

Cys Gly Thr Leu Ser Arg Ala Ser Gly Phe Leu Lys Trp Arg Glu Asn

145 150 155 160

Tyr Cys Asn Leu Glu Leu Pro Tyr Val Cys Lys Phe Lys Ala

165 170

<210> 2

<211> 175

<212> PRT

<213> Artificial sequence

<400> 2

Met Leu Pro Pro Met Ala Leu Pro Ser Val Ser Trp Met Leu Leu Ser

1 5 10 15

Cys Leu Met Leu Leu Ser Gln Val Gln Gly Glu Glu Pro Gln Arg Glu

20 25 30

Leu Pro Ser Ala Arg Ile Arg Cys Pro Lys Gly Ser Lys Ala Tyr Gly

35 40 45

Ser His Cys Tyr Ala Leu Phe Leu Ser Pro Lys Ser Trp Thr Asp Ala

50 55 60

Asp Leu Ala Cys Gln Lys Arg Pro Ser Gly Asn Leu Val Ser Val Leu

65 70 75 80

Ser Gly Ala Glu Gly Ser Phe Val Ser Ser Leu Val Lys Ser Ile Gly

85 90 95

Asn Ser Tyr Ser Tyr Val Trp Ile Gly Leu His Asp Pro Thr Gln Gly

100 105 110

Thr Glu Pro Asn Gly Glu Gly Trp Glu Trp Ser Ser Ser Asp Val Met

115 120 125

Asn Tyr Phe Ala Trp Glu Arg Asn Pro Ser Thr Ile Ser Ser Pro Gly

130 135 140

His Cys Ala Ser Leu Ser Arg Ser Thr Ala Phe Leu Arg Trp Lys Asp

145 150 155 160

Tyr Asn Cys Asn Val Arg Leu Pro Tyr Val Cys Lys Phe Thr Asp

165 170 175

<210> 3

<211> 790

<212> DNA

<213> Artificial sequence

<400> 3

acaccatcct agggatctgc aagacagaca agatgcttcc ccgtataacc atcaccatca 60

tgtcctggat gctgctctcc tgcctgatgc tcctttctca ggtgcaaggt gaagttgcca 120

agaaagatgc cccatcttca cgtagcagct gccccaaggg ctcccgtgcc tatggctcct 180

attgctatgc cttgtttagt gtatctaaaa actggtatga tgcagatatg gcctgccaaa 240

agaggccctc aggacatctt gtgtctgtgc tcagtggcgc tgaagcttcc ttcctgtcct 300

ccatgatcaa aagcagtggt aacagtggcc aatatgtatg gattgggctc catgacccga 360

cactgggcta tgaacccaac agaggtggat gggagtggag caatgctgat gtgatgaatt 420

acatcaactg ggagacgaat ccttcctctt cctcaggcaa tcactgtggt accctgtcaa 480

gagcctcagg atttctgaag tggagagaga attattgtaa cttagagtta ccctatgtct 540

gcaaattcaa ggcctagagt acagaattga catcatgggt tgatatatac ctagccacaa 600

gcaagatccc aaatgtgaag acagatgaaa aagggaacat tctccccaca atcgaaatct 660

gacctcctga ttttctcctt ctctggccct tccctcctct catttgagga actctgtgtg 720

cactatggcc ttaattctca aagcataata ttaaagtata taactcatgc tcaaaaaaaa 780

aaaaaaaaaa 790

<210> 4

<211> 527

<212> DNA

<213> Artificial sequence

<400> 4

atgctgcctc ccatggccct gcccagtgta tcttggatgc tgctttcctg cctcatgctg 60

ctgtctcagg ttcaaggtga agaaccccag agggaactgc cctctgcacg gatccgctgt 120

cccaaaggct ccaaggccta tggctcccac tgtatgcctt gtttttgtca ccaaaatcct 180

ggacagatgc agatctggcc tgccagaagc ggccctctgg aaacctggtg tctgtgctca 240

gtggggctga gggatccttc gtgtcctccc tggtgaagag cattggtaac agctactcat 300

acgtctggat tgggctccat gaccccacac agggcaccga gcccaatgga gaaggttggg 360

agtggagtag cagtgatgtg atgaattact ttgcatggga gagaaatccc tccaccatct 420

caagccccgg ccactgtgcg agcctgtcga gaagcacagc atttctgagg tggaaagatt 480

ataactgtaa tgtgaggtta ccctatgtct gcaagttcac tgactag 527

<210> 5

<211> 576

<212> PRT

<213> Artificial sequence

<400> 5

Met Leu Tyr Leu Glu Thr Asn Ala Pro Ser Ser Tyr Ser Glu Pro Gln

1 5 10 15

Tyr Thr Ser Leu Gly Leu Leu Asn Ser Met Asp Gln Asn Gly Gly Ser

20 25 30

Thr Ser Thr Ser Pro Tyr Asn Asn Asp His Ala Gln Asn Asn Val Thr

35 40 45

Ala Pro Ser Pro Tyr Ala Gln Pro Ser Ser Thr Phe Glu Ala Leu Ser

50 55 60

Pro Ser Pro Ala Ile Pro Ser Asn Thr Asp Tyr Ala Gly Pro His Thr

65 70 75 80

Phe Asp Val Ser Phe Gln Gln Ser Ser Thr Ala Lys Ser Ala Thr Trp

85 90 95

Thr Tyr Ser Thr Glu Leu Lys Lys Leu Tyr Cys Gln Ile Ala Lys Thr

100 105 110

Cys Pro Ile Gln Ile Lys Val Leu Thr Asn Pro Pro Gln Gly Ala Val

115 120 125

Ile Arg Ala Met Pro Val Tyr Lys Lys Ala Glu His Val Thr Glu Val

130 135 140

Val Lys Arg Cys Pro Asn His Glu Leu Ser Arg Glu Phe Asn Asp Gly

145 150 155 160

Gln Ile Ala Pro Pro Ser His Leu Ile Arg Val Glu Gly Asn Ser His

165 170 175

Ala Gln Tyr Val Glu Asp Ser Ile Thr Gly Arg Gln Ser Val Leu Val

180 185 190

Pro Tyr Glu Pro Pro Gln Val Gly Thr Glu Phe Thr Thr Ile Leu Tyr

195 200 205

Asn Phe Met Cys Asn Ser Ser Cys Val Gly Gly Met Asn Arg Arg Pro

210 215 220

Ile Leu Ile Ile Val Thr Leu Glu Thr Arg Asp Gly Gln Val Leu Gly

225 230 235 240

Arg Arg Cys Phe Glu Ala Arg Ile Cys Ala Cys Pro Gly Arg Asp Arg

245 250 255

Lys Ala Asp Glu Asp Ser Ile Arg Lys Gln His Val Thr Asp Gly Thr

260 265 270

Lys Ser Ser Glu Ala Phe Arg Gln Ala Ser Ser His Leu Ser Gln Leu

275 280 285

Asn Ser Ile Lys Lys Arg Arg Ser Thr Asp Glu Glu Val Phe Cys Leu

290 295 300

Pro Ile Lys Gly Arg Glu Ile Tyr Glu Ile Leu Val Lys Ile Lys Glu

305 310 315 320

Ser Leu Glu Leu Met Gln Phe Leu Pro Gln Gln Thr Ile Glu Ser Tyr

325 330 335

Arg Gln Gln Gln Gln Asn Leu Leu Gln Lys Gln Ser Ser Leu Pro Pro

340 345 350

Gln Pro Ala Phe Gly Ser Ser Ser Pro Thr Leu Gly Lys Asn Lys Leu

355 360 365

Pro Ser Val Ser Gln Leu Ile Asn Pro Gln Gln Arg Asn Ala Leu Thr

370 375 380

Pro Ser Gly Met Pro Gly Gly Leu Thr Asp Met Thr Pro Pro Met Met

385 390 395 400

Gly Gly Pro Val Pro Met Asn Thr Asp Leu Ser Ser Leu Ser Pro Asn

405 410 415

Asn Pro Leu Gln Ser Gln Leu Gln Met Val Pro Ser Ser His Cys Thr

420 425 430

Pro Pro Pro Pro Tyr Pro Met Asp Asn Ser Ile Ser Ser Phe Leu Leu

435 440 445

Arg Leu Gly Cys Ser Ala Cys Leu Asp Tyr Phe Thr Ala Gln Gly Leu

450 455 460

Thr Asn Ile Tyr Gln Ile Glu Asn Tyr Asn Leu Glu Asp Leu Ser Arg

465 470 475 480

Leu Lys Ile Pro Thr Glu Phe Gln His Ile Ile Trp Lys Gly Ile Met

485 490 495

Glu Tyr Arg Gln Thr Met Glu Phe Ser Pro Pro Pro His Ile Leu Arg

500 505 510

Thr Ser Ser Gly Thr Ser Thr Val Ser Val Gly Ser Thr Glu Ala Arg

515 520 525

Gly Glu Arg Val Ile Asp Ala Val Arg Phe Thr Leu Arg Gln Thr Ile

530 535 540

Ser Phe Pro Pro Arg Asp Asp Trp Thr Asp Phe Ser Phe Asp Leu Ala

545 550 555 560

Pro Asp Ser Arg Arg Asn Lys Gln Gln Arg Ile Lys Glu Glu Gly Glu

565 570 575

<210> 6

<211> 625

<212> PRT

<213> Artificial sequence

<400> 6

Met Ser Gln Glu Ser Asp Asn Asn Lys Arg Leu Val Ala Leu Val Pro

1 5 10 15

Met Pro Ser Asp Pro Pro Phe Asn Thr Arg Arg Ala Tyr Thr Ser Glu

20 25 30

Asp Glu Ala Trp Lys Ser Tyr Leu Glu Asn Pro Leu Thr Ala Ala Thr

35 40 45

Lys Ala Met Met Ser Ile Asn Gly Asp Glu Asp Ser Ala Ala Ala Leu

50 55 60

Gly Leu Leu Tyr Asp Tyr Tyr Lys Val Pro Arg Asp Lys Arg Leu Leu

65 70 75 80

Ser Val Ser Lys Ala Ser Asp Ser Gln Glu Asp Gln Asp Lys Arg Asn

85 90 95

Cys Leu Gly Thr Ser Glu Ala Gln Ile Asn Leu Ser Gly Gly Glu Asn

100 105 110

Arg Val Gln Val Leu Lys Thr Val Pro Val Asn Leu Cys Leu Ser Gln

115 120 125

Asp His Met Glu Asn Ser Lys Arg Glu Gln Tyr Ser Val Ser Ile Thr

130 135 140

Glu Ser Ser Ala Val Ile Pro Val Ser Gly Ile Thr Val Val Lys Ala

145 150 155 160

Glu Asp Phe Thr Pro Val Phe Met Ala Pro Pro Val His Tyr Pro Arg

165 170 175

Ala Asp Ser Glu Glu Gln Arg Val Val Ile Phe Glu Gln Thr Gln Tyr

180 185 190

Asp Leu Pro Ser Ile Ala Ser His Ser Ser Tyr Leu Lys Asp Asp Gln

195 200 205

Arg Ser Thr Pro Asp Ser Thr Tyr Ser Glu Ser Phe Lys Asp Gly Ala

210 215 220

Ser Glu Lys Phe Arg Ser Thr Ser Val Gly Ala Asp Glu Tyr Thr Tyr

225 230 235 240

Asp Gln Thr Gly Ser Gly Thr Phe Gln Tyr Thr Leu Glu Ala Thr Lys

245 250 255

Ser Leu Arg Gln Lys Gln Gly Glu Gly Pro Met Thr Tyr Leu Asn Lys

260 265 270

Gly Gln Phe Tyr Ala Ile Thr Leu Ser Glu Thr Gly Asp Asn Lys Cys

275 280 285

Phe Arg His Pro Ile Ser Lys Val Arg Ser Val Val Met Val Val Phe

290 295 300

Ser Glu Asp Lys Asn Arg Asp Glu Gln Leu Lys Tyr Trp Lys Tyr Trp

305 310 315 320

His Ser Arg Gln His Thr Ala Lys Gln Arg Val Leu Asp Ile Ala Asp

325 330 335

Tyr Lys Glu Ser Phe Asn Thr Ile Gly Asn Ile Glu Glu Ile Ala Tyr

340 345 350

Asn Ala Val Ser Phe Thr Trp Asp Val Asn Glu Glu Ala Lys Ile Phe

355 360 365

Ile Thr Val Asn Cys Leu Ser Thr Asp Phe Ser Ser Gln Lys Gly Val

370 375 380

Lys Gly Leu Pro Leu Met Ile Gln Ile Asp Thr Tyr Ser Tyr Asn Asn

385 390 395 400

Arg Ser Asn Lys Pro Ile His Arg Ala Tyr Cys Gln Ile Lys Val Phe

405 410 415

Cys Asp Lys Gly Ala Glu Arg Lys Ile Arg Asp Glu Glu Arg Lys Gln

420 425 430

Asn Arg Lys Lys Gly Lys Gly Gln Ala Ser Gln Ala Gln Cys Asn Asn

435 440 445

Ser Ser Asp Gly Lys Met Ala Ala Ile Pro Leu Gln Lys Lys Ser Asp

450 455 460

Ile Thr Tyr Phe Lys Thr Met Pro Asp Leu His Ser Gln Pro Val Leu

465 470 475 480

Phe Ile Pro Asp Val His Phe Ala Asn Leu Gln Arg Thr Gly Gln Val

485 490 495

Tyr Tyr Asn Thr Asp Asp Glu Arg Glu Gly Ser Ser Val Leu Val Lys

500 505 510

Arg Met Phe Arg Pro Met Glu Glu Glu Phe Gly Pro Thr Pro Ser Lys

515 520 525

Gln Ile Lys Glu Glu Asn Val Lys Arg Val Leu Leu Tyr Val Arg Lys

530 535 540

Glu Asn Asp Asp Val Phe Asp Ala Leu Met Leu Lys Ser Pro Thr Val

545 550 555 560

Lys Gly Leu Met Glu Ala Leu Ser Glu Lys Tyr Gly Leu Pro Val Glu

565 570 575

Lys Ile Thr Lys Leu Tyr Lys Lys Ser Lys Lys Gly Ile Leu Val Asn

580 585 590

Met Asp Asp Asn Ile Ile Glu His Tyr Ser Asn Glu Asp Thr Phe Ile

595 600 605

Leu Asn Met Glu Ser Met Val Glu Gly Phe Lys Ile Thr Leu Met Glu

610 615 620

Ile

625

<210> 7

<211> 431

<212> PRT

<213> Artificial sequence

<400> 7

Met Leu Val His Ser Phe Ser Ala Met Asp Arg His Asp Gly Thr Ser

1 5 10 15

Asn Gly Thr Ala Arg Leu Pro Gln Leu Gly Thr Val Gly Gln Ser Pro

20 25 30

Tyr Thr Ser Ala Pro Pro Leu Ser His Thr Pro Asn Ala Asp Phe Gln

35 40 45

Pro Pro Tyr Phe Pro Pro Pro Tyr Gln Pro Ile Tyr Pro Gln Ser Gln

50 55 60

Asp Pro Tyr Ser His Val Asn Asp Pro Tyr Ser Leu Asn Pro Leu His

65 70 75 80

Ala Gln Pro Gln Pro Gln His Pro Gly Trp Pro Gly Gln Arg Gln Ser

85 90 95

Gln Glu Ser Gly Leu Leu His Thr His Arg Gly Leu Pro His Gln Leu

100 105 110

Ser Gly Leu Asp Pro Arg Arg Asp Tyr Arg Arg His Glu Asp Leu Leu

115 120 125

His Gly Pro His Gly Leu Gly Ser Gly Leu Gly Asp Leu Pro Ile His

130 135 140

Ser Leu Pro His Ala Ile Glu Asp Val Pro His Val Glu Asp Pro Gly

145 150 155 160

Ile Asn Ile Pro Asp Gln Thr Val Ile Lys Lys Gly Pro Val Ser Leu

165 170 175

Ser Lys Ser Asn Ser Asn Ala Val Ser Ala Ile Pro Ile Asn Lys Asp

180 185 190

Asn Leu Phe Gly Gly Val Val Asn Pro Asn Glu Val Phe Cys Ser Val

195 200 205

Pro Gly Arg Leu Ser Leu Leu Ser Ser Thr Ser Lys Tyr Lys Val Thr

210 215 220

Val Ala Glu Val Gln Arg Arg Leu Ser Pro Pro Glu Cys Leu Asn Ala

225 230 235 240

Ser Leu Leu Gly Gly Val Leu Arg Arg Ala Lys Ser Lys Asn Gly Gly

245 250 255

Arg Ser Leu Arg Glu Lys Leu Asp Lys Ile Gly Leu Asn Leu Pro Ala

260 265 270

Gly Arg Arg Lys Ala Ala Asn Val Thr Leu Leu Thr Ser Leu Val Glu

275 280 285

Gly Glu Ala Val His Leu Ala Arg Asp Phe Gly Tyr Val Cys Glu Thr

290 295 300

Glu Phe Pro Ala Lys Ala Val Ala Glu Phe Leu Asn Arg Gln His Ser

305 310 315 320

Asp Pro Asn Glu Gln Val Ala Arg Lys Asn Met Leu Leu Ala Thr Lys

325 330 335

Gln Ile Cys Lys Glu Phe Thr Asp Leu Leu Ala Gln Asp Arg Ser Pro

340 345 350

Leu Gly Asn Ser Arg Pro Asn Pro Ile Leu Glu Pro Gly Ile Gln Ser

355 360 365

Cys Leu Thr His Phe Asn Leu Ile Ser His Gly Phe Gly Ser Pro Ala

370 375 380

Val Cys Ala Ala Val Thr Ala Leu Gln Asn Tyr Leu Thr Glu Ala Leu

385 390 395 400

Lys Ala Met Asp Lys Met Tyr Leu Ser Asn Asn Pro Asn Ser His Thr

405 410 415

Asp Asn Ser Ala Lys Ser Ser Asp Lys Glu Glu Lys His Arg Lys

420 425 430

<210> 8

<211> 454

<212> PRT

<213> Artificial sequence

<400> 8

Leu Asp Phe Leu Trp Ala Leu Glu Thr Pro Gln Thr Ala Thr Thr Met

1 5 10 15

Pro Leu Asn Val Asn Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp

20 25 30

Ser Val Gln Pro Tyr Phe Ile Cys Asp Glu Glu Glu Asn Phe Tyr His

35 40 45

Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp Ile

50 55 60

Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser Arg

65 70 75 80

Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Ala Thr Ser Phe

85 90 95

Ser Pro Arg Glu Asp Asp Asp Gly Gly Gly Gly Asn Phe Ser Thr Ala

100 105 110

Asp Gln Leu Glu Met Met Thr Glu Leu Leu Gly Gly Asp Met Val Asn

115 120 125

Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile

130 135 140

Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu

145 150 155 160

Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Thr

165 170 175

Ser Leu Ser Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu

180 185 190

Tyr Leu Gln Asp Leu Thr Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser

195 200 205

Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys

210 215 220

Thr Ser Ser Asp Ser Thr Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu

225 230 235 240

Ser Ser Glu Ser Ser Pro Arg Ala Ser Pro Glu Pro Leu Val Leu His

245 250 255

Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu

260 265 270

Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Thr Pro

275 280 285

Ala Lys Arg Ser Glu Ser Gly Ser Ser Pro Ser Arg Gly His Ser Lys

290 295 300

Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His

305 310 315 320

Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala

325 330 335

Ala Lys Arg Ala Lys Leu Asp Ser Gly Arg Val Leu Lys Gln Ile Ser

340 345 350

Asn Asn Arg Lys Cys Ser Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn

355 360 365

Asp Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu

370 375 380

Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu

385 390 395 400

Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala

405 410 415

Tyr Ile Leu Ser Ile Gln Ala Asp Glu His Lys Leu Thr Ser Glu Lys

420 425 430

Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln

435 440 445

Leu Arg Asn Ser Gly Ala

450

<210> 9

<211> 1728

<212> DNA

<213> Artificial sequence

<400> 9

atgttgtacc tggagaccaa tgctccctca tcctacagcg agcctcagta tacaagcctg 60

gggcttctca acagcatgga tcaaaatggg ggctccacat ccaccagccc ctacaacaac 120

gatcatgctc aaaacaatgt gacggcacca tcgccctatg cccagcccag ctccaccttt 180

gaagccctct ctccatcacc tgccatccca tccaacacag actacgctgg cccacacacc 240

tttgacgtgt cctttcagca gtccagcaca gccaaatctg caacctggac gtactccaca 300

gagttgaaga agctgtactg tcagattgcc aaaacatgtc ctattcaaat caaggtccta 360

accaacccac cccagggtgc agtcatacgg gccatgcctg tctataagaa agcagagcat 420

gttactgagg tggtcaaacg atgcccgaac cacgagctga gcagagagtt caatgacggc 480

caaatagccc cacccagtca ccttattagg gttgagggga acagtcacgc tcaatacgtg 540

gaggattcca ttactgggcg tcaaagtgtg ctggtgcctt atgagccccc tcaggtgggc 600

acagagttca caaccatctt gtacaatttc atgtgtaact ccagctgtgt tggcggaatg 660

aacaggcggc caattctcat cattgtcaca ttggaaacca gagatggtca ggttttgggt 720

cgacggtgtt ttgaggctcg tatctgtgcg tgccctggac gtgaccgcaa agctgacgag 780

gacagcatcc gcaaacagca cgtcaccgat ggcacaaaga gcagtgaggc tttccgtcaa 840

gcctcttctc acctatccca gctaaactcc atcaagaaac gaagatctac agacgaggaa 900

tgttttgtct gcctattaaa ggccgtgaaa tctatgagat tttggtgaaa atcaaagagt 960

ctttggaact catgcagttc ctccctcagc agactattga gtcatacaga cagcagcagc 1020

agaacctact gcagaaacag agctctctgc caccccagcc tgcattcggc tccagctcgc 1080

ccaccctcgg caagaacaaa ctgccctctg tcagtcaact catcaacccc caacaacgca 1140

acgctctcac cccatccggc atgccaggag gactcactga catgacccct cctatgatgg 1200

gtggccctgt tcctatgaac acagacctga gctccttgag ccccaacaac cccctccagt 1260

cacagttaaa atggtgcctt catcccactg cacacctcca ccaccctacc cgatggacaa 1320

cagcatttcc agtttccgtt gaggctgggc tgctcggcct gtttggacta tttcacagcc 1380

caaggactga ccaatatcta ccagattgag aattataact tagaggactt gtccaggctg 1440

aagatcccca cagaattcca gcacatcatc tggaaaggca tcatggagta ccggcagacc 1500

atggagtttt ctccccctcc ccacatcttg cgcacctcta gcgggacatc cactgtcagc 1560

gtgggctcca ctgaagcccg aggcgagcgc gtgatcgacg ccgtgcgctt caccctccgt 1620

cagaccatct ccttccctcc gcgggatgac tggaccgatt tctccttcga tctggcccct 1680

gattcacgcc gcaacaagca gcagcgcatc aaggaggaag gagaatga 1728

<210> 10

<211> 1878

<212> DNA

<213> Artificial sequence

<400> 10

atgtcacaag agtcggacaa taataaaaga ctagtggcct tagtgcccat gcccagtgac 60

cctcccttca acacccgaag agcctacaca agtgaggatg aggcctggaa gtcatatctg 120

gagaaccccc tgactgcggc caccaaggcg atgatgagca tcaacgggga cgaggacagt 180

gctgccgccc tgggcctgct ctatgactac tacaaggttc ctcgagacaa gagacttctg 240

tctgtgagca aagcaagtga cagccaagaa gaccaggata aaagaaactg ccttggcacc 300

agtgaagccc agatcaattt gagcggaggc gagaacagag tgcaggttct gaagactgtc 360

ccggtgaacc tctgtctaag tcaagaccac atggagaatt cgaagcgcga gcagtacagt 420

gtatccatca ccgagagctc tgccgtcatc cccgtgtcag gcatcaccgt ggtgaaagcc 480

gaggatttca caccggtgtt catggcgccc ccggtgcact atccccgcgc ggacagtgag 540

gagcagcgcg tggttatctt tgaacagact cagtacgacc tgccctccat agccagccac 600

agctcctatc tcaaggacga ccagcgcagc acgccggaca gcacctacag cgagagcttt 660

aaggacggcg cctcggagaa atttcggagt acttctgttg gtgctgacga gtatacatat 720

gaccagacgg gaagtggtac atttcagtac accctggaag ccaccaaatc tctccgtcag 780

aagcaggggg agggccccat gacctacctc aacaaaggac aattctatgc cataacactc 840

agtgagactg gagacaacaa atgcttccga caccccatca gcaaagtcag gagtgtggtg 900

atggtggtct ttagtgaaga caaaaaccga gatgagcagc tgaaatactg gaagtactgg 960

cactcccggc agcacactgc caagcagagg gtccttgaca ttgctgatta caaggagagc 1020

ttcaacacca tcgggaacat tgaagagatc gcatacaatg ctgtttcctt cacctgggat 1080

gtgaacgagg aggcaaagat ttttatcacc gtgaattgcc tgagtacaga tttctcctcc 1140

caaaagggtg taaaaggact tcccctgatg attcagatcg acacgtacag ctacaacaac 1200

cgcagcaata aacccatcca cagagcatac tgccagatca aggtcttctg tgacaaggga 1260

gcagaaagaa aaatccggga tgaagagaga aagcagaaca ggaagaaagg gaagggccag 1320

gcctctcaag cccagtgcaa caactcctct gatgggaaga tggccgccat accgttacag 1380

aagaagagtg acatcacgta cttcaaaacc atgcccgacc tgcactcaca gcctgtgctc 1440

ttcataccag atgttcactt tgcaaaccta cagaggaccg gacaggttta ttacaacaca 1500

gacgatgagc gagaaggcag cagcgtcctt gttaagcgga tgttcaggcc catggaagag 1560

gagtttggtc caacaccgtc taagcagatc aaagaagaaa acgtaaaacg agtgctttta 1620

tatgtgagga aggagaacga tgacgtcttc gatgctctga tgctgaaatc acccacggtg 1680

aagggtctga tggaagcgct gtctgagaag tatgggctgc cagtggagaa aatcacaaag 1740

ctttataaga agagcaaaaa gggcatcctg gtcaacatgg atgacaacat cattgagcac 1800

tattcaaatg aggacacctt catcctcaac atggagagca tggtggaagg cttcaagatc 1860

acgctgatgg agatctga 1878

<210> 11

<211> 1296

<212> DNA

<213> Artificial sequence

<400> 11

atgttagttc acagtttttc agctatggac cgtcacgacg gcaccagcaa cgggacggca 60

cggttgcccc agctgggcac tgtaggtcaa tctccctaca ccagcgcccc gccgctgtcc 120

cacaccccta atgccgactt ccagcctccc tacttccccc cgccctacca gcctatctac 180

ccccagtcgc aagatcctta ctcccacgtc aacgacccct acagcctgaa tcctctgcac 240

gcccagccgc agccgcagca cccgggctgg cccggtcaga ggcagagcca ggaatctggg 300

ctcttacaca cacaccgggg cttaccccac caactgtcgg gcctggaccc tcgcagggac 360

tatcggcggc acgaggacct cttgcacggc ccgcacgggc tcggctctgg gctcggggac 420

ctcccgatcc actccttacc tcacgccatc gaggacgtcc cgcacgtaga agacccgggt 480

attaacatcc cagatcaaac tgtaattaag aaaggccccg tgtccctgtc caagtccaac 540

agcaatgctg tctccgccat ccctattaac aaggacaacc tcttcggtgg cgtggtaaac 600

cccaacgaag tcttctgttc agttccgggt cgcctgtcgc tcctcagctc cacctcgaag 660

tacaaggtca cggtggcgga agtacagcgg cgcctctcac cgcccgagtg tcttaacgcg 720

tcgctcctgg gcggagtact gcggagagcg aagtctaaga atggagggag atctttaaga 780

gaaaaactgg acaagatagg attgaatctg ccagcaggga gacgtaaagc tgccaacgtt 840

accctcctca cgtcactagt ggaaggagaa gccgtccacc tagccaggga ctttgggtac 900

gtgtgcgaaa ctgaatttcc tgccaaagca gtagcagaat ttctcaaccg acaacattcc 960

gatcccaatg agcaagtggc aagaaaaaac atgctcctgg ccacaaaaca gatctgcaaa 1020

gagttcactg acctgctggc tcaggaccga tctcccctgg ggaactcgcg gcccaatcct 1080

atcctggagc ctggcatcca gagttgcttg acccacttca acctcatctc ccatggcttc 1140

ggcagcccgg cggtgtgtgc agcggttacg gccctgcaga actatctcac cgaggccctc 1200

aaggccatgg acaaaatgta cctcagcaac aaccccaaca gccacacgga caacagcgcc 1260

aaaagcagtg acaaagaaga gaaacacaga aagtga 1296

<210> 12

<211> 1365

<212> DNA

<213> Artificial sequence

<400> 12

ctggatttcc tttgggcgtt ggaaaccccg cagacagcca cgacgatgcc cctcaacgtg 60

aacttcacca acaggaacta tgacctcgac tacgactccg tacagcccta tttcatctgc 120

gacgaggaag agaatttcta tcaccagcaa cagcagagcg agctgcagcc gcccgcgccc 180

agtgaggata tctggaagaa attcgagctg cttcccaccc cgcccctgtc cccgagccgc 240

cgctccgggc tctgctctcc atcctatgtt gcggtcgcta cgtccttctc cccaagggaa 300

gacgatgacg gcggcggtgg caacttctcc accgccgatc agctggagat gatgaccgag 360

ttacttggag gagacatggt gaaccagagc ttcatctgcg atcctgacga cgagaccttc 420

atcaagaaca tcatcatcca ggactgtatg tggagcggtt tctcagccgc tgccaagctg 480

gtctcggaga agctggcctc ctaccaggct gcgcgcaaag acagcaccag cctgagcccc 540

gcccgcgggc acagcgtctg ctccacctcc agcctgtacc tgcaggacct caccgccgcc 600

gcgtccgagt gcattgaccc ctcagtggtc tttccctacc cgctcaacga cagcagctcg 660

cccaaatcct gtacctcgtc cgattccacg gccttctctc cttcctcgga ctcgctgctg 720

tcctccgagt cctccccacg ggccagccct gagcccctag tgctgcatga ggagacaccg 780

cccaccacca gcagcgactc tgaagaagag caagaagatg aggaagaaat tgatgtggtg 840

tctgtggaga agaggcaaac ccctgccaag aggtcggagt cgggctcatc tccatcccga 900

ggccacagca aacctccgca cagcccactg gtcctcaaga ggtgccacgt ctccactcac 960

cagcacaact acgccgcacc cccctccaca aggaaggact atccagctgc caagagggcc 1020

aagttggaca gtggcagggt cctgaagcag atcagcaaca accgcaagtg ctccagcccc 1080

aggtcctcag acacggagga aaacgacaag aggcggacac acaacgtctt ggaacgtcag 1140

aggaggaacg agctgaagcg cagctttttt gccctgcgtg accagatccc tgaattggaa 1200

aacaacgaaa aggcccccaa ggtagtgatc ctcaaaaaag ccaccgccta catcctgtcc 1260

attcaagcag acgagcacaa gctcacctct gaaaaggact tattgaggaa acgacgagaa 1320

cagttgaaac acaaactcga acagcttcga aactctggtg cataa 1365

Claims (10)

1. A REG 3A-photosensitive hydrogel, characterized in that the amino acid sequence of REG3A in said REG 3A-photosensitive hydrogel is shown as SEQ ID NO. 1-2, and the nucleotide sequence thereof is shown as SEQ ID NO. 3-4.

2. A REG3A-DGTM photosensitive hydrogel composition comprising the REG3A-DGTM photosensitive hydrogel according to claim 1 and DGTM factors that induce epidermal regeneration of a wound surface, wherein the DGTM factors comprise DNP63A, GRHL2, TFAP2A and C-Myc.

3. The REG3A-DGTM photosensitive hydrogel composition according to claim 2, wherein the DGTM factor protein has the amino acid sequence shown in SEQ ID No. 5-8 and the nucleotide sequence shown in SEQ ID No. 9-12.

4. The REG3A-DGTM photosensitive hydrogel composition of claim 2, wherein said DGTM factor induces reprogramming of mesenchymal cells into keratinocytes.

5. A pharmaceutical composition or reagent or kit comprising DGTM factor and/or REG 3A-photosensitive hydrogel according to claim 1 and/or REG3A-DGTM photosensitive hydrogel composition according to claim 2 or 3.

6. A preparation method of REG 3A-photosensitive hydrogel is characterized in that the preparation method of REG 3A-photosensitive hydrogel is that REG3A protein and photosensitive hydrogel are mixed and incubated on wound surface, and then are irradiated by ultraviolet lamp for 10-30s and then are solidified.

7. Use of REG 3A-photosensitive hydrogel according to claim 1, REG3A-DGTM photosensitive hydrogel composition according to claim 2, or pharmaceutical composition or reagent or kit according to claim 5 for the preparation of a medicament for the treatment of large area skin wounds.

8. Use of the REG 3A-photosensitive hydrogel of claim 1, the REG3A-DGTM photosensitive hydrogel composition of claim 2, or the pharmaceutical composition or the reagent or the kit of claim 5 for the preparation of a medicament for inhibiting wound cytokine production.

9. Use of REG 3A-photosensitive hydrogel of claim 1, REG3A-DGTM photosensitive hydrogel composition of claim 2, or pharmaceutical composition or reagent or kit of claim 5 for the preparation of a medicament for promoting keratinocyte proliferation.

10. The REG 3A-photosensitive hydrogel according to claim 1, the REG3A-DGTM photosensitive hydrogel composition according to claim 2, the pharmaceutical composition or the reagent or the kit according to claim 5 for use in a method for promoting healing of large-area skin wounds, wherein the REG 3A-photosensitive hydrogel and/or the REG3A-DGTM photosensitive hydrogel composition is administered to a subject individual.

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