CN114591891A

CN114591891A - Construction method of cow interphalangeal skin explant model

Info

Publication number: CN114591891A
Application number: CN202210401138.7A
Authority: CN
Inventors: 郑家三; 葛延松; 张鹤飞; 周志新; 孙悦; 杨春雪; 岳洋; 范春玲
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2022-04-18
Filing date: 2022-04-18
Publication date: 2022-06-07

Abstract

The invention provides a method for constructing a milk cow inter-toe skin explant model, and belongs to the technical field of skin explant models. The model construction method comprises the following steps: immersing the interphalangeal skin of the dairy cow in a transport culture medium for transport, cleaning the interphalangeal skin of the dairy cow by using a washing culture medium after the transport is finished, and culturing the cleaned interphalangeal skin of the dairy cow in a cell chamber containing a tissue culture medium to finish the model construction; and finally, carrying out feasibility evaluation on the cow interdigital skin explant model. The 3D cow interphalangeal skin model constructed by the method accords with the 3R principle of experimental animals such as reduction, substitution and optimization, and meanwhile, the constructed model can maintain good tissue structure integrity, fully simulates the cow interphalangeal skin in-vivo environment, and has important significance for deeply researching the invasion action mechanism of pathogenic microorganisms of the cow interphalangeal skin.

Description

Construction method of cow interphalangeal skin explant model

Technical Field

The invention relates to the technical field of skin explant models, in particular to a construction method of a cow interphalangeal skin explant model.

Background

With the rapid development of the livestock breeding industry, cow foot and limb diseases become the third disease which endangers the healthy breeding of cows after mastitis and breeding disorder, and the cow foot and limb diseases are the general term for the pathological changes of the hoof and limb parts of the cows caused by various poor breeding factors such as environment, management, nutrition and physiology. According to the statistics of the food and agriculture organization of the United nations, the incidence rate of cow foot and limb diseases in developed countries is 15%; the incidence rate in developing countries reaches 30-40%. The acropodopathy can cause the reduction of the production and reproduction performance of the dairy cows, the shortening of the utilization years, the increase of treatment cost and the like, and brings great economic loss to the dairy cow industry.

Common infectious diseases of the milk cow interdigital skin comprise foot rot, hoof dermatitis, interdigital dermatitis and the like. The hoof skin of sick cows often shows necrosis and dehiscence, inflammation spreads from toe to toe in the skin with systemic symptoms, and cow toe infectious hoof has become an important group of diseases affecting the health of cow hooves. The inflammatory pathogenesis of the diseases is reported less at home and abroad, and the research on the interphalangeal skin of the dairy cows is mainly limited by the 3R (Replacement, Reduction, reference) principle of experimental animals and the ethics and other factors. The cow interdigital skin explant model can meet the research requirements of cow interdigital skin infectious diseases and also meet the ethical requirements of experimental animals, so that the invention can promote the in vitro test deep research of cow interdigital skin infectious mechanisms and further promote the sustainable development of healthy cow breeding.

Disclosure of Invention

The invention aims to provide a method for constructing a milk cow inter-toe skin explant model, the milk cow inter-toe skin explant model constructed by the method has the characteristics of high stability and high repeatability, and in addition, the construction of the model can greatly reduce the use frequency of experimental animals.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for constructing an explant model of skin between toes of a dairy cow, which comprises the following steps:

immersing the interphalangeal skin of the dairy cow in a transportation culture medium for transportation, cleaning the interphalangeal skin of the dairy cow by using a washing culture medium after transportation is finished, culturing the cleaned interphalangeal skin of the dairy cow in a cell chamber containing a tissue culture medium, finishing model construction, and finally performing feasibility evaluation on an explant model of the interphalangeal skin of the dairy cow;

the components of the transport culture medium comprise a DMEM/F-12 culture medium, a penicillin-streptomycin-amphotericin B solution, gentamicin sulfate and L-glutamine;

the components of the washing culture medium comprise a DMEM/F-12 culture medium, a penicillin-streptomycin-amphotericin B solution and gentamicin sulfate;

the components of the tissue culture medium comprise a high-sugar DMEM culture medium, a DMEM/F-12 culture medium, a penicillin-streptomycin-amphotericin B solution, gentamicin sulfate, L-glutamine and fetal calf serum.

Preferably, in the transport culture medium, the concentration of gentamicin sulfate is 4-6 mug/mL, and the volume ratio of the DMEM/F-12 culture medium, the penicillin-streptomycin-amphotericin B solution and the L-glutamine is (97.5-98.5): (0.9-1.1): (0.9-1.1).

Preferably, in the washing culture medium, the concentration of gentamicin sulfate is 1-3 mug/mL, and the volume ratio of the DMEM/F-12 culture medium to the penicillin-streptomycin-amphotericin B solution is (98.5-99.0): (0.9-1.1).

Preferably, in the tissue culture medium, the volume ratio of the high-glucose DMEM medium, the DMEM/F-12 medium, the penicillin-streptomycin-amphotericin B solution, the L-glutamine and the fetal calf serum is (63-66): (21-22): (0.9-1.1): (0.9-1.1): (9-11), wherein the concentration of the gentamicin sulfate is 4-6 mug/mL.

Preferably, the cleaning times are 2-4 times, and each time lasts for 10-20 min.

Preferably, the cell chamber comprises an inner chamber and an outer chamber, and the content of the tissue culture medium added into the outer chamber is 1-3 mL.

Preferably, the cell chamber comprises an inner chamber and an outer chamber, and the inner chamber is filled with a tissue culture medium to the junction of the epidermis and the dermis of the interphalangeal skin of the cow.

Preferably, before the interphalangeal skin of the cow is immersed in the transport culture medium, the dirt on the surface of the hoof of the cow is washed by clear water, and then the surface skin of the hoof of the cow is washed by chlorhexidine gluconate with the mass fraction of 1-3%.

Preferably, the feasibility assessment is scored by: pathological tissue change of skin explants, expression of apoptosis-related protein Caspase-3, and apoptosis rate examination of TUNEL cell apoptosis.

Preferably, the criteria for feasibility assessment are that explant models with skin explant pathological tissue examination score < 3 points, apoptosis-related protein Caspase-3 expression positive composite score < 6 points, and apoptosis rate score < 3 points are considered feasible.

The invention has the following beneficial effects:

the invention provides a method for constructing a cow inter-toe skin explant model for the first time, and the constructed model is a 3D cow inter-toe skin model. Compared with a cell model, the explant model can maintain the integrity of the tissue structure more greatly and simulate the skin in-vivo environment more truly, and meanwhile, the explant model meets the requirements of experimental animals such as reduction, substitution and optimization. The construction of the model lays an early foundation for deeply researching the interaction and cell signal conduction among skin cells, the adhesion and differentiation of keratinocytes and the change of skin barrier function.

Drawings

Fig. 1 is a graph of pathological changes (h.e × 200) of skin explants across the toes of cows at different periods;

FIG. 2 is a graph showing the expression of Caspase-3 protein (DAB X400) in interphalangeal skin explants of cows at different periods;

fig. 3 is a graph of apoptosis in interphalangeal skin explants of dairy cows at different periods, where red is TUNEL-stained apoptotic cells (on the right side of each graph) and blue is DAPI-stained total cells (on the left side of each graph), where unclear writing and lines in the graph do not affect the understanding of the technical content of the present invention.

Fig. 4 is an assembly diagram of a cow inter-toe skin explant model.

Detailed Description

In the invention, before the interphalangeal skin of the cow is immersed in the transportation culture medium, as a preferred embodiment, when the interphalangeal skin of the cow hoof is collected, the cow hoof is washed by clear water until no visible dirt exists on the surface, and then chlorhexidine gluconate with the mass fraction of 1-3% is used for killing pathogenic microorganisms on the surface of the cow hoof.

In the invention, collected cow hoof intertoe skin is fully cleaned and disinfected, and then the cow intertoe skin is collected under aseptic condition.

In the invention, after the interphalangeal skin of the dairy cow is collected, the interphalangeal skin of the dairy cow is soaked in a transport culture medium for transportation. In the transport culture medium, the concentration of gentamicin sulfate is preferably 4-6 mug/mL, and the volume ratio of the DMEM/F-12 culture medium, the penicillin-streptomycin-amphotericin B solution and the L-glutamine is preferably (97.5-98.5): (0.9-1.1): (0.9-1.1). The transportation is preferably cryogenic, such as by means of ice or organ preservation transport containers. The invention adopts the transport culture medium to provide nutrient substances required by skin tissues in short-time transport and maintain the activity of the tissues.

In the present invention, after the transport is completed, the interphalangeal skin of the cow is washed with a washing medium. In the washing culture medium, the concentration of gentamicin sulfate is preferably 1-3 mug/mL, and the volume ratio of the DMEM/F-12 culture medium to the penicillin-streptomycin-amphotericin B solution is preferably (98.5-99.0): (0.9-1.1). The number of times of cleaning is preferably 2-4, each time for 10-20 min, and more preferably 3, each time for 12-18 min. In the cleaning, as a preferred embodiment, the cleaning medium is preheated to 36-38 ℃ and cleaned at room temperature. The washing culture medium has the functions of inhibiting the growth of bacteria and fungi and preventing microbial contamination.

In the invention, the cleaned cow interphalangeal skin is cultured in a cell culture chamber containing a tissue culture medium to complete model construction. The cell culture chamber can be a 6-well plate, a 12-well plate or a 24-well plate. In the invention, the cell chamber comprises an inner chamber and an outer chamber, and the content of the tissue culture medium added into the outer chamber is preferably 1-3 mL. Because the isolated tissue lacks nutrients supplied by a blood system, in order to ensure the isolated tissue to obtain nutrients and oxygen, as a preferred embodiment, a tissue culture medium is added into the inner chamber to the junction of the epidermis and the dermis between the toes of the dairy cow to form a culture condition of being alternately exposed to a culture solution and a gas phase, and under the culture condition, the skin tissue absorbs the nutrients of the culture medium and simultaneously increases the contact area with the gas, so that more sufficient oxygen can be absorbed, and the in vivo physiological environment is more approximate. In the tissue culture medium, the volume ratio of the high-glucose DMEM medium, the DMEM/F-12 medium, the penicillin-streptomycin-amphotericin B solution, the L-glutamine and the fetal calf serum is preferably (63-66): (21-22): (0.9-1.1): (0.9-1.1): (9-11), the concentration of the gentamicin sulfate is preferably 4-6 mug/mL; the culture conditions are preferably 5% CO at 37 deg.C₂Culturing in a humidified incubator. In the present inventionIn the present invention, the medium is preferably replaced every 12 hours when cultured in the tissue culture medium. The tissue culture medium of the present invention can provide sufficient nutrients and basic substances for maintaining the survival of skin tissues and provide a suitable living environment for the skin tissues.

In the invention, the diameter of the interphalangeal skin of the cow in the transportation culture medium, the washing culture medium or the tissue culture medium is preferably less than or equal to 8 mm. The DMEM/F-12 medium is preferably DMEM/F-121:1 medium and the penicillin-streptomycin-amphotericin B solution is preferably penicillin-streptomycin-amphotericin B solution (100X) in transport, wash or tissue culture medium.

In the invention, skin tissues of nodes at different culture times are collected and fixed in 4% paraformaldehyde, and pathology and other evaluations are carried out, so that the feasibility of the cow interphalangeal skin explant model is finally judged. As a preferred embodiment, the feasibility assessment is scored by the following criteria: pathological tissue change of skin explants, expression of apoptosis-related protein Caspase-3 and apoptosis of TUNEL cells to check the apoptosis rate. The standard of feasibility evaluation is preferably that the explant model is considered feasible when the skin explant pathological tissue examination score is less than 3 points, the apoptosis-related protein Caspase-3 expression positive comprehensive score is less than 6 points and the apoptosis rate score is less than 3 points. As a preferred embodiment, the evaluation method of the cow interphalangeal skin explant model comprises the following steps: 1) scoring according to skin explant pathological tissue changes: epidermal cell degeneration (necrosis) and all the changes in epidermis and dermis described below, score 4 (tissue not viable); multifocal subfissure of epidermis, vacuolation of basal layer cells (epidermal degeneration), nuclear compaction and detachment of exocrine glands, leukocytes and epidermal cells, multifocal compaction of basal layer cells showing signs of autolysis accompanied by degeneration of neutrophils, score 3 (tissue impracticable); keratinocyte apoptosis, vacuolation of basal layer cells, epithelial dehiscence, signs of autolysis of the eccrine glands and peripheral nerves, and signs of autolysis accompanied by shedding of part of follicular epithelial cells, scored 2 (tissue feasible); scattered in apoptotic keratinocytes, scored 1 (tissue viable); normal tissue architecture and viability was observed, with occasional small numbers of exfoliated epithelial cells in the secretory glands scored 0 (tissue viable). 2) And (3) carrying out positive scoring according to the expression of an apoptosis-related protein Caspase-3: positive rate: the number of positive cells/total number of cells x 100%, reflecting the percentage of positive cells. Scoring the cell positive ratio: 0-5% of 0min, 6-15% of 1 min, 16-50% of 2min, 51-75% of 3min, and more than 75% of 4 min; positive cell staining intensity: the average positive intensity of the measurement area is 0, 1, 2 and 3 points, the negative is not colored, and the point is counted as 0 point; weak positive light yellow, 1 point is counted; the medium positive is brown yellow, and the score is 2; strong positive brown is counted for 3 points; positive scoring: the positive composite score is positive cell staining intensity score multiplied by positive cell ratio score, and the larger the data is, the stronger the composite positive intensity is. 3) Scoring by TUNEL apoptosis check apoptosis rate: and calculating the percentage of positive cells in 1000 cells in the field to the cells of the same type to be observed, wherein the apoptosis rate is the number of apoptotic cells/1000 multiplied by 100%. The apoptosis rate was scored: the apoptosis rate is 0 point to 0-5%, 1 point to 6-15% and 2 points to 16-50%, and the model is considered to be feasible; the apoptosis rate was in the range of 51% to 75% for score 3 and > 75% for score 4, and the model was considered not feasible. 4) Model feasibility scoring criteria: the feasibility of the model is comprehensively evaluated according to the pathological histology examination score, the positive comprehensive score and the apoptosis rate score. The explant model with skin explant pathological tissue examination score less than 3 points, apoptosis-related protein Caspase-3 expression positive comprehensive score less than 6 points and apoptosis rate score less than 3 points is considered to be feasible. By the evaluation method, the tissue structure of the cow interphalangeal skin explant constructed by the method is complete and the model is feasible when the tissue culture medium is cultured for 36 hours.

In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples of the present invention, the DMEM/F-121:1 medium was purchased from Biosharp brand under the trademark BL305A, manufactured by Langeco technologies, Inc.; the high-glucose DMEM is purchased from asharp brand under the flag of lange co technologies ltd, cat # BL 301A; the penicillin-streptomycin-amphotericin B solution (100x) is purchased from Biyuntian biotechnology limited, with the product number of C0224-100 mL; the L-glutamine is available from Beijing Sorbey science and technology, Inc., under the product number G0200; the gentamicin sulfate is purchased from Beijing Soilebao science and technology Limited company, and the product number is L1312-10 mL; the fetal calf serum was purchased from CLARK, Inc., cat # FB 15015.

Example 1

(one) test animal

The same healthy cow back hoof with a good hoof-shaped structure newly slaughtered by a slaughterhouse is used as a group of test samples, 6 back hoofs of three cows are collected, hoof dirt is treated under the same condition, the transportation mode is kept consistent in the whole test process, and ice transportation is adopted.

Construction of explant model of interphalangeal skin of dairy cow

1. Preparation of a culture medium:

firstly, preparation of a transport culture medium: in each 100mL of transport medium, 1mL of penicillin-streptomycin-amphotericin B solution (100X), 1mL of L-glutamine solution, 9 μ L of 5 μ g/mL gentamicin sulfate, and the balance of DMEM/F121:1 medium.

Preparation of washing culture medium: in each 100mL of washing medium, 1mL of penicillin-streptomycin-amphotericin B solution (100X), 4. mu.L of 2. mu.g/mL gentamicin sulfate, and the balance of DMEM/F-121:1 medium.

Preparation of tissue culture medium: in each 100mL of tissue culture medium, 1mL of penicillin-streptomycin-amphotericin B solution (100x), 1mL of L-glutamine solution, 10 μ L of 5 μ g/mL gentamicin sulfate, 10mL of fetal bovine serum, 65.9925mL of DMEM high-sugar medium and the balance of DMEM/F-121:1 medium.

2. Sample collection and transportation:

the method comprises the steps of collecting the back hoofs of the same healthy cow which is newly slaughtered in a slaughterhouse and has a good hoof-shaped structure as a group of test objects, collecting 6 back hoofs of 3 cows in total, and establishing three groups of controls. Washing the hoof of a freshly slaughtered cow with clear water until no visible dirt exists on the surface, and then disinfecting the skin surface with a chlorhexidine gluconate skin cleaning agent with the mass fraction of 2%. The whole interphalangeal skin was then aseptically harvested, immersed in the transport medium described above, and transported to the laboratory on ice.

3. Assembling of models

The explants were aseptically collected from the interphalangeal skin using an 8mm skin biopsy punch and immersed in wash medium preheated to 37 ℃, washed 3 times at room temperature for 15 min/time, the washed tissue was placed in the inner chamber of a 12-well cell culture chamber and 2mL of tissue medium was added from the outer chamber to the interface of the explant epidermis and dermis forming culture conditions that were alternately exposed to culture fluid and gas phase. Placing the assembled explant model at 37 deg.C and 5% CO₂The culture was carried out in a humidified incubator, 1 change of medium was carried out every 12 hours, and 2 skin explants were collected and fixed in 4% paraformaldehyde at 0, 12, 24, 36, 48 and 72 hours of culture.

4. Pathological tissue section preparation

The paraffin tissue section is prepared by adopting the national invention patent of the pathology subject group of the animal science and technology college of the university of eight agricultural reclamation of Heilongjiang, the patent application number 201110175687.9, and the patent name 'Green and Environment-friendly preparation technology of animal tissue paraffin section'.

1) Morphological examination of pathological tissues

Paraffin sections were routinely dewaxed to water, graded ethanol (100%, 95%, 85%, 75%) rehydrated for 3min per grade, distilled water for 2 min. Staining with hematoxylin staining solution for 15min, washing off loose color with distilled water for 5min, drying in an oven at 55 ℃, staining with eosin for 30s, quickly dehydrating with distilled water for 2-3 s, sealing with neutral gum, observing the tissue structure integrity of skin explants under a microscope, and scoring according to a developed histopathology scoring system (table 1).

TABLE 1 histopathology scoring table

2) Determination of Caspase-3 protein

The immunohistochemical method mainly comprises the following steps: paraffin sections were dewaxed conventionally to water, washed with running water, PBS washed 3min X3 times, and wiped dry. Repairing antigen with pancreatin, washing with PBS for 3min × 3 times, and wiping to dry. 3% of H₂O₂Endogenous peroxidase was digested, washed with PBS, blocked with serum from non-immune animals, followed by serum flinging, and primary antibody was added dropwise (Caspase-3 titer 1:400) at 4 ℃ overnight in a refrigerator. After the next day of PBS washing, streptavidin-peroxidase solution was added dropwise and incubated for 10 min. After being washed by PBS, DAB developing solution which is prepared freshly is dripped, and the developing is controlled under a microscope. Hematoxylin counterstain the cell nucleus for 30s, wash with running water, dry and seal for observation.

And (3) observing the positive expression rate of Caspase-3 protein in 3 groups of different visual fields of each tissue section, and recording the test result.

And (3) carrying out positive scoring according to the expression of an apoptosis-related protein Caspase-3: positive rate: the number of positive cells/total number of cells x 100%, reflecting the percentage of positive cells. Scoring the cell positive ratio: 0-5% of 0min, 6-15% of 1 min, 16-50% of 2min, 51-75% of 3min, and more than 75% of 4 min; positive cell staining intensity: the average positive intensity of the measurement area is 0, 1, 2 and 3 points, the negative is not colored, and the point is counted as 0 point; weak positive light yellow, 1 point is counted; the medium positive is brown yellow, and the score is 2; strong positive brown is counted for 3 points; positive scoring: the positive composite score is positive cell staining intensity score multiplied by positive cell ratio score, and the larger the data is, the stronger the composite positive intensity is.

3) TUNEL apoptosis assay

Paraffin wax slices are dewaxed conventionally to water, absolute ethanol for 5min, 90% ethanol for 2min, 70% ethanol for 2min, and distilled water for 2 min. 20. mu.g/mL protease K without DNase was added dropwise thereto and allowed to react at room temperature for 20 min. PBS was washed 3 times, 50. mu.L of prepared TUNEL assay was added dropwise to the samples and incubated at 37 ℃ in the dark for 60 min. After 3 times of PBS washing, the cells were dried, and were counterstained with DAPI at room temperature for 5min and washed 3 times with PBS. The cells were packed with neutral gum and observed under a fluorescent microscope, and the red cells were apoptotic cells and the blue cells were all cells.

And calculating the percentage of positive cells in 1000 cells in the field of a fluorescence microscope to the cells of the same type to be observed, wherein the apoptosis rate is the number of apoptotic cells/1000 multiplied by 100%. The apoptosis rate was scored: the apoptosis rate is 0 point when the cell is 0-5%, 1 point when the cell is 6-15%, and 2 points when the cell is 16-50%, and the model is considered to be feasible; the apoptosis rate was in the range of 51% to 75% for score 3 and > 75% for score 4, and the model was considered not feasible.

(III) evaluation of skin explant model

Model feasibility scoring criteria: the feasibility of the model is comprehensively evaluated according to the pathological histology examination score, the positive comprehensive score and the apoptosis rate score. The explant model with skin explant pathological tissue examination score less than 3 points, apoptosis-related protein Caspase-3 expression positive comprehensive score less than 6 points and apoptosis rate score less than 3 points is considered to be feasible.

(IV) examination of interphalangeal skin explants of cows

1. Histopathological examination

As can be seen from fig. 1, the epidermis of the skin tissue collected for 0h is complete, the structures of all layers are clear, the connective tissue of the dermis layer is regularly arranged, the subcutaneous tissue is positioned below the dermis, the muscle fibers of the muscle layer are closely arranged, and hair follicles, sebaceous glands, sweat glands and other skin accessory organs are scattered and distributed; the collagen fibers in the dermis layer of the skin tissue after 12 hours show signs of disorder, and the skin tissue after 24 hours can still maintain the complete epidermal structure; the dermis layer of the skin tissue after 36 hours is not good enough in shape, and the phenomenon of collagen fiber disorder is obvious; epithelial hyperkeratosis can be seen in skin tissues of 48h, focal acanthosis thickens, cells of the acanthosis generate degeneration and necrosis, cell nucleus is solidified and contracted, collagen fiber of the dermis is disordered, and a small amount of hair follicles and sweat glands are necrotic; the skin tissue at 76h can be seen to have severe exfoliation and deletion, degeneration of cells in a spinous layer, disappearance of cytoplasm, collagen fiber disorder in a dermis layer, necrosis of hair follicles, sebaceous glands and the like of skin accessory organs in different degrees.

Histology scores were performed on skin explants at each time period: 0h is counted as 0 min; the score is 0 in 12 h; the score is 1 in 24 hours; 2 minutes after 36 hours; the time is counted as 3 minutes after 48 hours; the 72h was counted as 4 minutes.

2. Caspase-3 protein expression assay

The immunohistochemical result shows that the Caspase-3 protein of the skin explants cultured for 12, 24, 36, 48 and 72h in vitro is positively expressed (see figure 2), and the positive expression rate difference among groups is obvious (the pH value is less than 0.01); compared with the control group of 0h skin explants, the positive expression difference of Caspase-3 protein in 12h and 24h skin explants is not significant (the pH value is more than 0.05) (see Table 2), and the statistical significance is not achieved; the positive expression differences of Caspase-3 protein in skin explants of 36h, 48h and 72h are significant (the pH value is less than 0.05) (see Table 2), and the statistical significance is achieved.

And (3) comprehensively scoring the Caspase-3 protein positive expression of the skin explant tissue slices of each time period: 0h is 0 min; the score is 0 in 12 h; the score is 2 in 24 hours; 2 minutes after 36 hours; the score is 2 when the time is 48 hours; the 72h is counted as 2 minutes.

TABLE 2 comparison of Positive rates of Caspase-3 at 12h, 24h, 36h, 48h and 72h for milk cow interphalangeal skin explants

3. TUNEL apoptosis assay

The apoptosis detection result shows that the Caspase-3 protein of the skin explants cultured for 12, 24, 36, 48 and 72h in vitro is positively expressed (figure 3), and the positive expression rate difference among groups is obvious (p is less than 0.01); the difference in apoptosis rate was not significant (with a ph > 0.05) in 12h and 24h skin explants compared to the control group of 0h skin explants (table 3), with no statistical significance; the differences in apoptosis rate among skin explants at 36h, 48h and 72h (p < 0.01) were significant (table 3), which was statistically significant.

Scoring the apoptosis rate of the skin explant tissue sections at each time period: 0h is 0 min; the score is 0 in 12 h; the score is 1 in 24 hours; the score is 1 in 36 h; the score is 2 when the time is 48 hours; the 72h is counted as 2 minutes.

TABLE 3 comparison of Positive rates of TUNEL between 12h, 24h, 36h, 48h and 72h for milk cow inter-digital skin explants

And (3) synthesizing the scoring results, wherein the Caspase-3 positive comprehensive scoring and the apoptosis scoring of the skin explant tissue slices in each time period are in a feasible range, but the pathological and histological scoring of the skin explant in 48h is more than 3 points, the tissue structure of the skin explant in 36h is complete, and the model is feasible, wherein the tissue structure of the skin explant in 24h is more complete than that in 36h, and the state of the skin explant is optimal.

Example 2

1. The construction method of the cow interdigital skin explant model comprises the following steps:

1) the preparation method of the culture medium comprises the following steps:

firstly, preparation of a transport culture medium: in each 100mL of transport medium, penicillin-streptomycin-amphotericin B solution (100X) is 0.9mL, L-glutamine solution is 1.1mL, 6. mu.g/mL gentamicin sulfate is 10. mu.L, and the balance is DMEM/F121:1 medium.

Preparation of washing culture medium: in each 100mL of washing medium, 0.9mL of penicillin-streptomycin-amphotericin B solution (100X), 6. mu.L of 3. mu.g/mL gentamicin sulfate, and the balance of DMEM/F-121:1 medium.

Preparation of tissue culture medium: in each 100mL of tissue culture medium, 0.9mL of penicillin-streptomycin-amphotericin B solution (100x), 1.1mL of L-glutamine solution, 10 μ L of 6 μ g/mL gentamicin sulfate, 11mL of fetal bovine serum, 66mL of DMEM high-sugar medium and the balance of DMEM/F-121:1 medium.

2) Sample collection and model assembly: the collected cow hooves are washed clean by clear water until no dirt exists on the surfaces, then chlorhexidine gluconate skin cleaning agents with the mass fraction of 3% are used for killing the skin surfaces, the interphalangeal skin of the cow cut off under the aseptic condition is immediately immersed into a prepared transportation culture medium, the milk cow interphalangeal skin is transported to a laboratory on ice, an 8mm skin biopsy puncher is used for cutting the interphalangeal skin of the cow into tissue blocks with the diameter of 8mm, and the tissue blocks are placed in a washing culture medium preheated to 37 ℃ to be washed for 2 times at room temperature for 20 min/time. Then culturing in 12-well cell culture chamber, adding 2mL culture solution from the outer chamber, and adding culture solution to the inner chamber to the junction of explant epidermis and dermis to form an alternate burstExposing to culture medium and gas phase culture conditions, placing 12-well cell culture dish at 37 deg.C and 5% CO₂Culturing in a humidified incubator to complete model construction, changing liquid once every 12h, collecting skin tissues at different time nodes, and fixing in 4% paraformaldehyde.

2. Evaluation of cow interdigital skin explant model:

1) scoring according to skin explant pathological tissue changes: epidermal cell degeneration (necrosis) and all the changes in epidermis and dermis described below, score 4 (tissue not viable); multifocal hypoepidermic fissuring, vacuolation of basal lamina cells (epidermal degeneration), nuclear pyknosis and detachment of exocrine glands, leukocytes and epidermal cells, multifocal pyknosis of basal lamina cells showing signs of autolysis accompanied by degeneration of neutrophils, scoring 3 points (tissue infeasible); keratinocyte apoptosis, vacuolation of basal layer cells, epithelial dehiscence, signs of autolysis of the eccrine glands and peripheral nerves, and signs of autolysis accompanied by shedding of part of follicular epithelial cells were scored 2 (tissue viable); scattered over apoptotic keratinocytes, scored 1 (tissue viable); normal tissue architecture and viability was observed, with occasional small numbers of exfoliated epithelial cells in the secretory glands scored 0 (tissue viable).

2) And (3) carrying out positive scoring according to the expression of an apoptosis-related protein Caspase-3: the positive rate: the number of positive cells/total number of cells x 100%, reflecting the percentage of positive cells. Scoring the cell positive ratio: 0-5% of 0min, 6-15% of 1 min, 16-50% of 2min, 51-75% of 3min, and more than 75% of 4 min; positive cell staining intensity: the average positive intensity of the measurement area is 0, 1, 2 and 3 points, the negative is not colored, and the point is counted as 0 point; weak positive light yellow, 1 point is counted; medium positive brown yellow, 2 points are counted; strong positive brown is counted for 3 points; positive scoring: the positive composite score is positive cell staining intensity score multiplied by positive cell ratio score, and the larger the data is, the stronger the composite positive intensity is.

3) Scoring by TUNEL apoptosis check apoptosis rate: and calculating the percentage of positive cells in 1000 cells in the field to the cells of the same type to be observed, wherein the apoptosis rate is the number of apoptotic cells/1000 multiplied by 100%. The apoptosis rate was scored: the apoptosis rate is 0 point to 0-5%, 1 point to 6-15% and 2 points to 16-50%, and the model is considered to be feasible; the apoptosis rate ranged from 51% to 75% for score 3 and > 75% for score 4, and the model was considered not feasible.

4) Model feasibility scoring criteria: the feasibility of the model is comprehensively evaluated according to the pathological histological examination score, the positive comprehensive score and the apoptosis rate score. The explant model with skin explant pathological tissue examination score less than 3 points, apoptosis-related protein Caspase-3 expression positive comprehensive score less than 6 points and apoptosis rate score less than 3 points is considered to be feasible.

Example 3

1) the preparation method of the culture medium comprises the following steps:

firstly, preparation of a transport culture medium: in each 100mL of transport medium, 1.1mL of penicillin-streptomycin-amphotericin B solution (100X), 0.9mL of L-glutamine solution, 8 μ L of 4 μ g/mL gentamicin sulfate, and the balance of DMEM/F121:1 medium.

Preparation of washing culture medium: in each 100mL of washing medium, 1.1mL of penicillin-streptomycin-amphotericin B solution (100X), 2. mu.L of 1. mu.g/mL gentamicin sulfate, and the balance of DMEM/F-121:1 medium.

Preparation of tissue culture medium: in each 100mL of tissue culture medium, 1.1mL of penicillin-streptomycin-amphotericin B solution (100x), 0.9mL of L-glutamine solution, 10 μ L of 4 μ g/mL gentamicin sulfate, 9mL of fetal bovine serum, 63mL of DMEM high-glucose medium and the balance of DMEM/F-121:1 medium.

2) Sample collection and model assembly: the collected cow hooves are washed clean by clear water until no visible dirt exists on the surfaces, then 1% chlorhexidine gluconate skin cleaner is used for disinfecting the skin surfaces, the interphalangeal skin of the cow which is cut off under the aseptic condition is immersed into a prepared transportation culture medium, the cow interphalangeal skin is transported to a laboratory on ice, an 8mm skin biopsy punch is used for cutting the interphalangeal skin of the cow into tissue blocks with the diameter of 8mm, and the tissue blocks are placed in a washing culture medium preheated to 37 ℃ for washing for 4 times at room temperature for 10 min/time. Then cultured in 12-well cell culture chambers,completing model construction, adding 2mL of culture solution from an outer chamber, adding the culture solution to the junction of the epidermis and the dermis of the explant in an inner chamber to form culture conditions of alternately exposing to the culture solution and a gas phase, placing a 12-hole cell culture dish at 37 ℃ and 5% CO₂Culturing in a humidified incubator, changing the culture solution once every 12h, collecting skin tissues at different time nodes, and fixing the skin tissues in 4% paraformaldehyde.

2. Evaluation of cow interdigital skin explant model:

1) scoring according to skin explant pathological tissue changes: epidermal cells degenerated (necrotic) and all the changes in the epidermis and dermis described below appeared, scored 4 (tissue not viable); multifocal subfissure of epidermis, vacuolation of basal layer cells (epidermal degeneration), nuclear compaction and detachment of exocrine glands, leukocytes and epidermal cells, multifocal compaction of basal layer cells showing signs of autolysis accompanied by degeneration of neutrophils, score 3 (tissue impracticable); keratinocyte apoptosis, vacuolation of basal layer cells, epithelial dehiscence, signs of autolysis of the eccrine glands and peripheral nerves, and signs of autolysis accompanied by shedding of part of follicular epithelial cells, scored 2 (tissue feasible); scattered over apoptotic keratinocytes, scored 1 (tissue viable); normal tissue architecture and viability was observed, with occasional small numbers of exfoliated epithelial cells in the secretory glands scored 0 (tissue viable).

2) Positive scoring was performed based on apoptosis-related protein Caspase-3 expression: positive rate: the number of positive cells/total number of cells x 100%, reflecting the percentage of positive cells. Scoring for cell positive rate: 0-5% of 0min, 6-15% of 1 min, 16-50% of 2min, 51-75% of 3min, and more than 75% of 4 min; positive cell staining intensity: the average positive intensity of the measurement area is 0, 1, 2 and 3 points, the negative is not colored, and the point is counted as 0 point; weak positive faint yellow, and 1 point is counted; medium positive brown yellow, 2 points are counted; strong positive brown is counted for 3 points; positive scoring: the positive composite score is positive cell staining intensity score multiplied by positive cell ratio score, and the larger the data is, the stronger the composite positive intensity is.

3) Scoring by TUNEL apoptosis check apoptosis rate: and calculating the percentage of positive cells in 1000 cells in the field to the cells of the same type to be observed, wherein the apoptosis rate is the number of apoptotic cells/1000 multiplied by 100%. The apoptosis rate was scored: the apoptosis rate is 0 point when the cell is 0-5%, 1 point when the cell is 6-15%, and 2 points when the cell is 16-50%, and the model is considered to be feasible; the apoptosis rate was in the range of 51% to 75% for score 3 and > 75% for score 4, and the model was considered not feasible.

4) Model feasibility scoring criteria: the feasibility of the model is comprehensively evaluated according to the pathological histology examination score, the positive comprehensive score and the apoptosis rate score. The explant model with skin explant pathological tissue examination score less than 3 points, apoptosis-related protein Caspase-3 expression positive comprehensive score less than 6 points and apoptosis rate score less than 3 points is considered to be feasible.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing an explant model of skin between toes of a cow is characterized by comprising the following steps:

immersing the interphalangeal skin of the dairy cow in a transportation culture medium for transportation, cleaning the interphalangeal skin of the dairy cow by using a washing culture medium after transportation is finished, culturing the cleaned interphalangeal skin of the dairy cow in a cell chamber containing a tissue culture medium to finish model construction, and finally performing feasibility evaluation on an explant model of the interphalangeal skin of the dairy cow;

2. The construction method according to claim 1, wherein the concentration of gentamicin sulfate in the transport medium is 4-6 μ g/mL, and the volume ratio of the DMEM/F-12 medium, the penicillin-streptomycin-amphotericin B solution and the L-glutamine is (97.5-98.5):

(0.9～1.1)：(0.9～1.1)。

3. the construction method according to claim 1, wherein the concentration of gentamicin sulfate in the washing culture medium is 1-3 μ g/mL, and the volume ratio of the DMEM/F-12 culture medium to the penicillin-streptomycin-amphotericin B solution is (98.5-99.0): (0.9-1.1).

4. The construction method according to claim 1, wherein in the tissue culture medium, the volume ratio of the high-glucose DMEM medium, the DMEM/F-12 medium, the penicillin-streptomycin-amphotericin B solution, the L-glutamine and the fetal bovine serum is (63-66): (21-22): (0.9-1.1): (0.9-1.1): (9-11), wherein the concentration of the gentamicin sulfate is 4-6 mug/mL.

5. The construction method according to claim 1, wherein the number of cleaning is 2-4 times, each time for 10-20 min.

6. The method of claim 1, wherein the cell chamber comprises an inner chamber and an outer chamber, and the outer chamber is filled with 1-3 mL of tissue culture medium.

7. The method of claim 1, wherein the cell chamber comprises an inner chamber and an outer chamber, and the inner chamber is filled with tissue culture medium to the junction between the epidermis and the dermis of the interphalangeal skin of the cow.

8. The construction method according to claim 1, wherein before the interphalangeal skin of the cow is immersed in the transport medium, the dirt on the surface of the hoof of the cow is washed with clear water, and then the surface skin of the hoof of the cow is washed with chlorhexidine gluconate with a mass fraction of 1-3%.

9. The construction method of claim 1, wherein the feasibility assessment is scored by: pathological tissue change of skin explants, expression of apoptosis-related protein Caspase-3, and apoptosis rate examination of TUNEL cell apoptosis.

10. The construction method of claim 9, wherein the feasibility assessment criteria are that the explant model is considered feasible when the skin explant pathological tissue examination score is less than 3 points, the apoptosis-related protein Caspase-3 expression positive comprehensive score is less than 6 points, and the apoptosis rate score is less than 3 points.