Method for constructing animal model of common acne-like skin lesion caused by propionibacterium acnes biofilm

Abstract

The invention relates to a method for constructing an animal model of acne-like skin lesion caused by a propionibacterium acnes biofilm, which comprises the following steps of: constructing the propionibacterium acnes biofilm in vitro, and intracutaneously injecting a propionibacterium acnes biofilm suspension into ear skin of an ICR mouse, so as to construct the animal model of acne-like skin lesion caused by the propionibacterium acnes biofilm. The problems that an existing acne vulgaris animal model can only be used for evaluating some aspects of the pathogenesis of acne vulgaris, but cannot reveal the complexity of interaction of the acne vulgaris, the reactivity of local inflammation caused by propionibacterium acnes in a hair follicle sebaceous gland unit resident bacterium and free bacterium state is small, and the pathogenesis of the acne vulgaris cannot be evaluated are solved. The common acne inflammatory skin lesion is difficult to restore on an in-vitro animal model. The model established by the invention is simple and easy to operate, can be widely popularized and applied, and provides an experimental tool for research and development of common acne pathogenesis and medicines.

Description

Technical Field

[0001] The invention relates to a method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm. Background Art

[0002] Acne vulgaris is a chronic inflammatory pilosebaceous gland unit skin disease that is more common in adolescents but can persist into adulthood. The incidence rate among adolescents is as high as 93%, making it the eighth most common chronic disease in the world. It is prone to post-inflammatory erythema and pigmentation, which seriously affects the patient's appearance and physical and mental health (Eichenfield, DZ, Sprague, J., & Eichenfield, LF 2021. Management of Acne Vulgaris: A Review. JAMA, 326(20): 2055–2067). It is currently believed that the onset of acne vulgaris is mainly related to androgen-induced hyperplasia of the sebaceous glands, excessive secretion of sebum, abnormal keratinization of the hair follicle duct opening, proliferation of microorganisms such as Propionibacterium acnes, and immune inflammatory response. (Harper, JC 2020. Acnevulgaris: What's new in our 40th year. Journal of the American Academy of Dermatology, 82(2): 526–527).

[0003] At present, in vitro experiments for studying the pathogenesis of acne vulgaris and developing new therapeutic drugs for acne vulgaris are mainly focused on the cellular level. The establishment of animal models plays an important role in exploring the pathogenesis of acne vulgaris and drug development. However, there is currently no animal model that can cover all pathogenic factors of human acne vulgaris. Existing animal models of acne vulgaris, such as Syrian hamsters, rabbit ears, rhinoceros mice, HR-1 mice, etc., can only be used to explore certain links in the pathogenesis of acne vulgaris, but cannot reveal the complexity of its interactions. (Kanwar, IL, Haider, T., Kumari, A., Dubey,S., Jain, P., et al. 2018. Models for acne: A comprehensive study. Drug Discoveries & Therapeutics, 12(6): 329–340). Summary of the invention

[0004] The technical problem to be solved by the present invention is that most of the existing animal models of common acne are caused by external chemical substances, and Propionibacterium acnes is a resident bacterium of the hair follicles and sebaceous glands. At present, some animal models are injected with Propionibacterium acnes suspension instead of biofilm, which makes it difficult to simulate the formation of inflammatory skin lesions of common acne in in vitro animal models.

[0005] The applicant believes through research that Propionibacterium acnes is a resident bacterium that parasitizes in the human hair follicle sebaceous gland unit. The change of Propionibacterium acnes subtype and the formation of biofilm often induce the onset of acne vulgaris. In fact, Propionibacterium acnes biofilm can be seen in the hair follicle sebaceous gland unit in the skin lesions of acne patients and in the surrounding normal skin. Once the biofilm is formed, not only the pathogenicity increases, but also its resistance to antimicrobial drugs increases. In fact, it is the Propionibacterium acnes biofilm that plays a key pathogenic role in the onset of acne vulgaris, rather than the Propionibacterium acnes itself that colonizes in the normal hair follicle sebaceous gland unit.

[0006] Based on the above research, the present invention provides a method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm, by constructing a Propionibacterium acnes biofilm in vitro, and injecting the Propionibacterium acnes biofilm suspension intradermally into the ear skin of ICR mice. The specific steps of the method are as follows: 1. Use the standard strain of Propionibacterium acnes ATCC11827 to continuously culture in vitro on a cell slide pre-placed in a 24-well plate to construct a Propionibacterium acnes biofilm, and collect the biofilm suspension.

[0007] 2. Use female SPF-level ICR mice, inject the Propionibacterium acnes biofilm constructed in vitro into the ear skin of the mice, and put them into cages for further feeding after they wake up.

[0008] 3. After intradermal injection of Propionibacterium acnes biofilm to form an inflammatory reaction visible to the naked eye in the injection area, the morphology of the mouse ear experimental area was observed, and the tissue of the experimental area was excised for HE staining and immunohistochemical staining. When inflammatory erythema papules and pustules similar to acne vulgaris and corresponding histopathological characteristics appeared in the mouse ear experimental area, the immunohistochemistry of the lesion tissue showed an increase in the expression of IL-1β compared with the control group injected with PBS by the same technique, indicating that the animal model of acne vulgaris-like lesions caused by Propionibacterium acnes biofilm was successfully constructed.

[0009] The applicant found through repeated preliminary experiments that: In the above step 1), it is appropriate to select the 8th day of in vitro culture of the Propionibacterium acnes biofilm. If the time is too short, the Propionibacterium acnes will not form a biofilm, and if the time is too long, the in vitro Propionibacterium acnes biofilm will disintegrate.

[0010] In the above step 1), the concentration of the P. acnes biofilm suspension was adjusted to 2×10 8CFU / ml.

[0011] In the above step 2), it is appropriate to use ICR mice of 6-8 weeks of age.

[0012] In step 2) above, the intradermal injection site should be the middle and posterior 2 / 3 of the mouse ear near the ear root, avoiding visible blood vessels. Injection too close to the outer edge will penetrate the skin, and injection too close to the inner side will have difficulty penetrating.

[0013] In the above step 2), if the injection volume of Propionibacterium acnes biofilm is too large, it will cause damage to the skin tissue structure, and if the injection volume is too small, it will be difficult to form an inflammatory response visible to the naked eye. Therefore, the injection volume is preferably 0.1 ml.

[0014] In the above step 3), if the Propionibacterium acnes biofilm infects the mouse ears for too long, it will cause damage to the skin tissue structure, and if the time is too short, it is difficult to form an inflammatory response visible to the naked eye. Based on this, the infection time is 48 hours, that is, the morphology of the experimental area of ​​the mouse ears is observed after 48 hours, and the tissue of the experimental area is excised for HE staining and immunohistochemical staining.

[0015] Compared with the prior art, the present invention has the following advantages and progress: 1. The present invention uses ICR mice as an animal model. The mouse ear skin is divided into epidermis, dermis and subcutaneous tissue, and contains skin appendages, such as hair follicles and sebaceous glands. The mouse ear skin is thin and the hair is short, making it easier to observe the formation of skin lesions.

[0016] 2. The step of preparing Propionibacterium acnes biofilm can simulate the pathogenic Propionibacterium acnes biofilm and extracellular matrix components in the hair follicle sebaceous gland unit of patients with acne vulgaris, thereby achieving the purpose of an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm.

[0017] 3. The present invention can successfully observe the clinical skin lesions of papules and pustules in the mouse animal model of the present invention and those in patients with acne vulgaris by intradermal injection. In addition, pathological characteristics such as follicular keratin plugs, neutrophil aggregation, dermal edema, etc. similar to those in patients with acne vulgaris can be observed in the mouse tissue pathology, which meets the requirements of an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm and can reflect highly similar clinical and histopathological manifestations.

[0018] 4. The present invention can replicate the clinical manifestations and histopathological manifestations of papulopustular lesions of patients with acne vulgaris in animals, and can be used for long-term drug development and disease mechanism exploration and other related research on animal models of acne vulgaris. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the biofilm structure of Propionibacterium acnes in vitro under a confocal microscope (magnification 40×).

[0020] Figure 2 The left picture shows that both the left and right ears of the mouse were injected with PBS. The right picture shows that the left ear of the mouse was injected with Propionibacterium acnes biofilm suspension, and the right ear was injected with PBS.

[0021] Figure 3 The left figure shows the pathological manifestation of skin lesions in the experimental area of ​​the ear of ICR mice 48 hours after intradermal injection of PBS. The right figure shows the pathological tissue manifestation of pustular skin lesions in the experimental area of ​​the ear of ICR mice 48 hours after intradermal injection of Propionibacterium acnes biofilm suspension (HE, ×40). Compared with the control group, the epidermis and the opening of the sebaceous glands of the hair follicles in the model group were hyperkeratotic, the sebaceous glands were hypertrophic, a large number of inflammatory cells infiltrated in the dermis, the interstitial space was edematous, and neutrophils aggregated to form a pus cavity structure.

[0022] Figure 4 The left figure shows the ear thickness of ICR mice 48 hours after intradermal injection of Propionibacterium acnes biofilm suspension, and the right figure shows the ear weight of ICR mice.

[0023] Figure 5 The left picture shows the expression of IL-1β in the ear experimental area of ​​ICR mice 48 hours after intradermal injection of PBS into the mouse ear. The right picture shows the expression of IL-1β in the ear experimental area of ​​ICR mice 48 hours after intradermal injection of Propionibacterium acnes biofilm suspension (IHC, ×200).

[0024] Figure 6 This is the average optical density of IL-1β in the ear experimental area of ​​ICR mice 48 hours after intradermal injection of Propionibacterium acnes biofilm suspension (P<0.05). DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0026] Construction of an animal model of acne vulgaris-like lesions caused by Propionibacterium acnes biofilm Methods and Materials: 1. In vitro preparation of Propionibacterium acnes biofilm suspension The Propionibacterium acnes strain (ATCC 11827, purchased from Guangdong Provincial Microbiological Culture Collection Center) frozen at -80°C was thawed at room temperature and inoculated on the surface of Columbia blood agar medium. After being cultured in an anaerobic culture device at 37°C for 48 hours, a single colony was picked and placed in 50 ml of Brain-Heart Infusion Broth (BHI medium) for anaerobic shaking culture at 37°C for 48 hours. The concentration of the bacterial suspension was adjusted to 1×108 Place a sterile cell slide in a 24-well cell culture plate and add the pre-prepared concentration of 1×10 8 1 ml of the bacterial suspension of CFUs / ml was pipetted into each well and cultured in an anaerobic culture device at 37°C. During the culture in the anaerobic culture device, 500 ul of the original BHI medium in the 24-well plate was aspirated every 1-2 days and 500 ul of fresh BHI medium was added. On the 8th day, the supernatant in the 24-well plate was removed and one well was randomly selected and the dye LIVE / DEADBacLight was used to TM Bacterial Viability Kit, stain the Propionibacterium acnes biofilm on the slide according to the instructions, and observe it under a confocal microscope. If a variety of morphological, crisscrossing channels and a three-dimensional honeycomb biofilm structure are seen, the biofilm is successfully constructed. Add 1 ml of PBS to each remaining well in the above 24-well plate, pipetting and mixing, and the Propionibacterium acnes biofilm suspension is obtained. Use the continuous plate count method and use PBS to adjust the concentration of the biofilm suspension to a final concentration of 2×10 8 CFUs / ml.

[0027] 2. Model establishment: ① Select healthy 6-8 week old female SPF grade ICR mice ② Select the injection site, the injection site is the ear of the ICR mouse, and disinfect the experimental skin area with 75% alcohol ③ Transfer the Propionibacterium acnes biofilm suspension obtained in step 1 to a 2ml skin test syringe, exhaust the air in the 2ml skin test syringe containing the Propionibacterium acnes biofilm suspension, tighten the local skin of the mouse ear with one hand, hold the syringe needle with the bevel upward with the other hand, and pierce the skin of the experimental area at 10-15° with the skin. When the bevel of the needle completely enters the skin, lay the syringe flat; fix the needle with the hand that tightens the skin, inject 0.1ml of Propionibacterium acnes biofilm suspension, form a skin mound locally, and quickly remove the needle. Do not press the local area. The control group was injected with the same injection technique, and the injection liquid was changed to 0.1ml PBS.

[0028] 3. Model evaluation: ① Observation of skin lesions The changes of skin lesions at the injection site were observed 48 hours after injection of Propionibacterium acnes biofilm suspension.

[0029] During the observation process, it was found that 48 hours after the ICR mice in the experimental group were infected with Propionibacterium acnes biofilm, papules and pustules on the basis of inflammatory erythema were observed, and the surrounding skin capillaries were inflammatoryly dilated. In the control group, no inflammatory erythema papules and pustular skin lesions appeared in the experimental area 48 hours after intradermal injection, and there was no capillary dilation in the surrounding skin. The thickest part of the mouse ear lesion area was selected for measurement, which was used as the mouse ear thickness; the mouse ear tissue was drilled in the center of the mouse ear with a corneal trephine with a diameter of 0.6 mm and weighed with the injection point as the center, which was used as the mouse ear weight.

[0030] ② Histopathological observation The skin lesion tissues in the experimental area of ​​ICR mice were removed for pathological examination, and the obtained tissues were fixed, embedded, and subjected to routine HE staining and observation under a microscope.

[0031] During microscopic observation, pathological sections of the tissues showed that 48 hours after the ears of the ICR mice in the experimental group were infected with Propionibacterium acnes biofilm, the epidermal stratum corneum was thickened, the openings of the hair follicles and sebaceous glands were hyperkeratinized, inflammatory cells infiltrated in the dermis, and neutrophils aggregated to form a pus cavity structure.

[0032] ③ Immunohistochemistry to detect the expression level of IL-1β in skin tissue The skin lesion tissue of the experimental area of ​​ICR mice was removed, dehydrated, embedded, sliced, stained, counterstained, transparent and then sealed. The part to be analyzed in each slice was photographed under a microscope, and image analysis was performed to determine the average optical density of IL-1β in the tissue.

[0033] During the observation under the microscope, the histopathological sections showed that the expression of IL-1β in the ears of ICR mice in the experimental group increased 48 hours after subcutaneous infection with Propionibacterium acnes biofilm, and the average optical density was statistically different from that in the control group (P<0.05).

[0034] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm, characterized in that it comprises the following steps: 1) Using the standard strain of Propionibacterium acnes ATCC11827, continuous culture was performed in vitro on a cell slide pre-placed in a 24-well plate to construct a Propionibacterium acnes biofilm, and the biofilm suspension was collected; 2) Female SPF ICR mice were used, and the pre-constructed in vitro Propionibacterium acnes biofilm was intradermally injected into the mouse ear skin. After the mice woke up, they were placed in cages and continued to be raised; 3) After intradermal injection of Propionibacterium acnes biofilm to form an inflammatory reaction visible to the naked eye in the injection area, the tissues in the experimental area were excised for HE staining and immunohistochemical staining. When inflammatory erythematous papules and pustules similar to acne vulgaris and corresponding histopathological characteristics appeared in the experimental area of ​​the mouse ears, immunohistochemistry of the lesion tissue showed an increase in the expression of IL-1β compared with the control group injected with PBS by the same technique, indicating that the animal model of acne vulgaris-like lesions caused by Propionibacterium acnes biofilm was successfully established.

2. The method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm according to claim 1, characterized in that In step 1), the Propionibacterium acnes biofilm cultured in vitro for 8 days was selected.

3. The method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm according to claim 1, characterized in that In step 1), the concentration of the P. acnes biofilm suspension was adjusted to 2×10 8 CFU / ml.

4. The method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm according to claim 1, characterized in that In step 2), ICR mice of 6-8 weeks of age were selected.

5. The method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm according to claim 1, characterized in that In step 2), the intradermal injection site is selected in the middle and posterior 2 / 3 of the mouse ear near the ear base.

6. The method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm according to claim 1, characterized in that In step 2), the injection volume of the Propionibacterium acnes biofilm is 0.1 ml.

7. The method for constructing an animal model of acne vulgaris-like skin lesions caused by Propionibacterium acnes biofilm according to claim 1, characterized in that In step 3), it takes 48 hours for the injection area to form a visible inflammatory reaction, that is, the morphology of the mouse ear experimental area is observed again after 48 hours, and the tissue of the experimental area is removed for HE staining and immunohistochemical staining.

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