CN112425560A - Skin hypertrophic scar animal model and construction method thereof - Google Patents
Skin hypertrophic scar animal model and construction method thereof Download PDFInfo
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- 229960001561 bleomycin Drugs 0.000 claims abstract description 18
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 claims abstract description 18
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- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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Abstract
The invention provides a construction method of a skin hypertrophic scar animal model, which comprises the following steps: the abdomen of the mouse is unhaired, and the bleomycin solution is intermittently and subcutaneously administered; wherein the concentration of the bleomycin solution is 0.2-0.8 mg/mL. The construction method of the invention can form the skin hypertrophic scar on the skin of the mouse, the scar is not easy to fade, the long-term observation is convenient, the model building stability is good, the repeatability is strong, the cost is low, the research on the pathogenesis of the hypertrophic scar and the screening and evaluation of scar treatment medicines are facilitated, and the application prospect is good.
Description
Technical Field
The present invention relates to the field of medicine. In particular, the invention relates to a skin hypertrophic scar animal model and a construction method thereof.
Background
Normal skin undergoes various traumas and rapidly undergoes a repair, the result of which is divided into skin regeneration and skin scar according to the final form. Among them, scars are subdivided into normal scars and pathologic scars. Pathological scars are mostly caused by an imbalance between anabolism and catabolism of collagen in the tissue during repair. Pathological scars can be further divided into hypertrophic scars, keloids, atrophic scars and scar cancer according to the difference of anatomical forms, wherein the hypertrophic scars are the most common type in clinic. Various types of scars affect the appearance and normal function of the skin to varying degrees, causing a significant physical or psychological burden on the patient. For a long time, the research on the pathogenesis of hypertrophic scars is not completely clear, and the treatment method through operation or non-operation cannot be completed, so that the establishment of animal models of related diseases and the acceleration of basic research on hypertrophic scars and the discovery of treatment medicines are urgently needed.
At present, the hypertrophic scar animal model mainly comprises the following types:
firstly, a human tissue nude mouse transplantation model: after the epithelium and subcutaneous fat of the scar tissue of the human skin are removed, the scar tissue of the human skin is transplanted to the back subcutaneous part of a nude mouse in an operation mode, and the transplanted scar tissue of the human can survive under the skin of the nude mouse and keep the original pathological characteristics and scar characteristics. However, the model cannot completely simulate the growth and the changing environment of the human hypertrophic scar, and has great limitation on the research of the pathological process of the scar. Moreover, the implanted scar is not generated by the animal on the body of a nude mouse, and the application of different environmental influence models is realized. Secondly, after the transplanted tissue survives for a certain time, the situation of atrophy and absorption appears, and the situation is obviously different from the persistent hyperplasia on the human body. Again, nude mice are non-immune and do not provide accurate feedback when studying the correlation of immunity and scarring.
II, rabbit ear hypertrophic scar model: the rabbit ear ventral wound and the rabbit ear cartilage are removed for healing, so that a stable and durable hypertrophic scar can be formed spontaneously, and the success rate is 69%. Histological staining, and finding that the scar tissue has the characteristics of human hypertrophic scars. However, the rabbit ear model exposes cartilage of the rabbit ear, so that the conditions of the base of the wound surface are different from those of the human wound surface, and the cartilage of the rabbit is also proliferated. These phenomena indicate that hypertrophic scars on rabbit ears are very different from hypertrophic scars on human ears.
Thirdly, a porcine hypertrophic scar model: the skin of the female Duroc pig is similar to the cone structure of human skin, and various indexes in the wound healing process have higher similarity with the hypertrophic scar of a human. However, the scar model of the pig is not reddened and raised in appearance, and the deep skin of the pig needs about three months for healing, and the modeling time is long. In addition, the feeding and management cost of the Duroc pigs is high, and the wide application of the model is limited.
Fourthly, a drug-induced mouse hypertrophic scar model: bleomycin has long been used to establish animal models of pulmonary fibrosis, scleroderma, and the like. When BALB/c mice are used and a micropump containing bleomycin is implanted subcutaneously, the skin can be induced to generate hypertrophic scars, and the characteristics of human skin hypertrophic scars are achieved. However, since such skin is not an evolution, the formation process is far from that of human skin hypertrophic scars. In addition, after bleomycin administration is stopped, the scar may subside, which is not conducive to long-term observation. On the other hand, when a large amount of mice are molded, a large amount of micropumps are needed, and the cost is relatively high.
Fifth, the mouse skin wound stretch model: a skin retractor is arranged on the back of the mouse, and mechanical tension is applied for 10 days to induce generation of hypertrophic scars when the incision wound on the back of the mouse is healed. The model has the characteristics of hyperplastic scars of human skin to a certain extent. However, as the mouse moves actively, tension is often damaged and falls off in the modeling process, so that the model establishment is unstable, and the repeatability of experimental research is influenced.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a construction method of a skin hypertrophic scar animal model, the skin hypertrophic scar animal model and a method for screening medicines for treating the skin hypertrophic scar, the skin hypertrophic scar can be formed on the skin of a mouse by using the construction method, the scar is not easy to fade, the long-term observation is convenient, the model establishment stability is good, the repeatability is strong, the cost is low, the research on the pathogenesis of the hypertrophic scar and the screening and evaluation of medicines for treating the scar are facilitated, and the application prospect is good.
In one aspect of the invention, the invention provides a construction method of a skin hypertrophic scar animal model. According to an embodiment of the invention, the method comprises: the method comprises the following steps of unhairing the abdomen of a mouse, and intermittently and subcutaneously administering a bleomycin solution, wherein the concentration of the bleomycin solution is 0.2-0.8 mg/mL.
At present, in the process of establishing a mouse hypertrophic scar model induced by adopting a medicament, a micropump containing bleomycin needs to be implanted subcutaneously to form scars by uninterrupted administration, but after the administration is stopped, the scars are easy to fade, the long-term observation is not facilitated, and skin injury can be caused by the operation of implanting the micropump.
The inventor of the invention finds that hypertrophic scars can be formed by controlling the concentration of the bleomycin solution to be 0.2-0.8 mg/mL and adopting an intermittent administration mode, the scars are not easy to fade, a micro pump is not needed to be implanted to achieve long-term uninterrupted administration, and the model establishment cost is reduced. And the model establishment stability is good, and the repeatability is strong. The animal model is beneficial to research on pathogenesis of hypertrophic scars and screening and evaluating scar treatment medicines, and has good application prospect.
According to the embodiment of the invention, the construction method of the skin hypertrophic scar animal model can also have the following additional technical characteristics:
according to the embodiment of the invention, the dosage of the bleomycin solution is 80-120 mu L each time. When 80-120 mu L of bleomycin solution with the concentration of 0.2-0.8 mg/mL is administered every time, hypertrophic scars can be effectively formed, the scars are not easy to fade away, and the cost for constructing the model is low.
According to an embodiment of the invention, the intermittent subcutaneous administration of the bleomycin solution is 2-4 times per week and the continuous administration is 4-6 weeks. When 80-120 mu L of bleomycin solution of 0.2-0.8 mg/mL is administered every time and 2-4 times per week for 4-6 weeks, hypertrophic scars can be formed and are not easy to fade.
According to an embodiment of the invention, the mouse is male. The model is constructed by adopting a male mouse, so that the hypertrophic scar is easy to form and is not easy to fade.
According to an embodiment of the invention, the mouse is 4-6 weeks old. A model is constructed by adopting a mouse with the age of 4-6 weeks, so that hypertrophic scars are easy to form and are not easy to fade.
According to an embodiment of the present invention, the right abdomen of the mouse was depilated. The depilation is convenient to administer, and hyperplastic scar is easily formed in the right abdomen and the scar is not easy to subside.
In yet another aspect of the invention, the invention provides an animal model of skin hypertrophic scars. According to the embodiment of the invention, the skin hypertrophic scar animal model is obtained by the construction method of the skin hypertrophic scar animal model. Therefore, the hyperplastic scar formed in the skin hyperplastic scar animal model according to the embodiment of the invention is not easy to fade, and is beneficial to long-term observation. The animal model is beneficial to research on pathogenesis of hypertrophic scars and screening and evaluating scar treatment medicines, and has good application prospect.
In yet another aspect, the present invention provides a method for screening drugs for treating hypertrophic scars on skin. According to an embodiment of the invention, the method comprises: and (3) administering a candidate drug to the skin hypertrophic scar animal model, and determining whether the candidate drug is a target drug or not based on the change of the skin hypertrophic scar after culturing for a preset time. As described above, the hypertrophic scar formed in the skin hypertrophic scar animal model of the invention is not easy to subside, which is beneficial for long-term observation. Therefore, the candidate drug is given to the animal model, and after the animal model is cultured for a period of time, the change of the scar is observed, so that whether the candidate drug is the target drug or not can be accurately determined. For example, if the scar becomes smaller, the drug candidate may be judged to be the target drug for treating hypertrophic scars of the skin.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows an electron micrograph of HE staining of a normal mouse and a model mouse according to one embodiment of the present invention;
FIG. 2 shows MASSON staining electron micrographs of normal and model mice according to one embodiment of the present invention;
FIG. 3 shows a schematic representation of mRNA expression analysis of α -SMA according to one embodiment of the invention;
FIG. 4 shows a schematic representation of an mRNA expression analysis of collagen-1 according to one embodiment of the present invention;
FIG. 5 shows a schematic representation of mRNA expression analysis of Collagen-3 according to one embodiment of the present invention.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Healthy SPF male DBA mice with the body mass of 20 +/-2 g are selected and bred in an SPF animal house, wherein the mice are 30, 4 weeks old. In an environment with constant temperature and humidity, the administration was started after 1 week of acclimatization. On the first day, mice were anesthetized and then depilated on the right flank, followed by subcutaneous injection of 100 μ l of 0.5mg/ml bleomycin. Injections were administered every other day for five weeks. During which the skin scarring was continuously observed. After the time of arrival, the injection site skin was collected.
1. The skin of each mouse was stained with HE and Mason's Trichome at the sampling site and examined for histopathological changes under light. FIG. 1 shows HE staining, and FIG. 2 shows Mason's Trichome staining. As shown in the figure, the skin tissue structure of a normal mouse is complete and is in a red dyeing wave shape, collagen fibers are thin and densely arranged, and part of fibers among and around the fibers can be seen to be drawn downwards and infiltrated by asexual cells; the thickening of the upper layer of the epidermis of the skin of the model mouse is obvious, the collagen deposition of the dermis layer is increased, the collagen bundles are thickened and irregularly arranged, the fat layer is replaced by connective tissues, and a large amount of brilliant star cells are infiltrated.
2. The final skin scarring status of all mice was counted. Of 30 of these mice, 27 developed hypertrophic scar pathological morphology with a success rate of 90%.
3. The mRNA expression levels of alpha-SMA, Collagen-1 and Collagen-3 in the skin of a normal mouse and the skin of a model mouse are measured by adopting an RT-PCR method. As shown in FIG. 3, FIG. 4 and FIG. 5, the mRNA expression of the model mouse skin for alpha-SMA, Collagen-1 and Collagen-3 is obviously higher than that of the normal mouse skin. The gene is a marker of hypertrophic scars, so that experimental results show that hypertrophic scars can be obtained by using the model disclosed by the invention.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A method for constructing an animal model of skin hypertrophic scar is characterized by comprising the following steps: the abdomen of the mouse is unhaired, and the bleomycin solution is intermittently and subcutaneously administered;
wherein the concentration of the bleomycin solution is 0.2-0.8 mg/mL.
2. The method according to claim 1, wherein the bleomycin solution is administered in an amount of 80 to 120 μ L per dose.
3. The method of claim 1, wherein the intermittent subcutaneous administration of the bleomycin solution is 2 to 4 times per week for 4 to 6 weeks.
4. The method of claim 1, wherein the mouse is male.
5. The method of claim 1, wherein the mouse is 4-6 weeks old.
6. The method of claim 1, wherein the right abdomen of the mouse is depilated.
7. An animal model of skin hypertrophic scars obtained by the method for constructing an animal model of skin hypertrophic scars according to any one of claims 1 to 6.
8. A method of screening for a drug for the treatment of hypertrophic scarring of skin, comprising:
administering a drug candidate to the skin hypertrophic scar animal model of claim 7, and determining whether the drug candidate is the target drug based on the change in skin hypertrophic scar after incubation for a predetermined period of time.
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Citations (5)
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|---|---|---|---|---|
| US20140072613A1 (en) * | 2012-09-10 | 2014-03-13 | Cynthia Lander | Compositions and Methods for Treating Cutaneous Scarring |
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| CA2916970A1 (en) * | 2016-01-08 | 2017-07-08 | Pharmascience Inc. | A smac mimetic compound for use in the treatment of proliferative diseases |
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| CN109288971A (en) * | 2018-11-28 | 2019-02-01 | 上海交通大学 | Dendrobium candidum leaf anti-proliferate scar application |
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2020
- 2020-10-27 CN CN202011162453.6A patent/CN112425560A/en active Pending
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|---|---|---|---|---|
| US20140072613A1 (en) * | 2012-09-10 | 2014-03-13 | Cynthia Lander | Compositions and Methods for Treating Cutaneous Scarring |
| CN104127403A (en) * | 2014-07-04 | 2014-11-05 | 上海交通大学医学院附属第九人民医院 | Application of flavonoid quercetin in inhibition of skin scar formation and skin fibration |
| CA2916970A1 (en) * | 2016-01-08 | 2017-07-08 | Pharmascience Inc. | A smac mimetic compound for use in the treatment of proliferative diseases |
| CN107617107A (en) * | 2017-09-18 | 2018-01-23 | 浙江大学 | A kind of anti-scar preparation of compound Angiotensin-Converting class |
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