CN112402592B

CN112402592B - Application of SPINK7 protein in preparation of medicine for promoting healing of excessive inflammatory wound surface

Info

Publication number: CN112402592B
Application number: CN202011403498.8A
Authority: CN
Inventors: 王涛; 粟永萍; 王军平; 赵娜; 龙爽; 汪国建
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2023-06-02
Anticipated expiration: 2040-12-04
Also published as: CN112402592A

Abstract

The invention relates to an application of SPINK7 protein in preparing a medicine for promoting healing of excessive inflammatory wounds, which can effectively inhibit skin wound excessive inflammatory reaction, promote healing of wounds, especially diabetic wounds, provide a way for promoting healing of chronic inflammatory wounds, especially diabetic wounds, and have good application prospects.

Description

Application of SPINK7 protein in preparation of medicine for promoting healing of excessive inflammatory wound surface

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of SPINK7 protein in preparation of a medicine for promoting healing of excessive inflammation wound surfaces.

Background

Inflammation is a defensive response of the body to injury, often beneficial, but excessive inflammatory response can lead to skin wound healing disorders. Skin wound healing is a complex and orderly dynamic repair process, and mainly consists of three phases with distinct characteristics and overlapping each other, namely an inflammatory reaction phase, a proliferation phase and a remodeling phase. Inflammatory response is a starting factor for smooth healing of skin wounds, but excessive and continuous inflammatory response can lead to difficult healing of wounds, and chronic difficult-to-heal wounds are formed. The healing difficulty of chronic inflammatory wounds, particularly diabetic wounds, is a troublesome worldwide medical problem, and no good treatment method exists at present; excessive inflammatory reactions result in limited proliferation of skin keratinocytes, failing to complete re-epithelialization; the proliferation and migration capacity of the fibroblast are reduced, and granulation tissue forms a barrier, so that the normal healing of wounds is inhibited. The chronic difficult-to-heal wound surface caused by excessive inflammatory reaction mainly comprises a diabetic wound surface, venous ulcer, pressure ulcer and the like, and particularly the diabetic difficult-to-heal wound surface represented by diabetic feet is typical. Of the diabetic patients, 15% have symptoms of difficult healing of wound surfaces, with up to 84% of patients being at risk of amputation due to failure of wound healing. Therefore, inhibiting inflammatory reaction of chronic refractory wounds represented by diabetic wounds has important significance for treating the refractory wounds.

Kazal-type serine protease inhibitor 7 (Serine protease inhibitor Kazal type7, SPINK 7) also known as esophageal cancer-associated gene 2 (ECRG 2), is a candidate cancer suppressor gene for esophageal cancer originally cloned and identified by Chinese scholars from esophageal tissues. SPINK7 is encoded by 258 bases, translated into a 9.23KD protein product containing 85 amino acids, secreted extracellularly, and its amino acid sequence is highly conserved in mammals such as any, mouse, rat, etc. The SPINK7 protein is expressed in the esophagus epithelium, oral mucosa and the like in a constitutive mode, and is also expressed in skin damage areas of immune diseases such as psoriasis, eczema and the like. The study shows that SPINK7 plays an important role in the regulation of cell proliferation and apoptosis, migration and invasion and other events, but the mechanism of action is not completely clear.

However, the role and mechanism of the protein SPINK7 in skin wound healing and chronic difficult-to-heal wound surface represented by diabetes wound surface are not reported at present, and whether the protein SPINK7 can be used as an intervention target for promoting healing of excessive inflammation wound surface is not clear.

Disclosure of Invention

The invention aims to provide application of SPINK7 protein in preparing a medicine for promoting healing of excessive inflammatory wounds, the application can effectively inhibit skin wound excessive inflammatory reaction, promote healing of wounds, particularly diabetic wounds, provide a way for promoting healing of chronic inflammatory wounds, particularly diabetic wounds, and have good application prospects.

The technical scheme of the invention is as follows:

application of SPINK7 protein in preparing medicine for promoting excessive inflammation wound healing is provided.

Application of SPINK7 protein in preparation of medicine for promoting diabetic wound healing

The SPINK7 protein can also be used as wound healing promoter or wound inflammation inhibitor in cosmetics and skin care products.

The medicine utilizes the SPINK7 protein to inhibit skin wound surface excessive inflammatory reaction and promote wound surface healing.

The wound surface is any one of chronic inflammation wound surface, pressure ulcer, venous ulcer, bedsore wound, burn wound and operation wound, and the chronic inflammation wound surface comprises diabetes wound and diabetes foot ulcer.

The SPINK7 protein inhibits the expression of inflammatory factor TNF-alpha and IL-6.

The excessive inflammation refers to the fact that in the inflammatory reaction of an organism, the persistent existence of inflammatory factors cannot be resolved, and the persistent high expression of the inflammatory factors such as IL-6, TNF-a and the like is usually shown, so that tissues are continuously damaged, and the inflammatory process is not prolonged.

A composition or kit comprising SPINK7 protein.

The amino acid sequence of the SPINK7 protein is shown as SEQ ID No. 1: MKITGGLLLLCTVVYFCSSSEAASLSPKKVDCSIYKKYPVVAIPCPITYLPVCGSDYITYGNECHLCT ESLKSNGRVQFLHDGSC.

The nucleotide sequence for encoding the SPINK7 protein is shown in SEQ ID No. 2: atgaagatcactgggggtctccttctgctctgtacagtggtctatttctgtagcagctcagaagctgctagtctgtctccaaaaaaagtggactgcagcatttacaagaagtatccagtggtggccatcccctgccccatcacatacctaccagtttgtggttctgactacatcacctatgggaatgaatgtcacttgtgtaccgagagcttgaaaagtaatggaagagttcagtttcttcacgatggaagttgctaa.

Recombinant vectors, recombinant microorganisms and/or transgenic cell lines containing nucleic acid molecules encoding the SPINK7 protein may likewise be used for the above-mentioned applications.

The composition or the kit is used for promoting healing of excessive inflammation wound surfaces; is used for reducing inflammatory response of diabetic wound and promoting wound healing; used as wound healing promoter or wound inflammation inhibitor; is used for inhibiting the expression of inflammatory factor TNF-alpha and IL-6 around wounds.

In the present invention, the SPINK7 protein may be derived from natural, synthetic, or recombinant, or may be obtained from synthetic or recombinant.

The compositions of the present invention comprise an effective amount of SPINK7 protein, together with one or more pharmaceutically acceptable any carrier, excipient, adjuvant, etc., including but not limited to diluents, binders, wetting agents, etc. It can be made into any dosage form suitable for human or animal, including but not limited to injection, liniment, corrosion inhibitor, etc. Which can be prepared according to known methods. The composition or kit contains SPINK7 protein in an amount of 0.1-99% by weight.

The beneficial effects are that:

the invention provides the application of the SPINK7 protein in inhibiting skin wound inflammation for the first time. The invention provides the SPINK7 protein for the first time, which can reduce the inflammatory response of the diabetic wound surface, effectively inhibit the excessive inflammatory response of the skin wound surface, and further promote the healing of the diabetic wound surface. Researches show that the expression level of proinflammatory cytokines such as IL-6, TNF-a and the like in inflammatory reaction can be obviously inhibited by infecting human keratinocyte strain HaCaT cells with slow viruses to overexpress SPINK7 protein; preparing skin wound surfaces by adopting SPINK7 knockout mice and corresponding control wild type mice, wherein the knockout mice are found to have delayed healing and are accompanied with significant up-regulation of the expressed IL-6 and TNF-a levels; further, the eukaryotic expression plasmid of the SPINK7 is used for up-regulating the level of the SPINK7 on the wound surface injection of the diabetic mice, so that the wound surface healing can be obviously promoted, and the expression level of proinflammatory factors such as IL-6, TNF-a and the like can be inhibited. The SPINK7 can inhibit wound inflammation, promote diabetic wound healing, and has good application prospect.

Drawings

FIG. 1 shows that stable clones of the HaCaT human keratinocytes infected by the SPINK7 lentivirus can highly express the SPINK7 protein through Western blot detection.

FIG. 2A is a graph showing that high expression of SPINK7 in HaCaT human keratinocytes can significantly inhibit the mRNA expression of IL-6 caused by combined stimulation of TNF-a and IFN-gamma.

FIG. 2B shows that high expression of SPINK7 in HaCaT human keratinocytes significantly inhibited the mRNA expression of TNF-a resulting from combined stimulation of TNF-a and IFN-gamma.

Fig. 3A is a general observation that SPINK7 knockdown resulted in delayed healing of skin wounds in mice.

Fig. 3B is a healing curve of the spike 7 knockout resulting in delayed healing of the skin wound of the mouse.

FIG. 3C is that SPINK7 knockdown resulted in significant up-regulation of mRNA for IL-6 in the skin lesions of mice.

FIG. 3D is that SPINK7 knockdown resulted in significant upregulation of mRNA for mouse skin wound TNF-a.

Fig. 4A is a general observation that SPINK7 promotes healing of diabetic skin wounds in mice.

Fig. 4B is a healing curve of SPINK7 promoting healing of diabetic skin wound in mice.

FIG. 4C is a graph showing that SPINK7 inhibits the mRNA expression of IL-6 in diabetic skin wounds.

FIG. 4D shows that SPINK7 inhibits mRNA expression of TNF-a in diabetic skin wounds.

Detailed Description

The present invention is specifically described below by way of examples, which are provided for further illustration of the present invention and are not to be construed as limiting the scope of the present invention.

Experimental procedures and conditions not explicitly described in the present invention were employed.

The invention uses commercially available analytically pure or biological grade products in addition to other reagents that are described as being of specific origin.

Example 1: SPINK7 inhibits inflammatory response of keratinocytes in vitro

3.1 experimental method:

and (3) cells: human keratinocyte strain HaCaT cells (purchased from ATCC) were cultured in DMEM high sugar medium (Gibco) containing 10% fetal bovine serum.

SPINK7 lentiviral infection stable clone selection:

HaCaT cells were plated overnight, and then lentiviral empty vectors, lenti-CMV and lenti-SPINK7 (Kirschner Biotechnology Co., ltd.) were infected at MOI (multiplicity of infection) of 200. After 24 hours, the culture medium is changed into a fresh culture medium, and the culture is continued for 48 hours. Then, puromycin (Biyundian corporation) was used for monoclonal screening, and after 7 days, the monoclonal culture was selected for expansion, and the subsequent experiments were performed.

Western blot detection of SPINK7 expression in HaCaT cells:

after establishing HaCaT cells stably infected with the SPINK7 lentivirus, extracting total proteins of the HaCaT cells and control cells by Lysis M (Roche), performing SDS-PAGE electrophoresis, performing membrane transfer, incubating by using a rabbit-derived SPINK7 antibody (Abcam), and detecting the expression condition of the SPINK7 in the HaCaT cells; beta-actin protein served as an internal reference.

TNF-a and IFN-gamma combined stimulation of HaCaT keratinocyte inflammation model:

HaCaT cells and blank cells, which express SPINK7 in lentivirus at 5×10, respectively ⁵ The individual cells/well were seeded into 6-well plates and cultured overnight. Then, the HaCaT keratinocytes were stimulated with TNF-a (Peprotech Co.) and IFN-gamma (Peprotech Co.) at final concentrations of 10ng/ml to prepare an inflammation model, and the supernatants were aspirated at 0h, 4h and 24h of the above cytokine combination, and the cells were lysed with 1ml of trizol (Invitrogen Co.).

Q-PCR detects the relative expression of inflammation-related molecules:

the cells lysed by the above-mentioned trizol were routinely extracted for total RNA. And (3) performing fluorescence quantitative PCR detection, wherein the method is the same as that before. The relevant primer information is as follows: IL-6F: TGGGAAATCGTGGGAAATGAG (SEQ ID No. 3), IL-6R: CTCTGAATGGACTCTGGCTTTG (SEQ ID No. 4); TNF-a F: CCCGGGCTCAGCCTCTTCTCATTC (SEQ ID No. 5), TNF-a R: GGATCCGGTGGTTTGCTACGACGT (SEQ ID No. 6); TBP F: AAGGGAGAATCATGGACCAG (SEQ ID No. 7), TBP R: CCGTAAGGCATCATTGGACT (SEQ ID No. 8). The results are expressed as how many times the expression of inflammatory molecules was compared to the control group 0h, taking the TBP gene as an internal reference.

3.2 experimental results:

the HaCaT clone stably infected by the SPINK7 lentivirus can be used for expressing the SPINK7 at a high protein level through Western blot detection, and the figure 1 is shown.

After the combination of TNF-a and IFN-gamma to stimulate the HaCaT cells with high expression of SPINK7 and the control cells thereof, detecting the expression of cytokines related to inflammation, which shows that the high expression of SPINK7 can obviously inhibit the transcriptional expression of IL-6, TNF-a and other molecules, see figures 2A and 2B, wherein, P <0.05; * P <0.01; * P <0.001.

Example 2: SPINK7 knockout causes excessive inflammatory response and inhibits wound healing of mice

2.1 experimental method:

animals: SPINK7 knockout mice and corresponding control wild type mice, 8-10 in each group. SPINK7 knockout mice were introduced from racing biotechnology company and bred in the experimental animal center of the Chinese people's free army medical university.

Establishment of a full-thickness skin defect wound surface model of a mouse:

mice were anesthetized by intraperitoneal injection with 1% sodium pentobarbital, shaved on the back, prepared with 75% alcohol and iodophor, and prepared with a skin biopsy device on the back skin with two wound surfaces of circular skin full-thickness defect 6mm in diameter.

Quantitative analysis of wound healing:

after the wound surface model is established, the wounds are recorded by photographing at equal points at the time of 0, 1, 3, 5, 7, 10 days and the like, the wound healing speed is calculated, and a time curve of the wound healing of the mice is drawn.

Quantitative PCR: the total RNA was extracted from the whole normal mouse skin for 0 day and wound tissue for 7 days, and the expression of IL-6 and TNF-a was examined.

2.2 experimental results:

as shown in fig. 3A, 3B: the wound healing of the SPINK7 knockout mice is obviously delayed compared with that of the control wild mice; as shown in fig. 3C, 3D: the expression level of IL-6 and TNF-a in wound tissues of the SPINK7 knockout mice is obviously increased compared with that of control wild mice in 7 days (P <0.05; P < 0.01). The SPINK7 is shown to be a key molecule for effectively inhibiting wound inflammation in vivo, and the deficiency of the SPINK7 can lead to excessive inflammatory reaction of the wound so as to delay wound healing.

Example 3: the partial injection of the SPINK7 eukaryotic expression plasmid into the wound surface inhibits the inflammatory reaction of the wound surface of the diabetic mice and promotes the healing of the wound surface of the diabetes

3.1 experimental method:

plasmid: eukaryotic expression plasmids for SPINK7 and control CMV empty plasmids were purchased from origin, and amplified and purified by conventional methods (molecular cloning guidelines, four edition 10, 2013).

Diabetic mice: c57BL/6 mice were continuously injected with 50mg/kg of streptozotocin (STZ, roche) for 5 days on a body weight basis. After stabilizing for 10 days, detecting the blood sugar of the mice, and if the blood sugar value is more than 20mmol/l, successfully modeling the diabetic mice.

Wound local plasmid injection: after preparing a circular full-thickness skin defect wound surface with the diameter of the back of the diabetic mice being 6mm, 10ug/100ul of purified plasmid is injected at 3 points of the wound.

The preparation of the wound healing curve and the quantitative PCR method after the wound is obtained are the same as before.

3.2 experimental results

As shown in fig. 4A, 4B: the wound healing of the diabetic mice can be obviously promoted after the SPINK7 eukaryotic expression plasmid is injected at the wound edge; as shown in fig. 4C, 4D: the expression level of IL-6 and TNF-a in diabetic wound tissues 7 days after the injection of the SPINK7 plasmid is obviously reduced compared with that of the control plasmid injection wound (P <0.05; P < 0.01). The method shows that the diabetes wound can be inflamed all the time by injecting the SPINK7 eukaryotic expression plasmid into the wound margin to improve the protein level of the SPINK7, and the healing of the diabetes wound is promoted.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

Sequence listing

<110> Chinese people's university of Legend army medical university

Application of <120> SPINK7 protein in preparation of medicine for promoting healing of excessive inflammatory wound surface

<141> 2020-12-04

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 85

<212> PRT

<213> SPINK7 protein (SPINK 7 protein)

<400> 1

Met Lys Ile Thr Gly Gly Leu Leu Leu Leu Cys Thr Val Val Tyr Phe

1 5 10 15

Cys Ser Ser Ser Glu Ala Ala Ser Leu Ser Pro Lys Lys Val Asp Cys

20 25 30

Ser Ile Tyr Lys Lys Tyr Pro Val Val Ala Ile Pro Cys Pro Ile Thr

35 40 45

Tyr Leu Pro Val Cys Gly Ser Asp Tyr Ile Thr Tyr Gly Asn Glu Cys

50 55 60

His Leu Cys Thr Glu Ser Leu Lys Ser Asn Gly Arg Val Gln Phe Leu

65 70 75 80

His Asp Gly Ser Cys

85

<210> 2

<211> 258

<212> DNA

<213> SPINK7 protein (SPINK 7 protein)

<400> 2

atgaagatca ctgggggtct ccttctgctc tgtacagtgg tctatttctg tagcagctca 60

gaagctgcta gtctgtctcc aaaaaaagtg gactgcagca tttacaagaa gtatccagtg 120

gtggccatcc cctgccccat cacataccta ccagtttgtg gttctgacta catcacctat 180

gggaatgaat gtcacttgtg taccgagagc ttgaaaagta atggaagagt tcagtttctt 240

cacgatggaa gttgctaa 258

<210> 3

<211> 20

<212> DNA

<213> IL-6 F(IL-6 F)

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tgggaaatcg tggaaatgag 20

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<213> IL-6 R(IL-6 R)

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ctctgaagga ctctggcttt g 21

<210> 5

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<213> TNF-a F(TNF-a F)

<400> 5

cccgggctca gcctcttctc attc 24

<210> 6

<211> 24

<212> DNA

<213> TNF-a R(TNF-a R)

<400> 6

ggatccggtg gtttgctacg acgt 24

<210> 7

<211> 20

<212> DNA

<213> TBP F(TBP F)

<400> 7

aagggagaat catggaccag 20

<210> 8

<211> 20

<212> DNA

<213> TBP R(TBP R)

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ccgtaaggca tcattggact 20

Claims

The application of the SPINK7 protein in preparing the medicines for promoting the healing of the excessive inflammatory wound surface is disclosed, and the amino acid sequence of the SPINK7 protein is shown as SEQ ID No. 1.
Application of SPINK7 protein in preparing medicine for promoting diabetic wound healing, wherein the amino acid sequence of SPINK7 protein is shown as SEQ ID No. 1.
3. Use according to claim 1 or 2, characterized in that: the medicine utilizes the SPINK7 protein to inhibit skin wound surface excessive inflammatory reaction and promote wound surface healing.
4. Use according to claim 1 or 2, characterized in that: the wound surface is any one of chronic inflammation wound surface, pressure ulcer, venous ulcer, bedsore wound, burn wound and operation wound.
5. The use according to claim 4, characterized in that: the chronic inflammatory wound surface comprises a diabetes wound and a diabetes foot ulcer.
6. The use according to claim 1 or 2, wherein the SPINK7 protein inhibits the expression of inflammatory factors TNF- α and IL-6.
7. The application of a recombinant vector, a recombinant microorganism and/or a transgenic cell line containing nucleic acid molecules for encoding the SPINK7 protein in the preparation of medicines for promoting the healing of excessive inflammatory wounds or promoting the healing of diabetic wounds, wherein the amino acid sequence of the SPINK7 protein is shown as SEQ ID No. 1.