KR20160133051A

KR20160133051A - Animal as models for wound, method of manufacturing thereof and method of analyzing efficacy of wound therapeutic agent using thereof

Info

Publication number: KR20160133051A
Application number: KR1020150065389A
Authority: KR
Inventors: 문성환; 정형민; 박순정
Original assignee: 건국대학교 산학협력단
Priority date: 2015-05-11
Filing date: 2015-05-11
Publication date: 2016-11-22
Also published as: KR101776236B1

Abstract

It is an object of the present invention to overcome limitations that occur when a skin recovery process uses a mouse different from a human skin cell as a model of wound treatment research and to provide a wound treatment that can be applied to wound treatment research in a realistic and economical way The present invention relates to a method for producing an animal for a model, and a method for analyzing the efficacy of a wound treatment agent using the same.
Accordingly, the present invention includes a permeable hydrogel of a natural material, which is inserted into a cylindrical body perpendicularly to the surface of the wound, and forms a thin film on the surface of the wound to suppress the lateral construction ability peculiar to the rodent epithelium Of the wound healing process.
According to the present invention, it is possible to produce an animal model for an animal which does not inhibit immunity because it does not cause an immune response, and it is possible to study an agent for wound healing using an animal for a model more similar to the environment of human wound have. In addition, the hydrogel is injected into the cylindrical body to form a thin film on the wound, thereby stably absorbing the sample and increasing the possibility of clinical application.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an animal for a wound disease model, a method for producing the same, and a method for analyzing the efficacy of a wound treatment agent using the same. BACKGROUND ART [0002]

The present invention relates to an animal for a wound disease model, a method for producing the same, and a method for analyzing the efficacy of a wound treatment agent using the same, and more particularly, The present invention relates to a method for preparing an animal for a disease model and analyzing the efficacy of a wound healing agent using the same.

A wound healing model resected by biopsy is often used to confirm the appearance of the skin's recovery from incision, excision, burn, and the like. These wounds represent a relatively rapid three-step process (cell migration, cell proliferation (granulation and reepithelialization), cell differentiation). Many previous studies have attempted to improve regeneration by drugs, growth factors, and hydrogels, but recent trends are focused on cell therapy. Endothelial cells (ECs) in adult multipotent cells have a simple process of isolation and are used for peripheral blood, bone marrow, adipose tissue and umbilical cord blood. And is attracting attention because it can be utilized from. Several studies have shown that transplantation of endothelial cells enhances the function of upper strata cells in the epithelium, thereby promoting wound healing. Despite these benefits, somatic endothelial cells are infinitely divisible and unable to proliferate, limiting their ability to slow down. Therefore, pluripotent cells, such as human embryonic stem cells, are preferred as cellular resources to overcome these limitations and obtain endothelial cells without limitation. In addition, the therapeutic effect of endothelial cells derived from human embryonic stem cells has been already known.

Several animal models have been designed to observe the wound healing process during cell therapy. Recent studies correspond to results derived from rodents with mammalian structures. However, unlike human rehabilitation methods such as re-epithelialization and tissue granulation, the skin of the rats has a unique panniculus carnosus layer, which causes the skin to slip and pull over the subcutaneous fascia, Lt; / RTI > This early primary closure mechanism can distort the degree of re-epithelization and therefore is difficult to assess for efficacy (Experimental dermatology 2012; 21: 581-585). Therefore, the recovery process should be confirmed in the state of secondary intention by deliberately exposing the cochlea to reveal granulation and re-epithelization. Previous studies have inserted silicone rings or splints to prevent migration from the surrounding epithelial cells in order to strongly inhibit the wound from closure (Journal of visualized experiments: JoVE 2013: e50265).

However, this approach only minimized keratinocyte migration as quickly as skin contractions. In order to prevent the epidermal cells from migrating, a model was developed to make the chimney shape on the wound and prevent the contraction of the skin, thereby prolonging the recovery period. Were clearly observed and measured. This is a new and simple approach that can be expected to be applied in terms of organizational analysis and cell therapy.

It is an object of the present invention to overcome limitations that occur when a skin recovery process uses a mouse different from a human skin cell as a model of wound treatment research and to provide a wound treatment that can be applied to wound treatment research in a realistic and economical way And a method for analyzing the efficacy of a wound healing agent using the same.

In order to accomplish the above object, the present invention provides a wound dressing formed on the skin of a mammal other than a human; A cylindrical body coupled perpendicularly to the wound surface and having an opening which is in intimate contact with the inside of the wound frame; And a hydrogel for forming a permeable membrane on the surface of the wound.

The animal for the wound disease model may not have been immunocompromised.

The animal for the wound disease model may be a rodent.

The raw material for preparing the hydrogel may be selected from the group including alginate, chitosan, citrus extract, and coconut extract.

The present invention also relates to a method for the treatment of cancer, comprising the steps of: 1) forming a wound on a skin of a mammal other than a human; 2) inserting one opening of the cylindrical body in close contact with the wound frame; And 3) treating the hydrogel on the wound body after the insertion of the cylindrical body to gel, thereby coating the body with a permeable membrane.

The present invention also provides a method for treating wound healing, comprising the steps of: 1) treating a wound-healing agent candidate to be continuously absorbed through a permeable membrane of an animal for a wound disease model prepared by the manufacturing method defined in the first aspect or its embodiment; 2) analyzing the degree of regeneration of cell tissue at the wound area of the animal over time; And 3) determining the wound healing efficacy of the candidate substance according to the analysis result.

According to the present invention as described above, a cylindrical body is inserted into the rim of the animal for the model to inhibit the horizontal construction ability peculiar to the rodent epithelium, thereby creating an environment similar to human re-epithelization. In addition, since an immune response does not occur, an animal for a model that does not inhibit immunity can be produced, and an animal model animal having similar conditions to the wound environment of a human body can be used to study a wound treatment agent.

According to the present invention, a hydrogel of a natural material having no toxicity, unlike a conventional matrigel, is injected into the cylindrical body to form a permeable membrane on the wound, thereby stably absorbing the sample, There is an effect that can be increased.

FIG. 1 is a graph showing the recovery of the epithelium of an animal for a 1 mm biopsy punch wound disease model (A) Preparation of an animal for a biopsy punch model; (B) Initial wound size measurement; (C) Recovery on days 0, 7 and 14 (D) comparison of the contraction of the wound; (E) tissue staining for confirmation of keratinocyte proliferation (black arrow, 10X).
(A) a biopsy punch; (B) forming a wound; (C) inserting a cylindrical body having a diameter of 12 mm into the rim of the wound; (D) (E) chimney-type model).
FIG. 3 shows the effect of suppressing the horizontal building capability of the chimney-type model.
FIG. 4 shows the results of immunostaining of the wound disease model and its samples from 1 week on (A) 1, 3, 7 and 14 days after wound infection; (B) The results of muscle layer staining of the samples (below, 10X); (C and D) presence of inflammatory cells identified by F4 / 80 staining). The reference is 50 μm.
FIG. 5 shows a comparison of the degree of epithelial binding between the control and hESC-EC groups after removal of the cylinders at 4 weeks (B and C) 4 (Black arrows), re-epithelization (black triangles) and collagen layers by CK6 and Masson's Trichrome staining on weekly samples; (D) continuous proliferation of keratinocytes identified by Ki67; (E) Presence of human endothelial cells confirmed by staining; (F) identification of hESC-ECs labeled with Dil (white arrow) and (G) human chromosome 17 (white arrow).
Figure 6 compares the recovery process of the biopsy punch model (left) versus the chimney model (right).
7 is a schematic view of applying an alginate gel permeable membrane to a chimney model animal.
Fig. 8 shows the results of comparison of the transmission efficiency of alginate gel permeation membranes having different concentrations of alginate.
9 shows an animal for a chimney model to which an alginate gel is applied in the presence of an immune function.

Hereinafter, the present invention will be described in detail.

The present invention relates to a wound formed on the skin of a mammal other than a human; A cylindrical body coupled perpendicularly to the wound surface and having an opening which is in intimate contact with the inside of the wound frame; And a hydrogel for forming a permeable membrane on the surface of the wound.

The animal for the wound disease model is characterized in that the immunity is not removed.

The animal for the wound disease model is characterized by being a rodent.

The raw material for producing the hydrogel is selected from the group consisting of alginate, chitosan, citrus extract, and coconut extract.

Biopsy punch and chimney animal for wound disease model

To prepare the animals for the biopsy punch model, Balb / C nude mice (6 weeks old, Orient bio Inc, Seoul, Korea) were inoculated with 40 μl of rumpun 40 mg / kg and ketamine 10 mg / kg Was injected into the abdominal cavity. Each mouse was disinfected with an alcohol swab and a 1 mm cut wound was formed at the center of the back surface using a conventional biopsy punch (Acuderm Inc., Fort Lauderdale, FL). It is desirable to excise the skin of a weakly punched mouse with scissors, because it can damage vessels and muscles when strongly punched. And covered over the wound using a transparent film (Opsite, Smith & Nephew Andover, Mass.) (FIG. 1A).

In order to manufacture an animal for a chimney-type model, a cylindrical body can be closely inserted into the inside of the rim of the wound. Then, in order to inhibit cell migration at the transplantation site, cultures of human embryonic stem cell derived endothelial cells (hESC-EC) (3 x 10 ⁵ cells), which are endothelial cells derived from human embryonic stem cells, The BD Matrigel was mixed and implanted through the opening of the cylindrical body and sealed with a transparent film. The main function of the transparent film is to prevent leakage of the cylindrical body or drying of the inside of the cylindrical body, and to prevent the inflow of foreign substances. All experiments were conducted under the approval of the animal care committee of Konkuk University (IACUC No. KU13125-1).

Insertion of a cylindrical body for manufacturing an animal for a chimney-type wound model

The cylinder was made by cutting the lid of a 1.7 ml microtube with scissors and making a hollow center in the center using a grinder. The cylindrical body was finished by sanding the cut edges until they were smooth (Fig. 2C). The size of the cylindrical body is preferably 12 mm in diameter and 10 mm in height. If the height is too low, there is a limitation on the amount of the substance to be injected, and if the height is too high, the skin of the mouse at the time of insertion hardly holds the weight of the substance injected into the cylindrical body and the inside thereof.

The cylindrical body of the chimney-like manufactured by micro tube after insertion in the wound, biocompatible medical bond is ^{Histoacryl ® L (B.Braun, Aesculap Ag} & Co. KG, Germany) by a cylindrical body with dissection implantation using the skin Can be bonded. As a result, the time required for manufacturing the animal for the model is shortened compared with the conventional method of sewing the silicon ring, and the cylindrical body can be effectively fixed to the wound area while the experiment is sufficiently performed in terms of durability.

Origin and characteristics of hESC-ECs

The undifferentiated hESCs (H9-hESC strain, human embryonic stem cell-derived endothelial cells) were incubated with mitogen-inactivated feeder cells in DMEM / F12 (50: 50%; Gibco BRL, Gaithersburg, MD) Lt; / RTI > DMEM / F12 media consisted of 20% (v / v) serum substitute (Gibco), 1% non-essential amino acid (Gibco), 100 mM beta-mercaptoethanol (Gibco) and basic fibroblast growth factor -FGF; bFGF) (Invitrogen, Grand Island, NY). The culture medium was changed on a daily basis and the hESCs were transferred to new culture-assisted cells once every 7 days using a pipette. Differentiation into human embryoid bodies (hEBs) was induced in DMEM / F12 culture medium containing 10% serum substitute containing BMP-4 (20 ng / ml) except for fibrocyte growth factor for 2 days. Thereafter, the human body was transferred to a Matrigel-coated plate and cultured in DMEM medium supplemented with 10% FBS (Gibco) for 10 days (up to 12 days after the start of differentiation). Cells were sorted on a FACS Vantage flow cytometer (BD Biosciences, Bedford, Mass.) Using mouse anti-human monoclonal CD31 antibody (BD Biosciences) to separate endothelial cells from differentiated cells. The sorted cells were cultured in collagen-coated medium with EGM-2 / MV medium (Lonza, Basel, Switzerland).

Skin wound size analysis

The wound area was digitally photographed using NIS-Elements BR 3.1 (Nikon, Japan) as a sample after 0, 3, 7 and 14 days. The size of the worn out wound was also measured at the same magnification.

Histological and immunochemical analysis

Samples of the wound disease model were prepared by euthanizing the mice and tissue and immunochemical assays were performed. After the sample was fixed with 4% PFA, the solution was washed with PBS 1h, 70% alcohol 1h, 80% alcohol 1h, 90% alcohol 1h, 95% alcohol 1h, 100% alcohol 1h, 100% ), Dehydrated, and then placed in paraffin to prepare a tissue slice having a thickness of 5 占 퐉. Tissue sections were stained with hematoxylin and eosin (H & E) and Masson's trichrome on slides to measure reepithelization and collagen layer thickness. Re-epithelialization was labeled with antibodies against cytokeratin 6 (CK6, Gene Tex, Irvine, CA). Hematoxylin (Sigma) was used as a contrast dye in the avidin-peroxidase system (ABC kit; Vector Laboratories, Burlingame, CA). Life technology used to identify hESC-ECs can fluorescently stain living cells as cell labeling reagents and FISH (CEP probe) was also used to identify hESC-ECs chromosome through staining. Another sample was placed in a frozen sectioned OCT compound to obtain tissue sections with a thickness of 15 μm and was first immersed in PBS to facilitate removal of OCT. This is to confirm cell proliferation by contrast dyeing of Ki67 (Thermo Scientific, Grand Island, NY) and DAPI.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Variation of Size of General Biopsy Punch Model

First, the biopsy punch model animals were used to compare the rim of the wound for 2 weeks and to confirm the suture of the wound. The wound was formed and the culture solution or hESC-ECs was implanted on the wound site and sealed with a film (Figs. 1A and 1B). No significant recovery effect was observed in both groups until 2 days had elapsed, but on day 3, the wound rapidly contracted, on day 7 the size of wound was reduced to half, and on day 14, suturing was performed (FIG. 1C , 1D). The shrinkage of the two groups occurred similarly and there was no significant difference. These results indicate that tissue samples from the hESC-EC group were completely shrunk using an active keratinocyte marker, Cytokeratin 6 (Gene Tex), and then stained (Fig. 1E, black arrow). Histological analysis confirmed the presence of an empty space beneath the skin layer newly formed by elongation of the epidermis due to the proliferation of keratinocytes (Fig. 1E, white arrow).

Example 2. Implantation of hESC-ECs into a chimney model

The manufacturing method of the animal for the chimney model is comparatively similar to that for the animal for the punch model (Figs. 2A and 2B). A non-toxic 1.7 ml microtube was processed to prepare a cylindrical body 10 mm high and 12 mm in diameter and inserted into the wound area. At the time of insertion, it is preferable to insert a histology bond at the contact site. Because the adhesive is used, it is possible to reduce the inconvenience of sewing using a surgical thread. To prevent cell migration, a liquid matrigel was mixed with the culture medium or cells and injected into the cylindrical body (Fig. 2C). Matrigel, which changes to gel form at room temperature, prevents cell and substrate damage. The top of the cylinder was sealed with a transparent film to prevent material from the outside from penetrating into the wound area (Fig. 2D, 2E). Under these conditions, two groups of control and hESC-EC were tested and compared. Since the cylindrical body remained fixed during the whole experiment period, it was confirmed that the horizontal construction ability of the wound was suppressed (FIG. 3).

Example 3. Histological analysis of a chimney-type model

In two-week wrist band measurements, it was found that shrinkage of the wounds was inhibited in both groups in which the cylindrical body was inserted to analyze the recovery of the open window (Fig. 4A). Histologic analysis was performed after the first week, confirming the presence of inflammatory cells and the onset of epithelial regeneration in the hESC-EC group (Fig. 4B, 4C, black arrow). The staining of F4 / 80 showed that a large amount of inflammatory cells were expressed in the hESC-EC group on the first day, but the expression of inflammatory cells in the control group continued (Fig. 4D), while slowly calming down to the seventh day. After four weeks, the cylinders were removed to perform additional tests on both groups. The newly formed keratinocyte layer in the control group was loosely adhered to the epithelium, and when pulled, the layer was separated. However, it was confirmed that the hESC-EC group was firmly and strongly bound and adhered well to the skin layer (FIG. 5A, white arrow) . Tissue regeneration capacity was analyzed using a method to examine the formation of keratinocytes and the thickness of the collagen layer. Re-epithelialization was observed by staining tissue samples with CK6 showing keratinocyte activity in the epithelial layer and only in the hESC-EC group, not the control group (Fig. 5B, black arrow). Masson's Trichrome staining analysis also showed that the collagen layer of the hESC-EC group was much thicker and less predominantly scar tissue formation (FIG. 5C, black arrow). Through the staining of Ki67 expressing cells capable of detecting keratin cells, continuous cell proliferation of the wound area was confirmed (Fig. 5D). In addition, staining with PECAM specific for human cells demonstrated that the transplanted hESC-ECs migrated to constitute capillary vessels appearing in tissue sections (FIG. 5E). In addition, it is known that hESC-ECs labeled with Dil can be included in lectin-detectable blood vessel wall cells (Fig. 5F) and hESC-ECs are implanted into the tissues via labeling by FISH (Fig. 5G).

Example 4. Effect and possibility of animal for chimney model

Compared to the existing wound disease model in which a silicone ring or a splint is installed, the present invention aims to accurately demonstrate the recovery process of a mechanism similar to a human body according to a histological viewpoint. Thus, an animal for a simple and effective set-up wound disease model that prevents the horizontal construction of the wound is presented (Fig. 6).

In this way, endothelial and collagen formation in mice can be organized in a histological similar to the human skin regeneration environment. Drug or cell therapy can also be performed under similar conditions. From the same viewpoint, the chimney-type model animal can simulate the re-epithelialization process of human skin with the mouse and analyze the regeneration effect of the treatment, thereby ensuring convenience and economy of the user.

Example 5: Carrier fabrication and application point exploration

In the case of transplanting cells into a chimney wound disease model, a configuration for forming a cell support by mixing with matrigel according to an embodiment of the present invention has been proposed. However, Matrigel is an extra cellular membeane (ECM) isolated from mouse cancer cells and has a limited application point. In order to compensate for this, a carrier has been devised to absorb desired substances such as proteins, cosmetics, and drugs as well as cells and culture media on the wound. As a delivery material, a hydrogel which can be easily gelated so as to form a permeable membrane on the surface of the wound region in the cylindrical body of the chimney model was used. The hydrogel is made of natural materials as a raw material and unlike the conventional matrigel, it is highly likely to be used in clinical applications.

As the raw material of the hydrogel to be used as a carrier, it is preferable to select alginate extracted from brown algae (Fig. 7). Alginate has a characteristic of gelation when it reacts with calcium, so the application method is simple. If this is used, it can be injected into the cylindrical body included in the chimney model to form a permeable membrane on the wound region. The alginate-gel permeable membrane thus formed has the advantage of verifying various types of samples. Alginate is generally used as an impression material, and it is easy to use because anyone can easily change the state into gel. In addition, various hydrogel membranes can be formed using natural materials such as chitosan, citrus extract, and coconut extract which can be gelled instead of alginate, thus being environmentally friendly.

In order to determine the concentration of the alginate gel which can be used most efficiently, the transmittance of 200 쨉 l trypan blue solution was compared for 24 hours with different concentrations of alginate, and 100 쨉 m alginate gel was most suitable for solution permeation (Fig. 8).

Example 6 Application of an animal for a wound disease model that does not inhibit immunity

Using endothelial cells derived from human embryonic stem cells on the wounds, it was possible to confirm that the effect of endothelial cells promotes skin regeneration. However, unlike previous studies, animals in chimney models do not experience immune responses, so mice that do not inhibit immunity can be tested.

Therefore, the chimney-type model to which the alginate gel of Example 5 was applied was implemented in a mouse having immunity rather than immunosuppressed mouse (Fig. 9). A cylindrical body was inserted into a C57B mouse (6 weeks old) according to the above animal manufacturing method for the chimney model, and alginate was injected therein to form a film. The permeation efficiency of the culture medium containing various useful proteins was verified by confirming the possibility that the culture solution could be repeatedly administered at intervals of 2 days and administered up to 14 days.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Wounds formed on the skin of mammals other than humans;
A cylindrical body coupled perpendicularly to the wound surface and having an opening which is in intimate contact with the inside of the wound frame; And
An animal for a wound disease model having a hydrogel for forming a permeable membrane on the surface of the wound.

The method according to claim 1,
Lt; RTI ID = 0.0 > 1, < / RTI > wherein the mammal is not immunocompromised.

The method according to claim 1,
Wherein the mammal is a rodent animal.

The method according to claim 1,
Wherein the raw material for producing the hydrogel is selected from the group consisting of alginate, chitosan, citrus extract, and coconut extract.

1) forming a wound on the skin of a mammal other than a human;
2) inserting one opening of the cylindrical body in close contact with the wound frame; And
3) A method for manufacturing an animal for a wound disease model, comprising the step of treating the wound body after the insertion of the cylindrical body with a hydrogel to form a gel, thereby coating the body with a permeable membrane.

1) treating the candidate substance for wound healing so that it can be continuously absorbed through the permeable membrane of the animal for the wound disease model of claim 1;
2) analyzing the degree of regeneration of cell tissue at the wound area of the animal over time; And
3) determining the efficacy of wound healing of the candidate substance according to the analysis result.