JP2004049228A - Morbidity model animal for sty and method for preparation of the same - Google Patents
Morbidity model animal for sty and method for preparation of the same Download PDFInfo
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- JP2004049228A JP2004049228A JP2003152420A JP2003152420A JP2004049228A JP 2004049228 A JP2004049228 A JP 2004049228A JP 2003152420 A JP2003152420 A JP 2003152420A JP 2003152420 A JP2003152420 A JP 2003152420A JP 2004049228 A JP2004049228 A JP 2004049228A
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- model animal
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、麦粒腫の病態モデル動物の作製方法およびその方法により作成された麦粒腫の病態モデル動物に関する。
【０００２】
【従来の技術】
麦粒腫（ものもらい）は、睫毛腺または瞼板腺の細菌感染による急性化膿性炎症であり、自覚症状および他覚的所見も顕著なことから、早期に治癒が望まれる疾患である。
麦粒腫の起炎菌は、黄色ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ）や表皮ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｅｐｉｄｅｒｍｉｄｉｓ）が大半を占めていることから、その治療には、ブドウ球菌に感受性のある抗菌薬を、症状に合わせて、局所または全身投与が行われる。
一方、麦粒腫などの外眼部感染症に対する薬効評価を行う上で、適切な動物感染モデルが存在することは重要である。外眼部感染症の病態モデルとして、例えば、角膜を傷付けた上で、角膜へ菌液を滴下して作製される緑膿菌角膜感染ウサギモデル（あたらしい眼科， Ｖｏｌ．１３， Ｎｏ．２： ２４９−２５３， １９９６）、結膜を傷つけた上で、黄色ブドウ球菌液を注入して作製される結膜炎モデルウサギ（Ｇｅｒｍａｎ Ｊ． Ｏｐｈｔｈａｌｏｍｏｌ， Ｖｏｌ．２： ４０９−４１１， １９９３）が知られている。
【０００３】
【発明が解決しようとする課題】
本発明の目的は、麦粒腫の病態モデル動物の作製方法およびその方法により作製された麦粒腫の病態モデル動物を提供することにある。
【０００４】
【課題を解決するための手段】
本発明者らは、外眼部感染症、とりわけ、麦粒腫の病態モデル動物の作製について鋭意検討した結果、シリカゲル含有カザミノ酸水溶液に懸濁したブドウ球菌を用いてヒトの麦粒腫に類似した病態を呈するウサギ感染モデルの作製することに成功し、本発明を完成するに至った。
【０００５】
【発明の実施の形態】
本発明の麦粒腫感染モデルの作製方法は、眼瞼結膜下に、シリカゲル含有カザミノ酸水溶液に懸濁した細菌を接種し、感染を惹起することを特徴とするものである。また、本発明の病態モデル動物は、眼瞼結膜下に、シリカゲル含有カザミノ酸水溶液に懸濁した細菌が接種され、感染が惹起されたヒト麦粒腫に類似する病態を呈する麦粒腫の病態モデル動物である。
【０００６】
本発明に使用される細菌は、麦粒腫の起炎菌であれば特に限定されないが、ブドウ球菌が好ましく、特に黄色ブドウ球菌または表皮ブドウ球菌が特に好ましい。本発明に使用される動物は、ヒトを除く哺乳類であれば、特に限定されないが、実験用に使用されるサル、イヌ、ウサギなどが挙げられ、特に、ウサギ日本白色在来種およびウサギニュージーランド白色種などの実験用ウサギが好ましい。
【０００７】
本発明の作製方法は、具体的には、（１）培養した細菌菌体を、（２）シリカゲル含有カザミノ酸溶液に懸濁し、（３）動物の眼瞼結膜下に接種し、（４）感染を惹起することを特徴とする方法である。この方法により、麦粒腫に類似の症状を呈する病態モデル動物を作製することができる。
本発明に使用されるシリカゲルの粒子は、１〜２００μであればよく、好ましくの２〜４５μの粒子サイズのものが好ましい。
シリカゲル含有カザミノ酸水溶液におけるシリカゲルの含量は、０．１〜２．０重量％、好ましくは、０．５〜１．５重量％である。また、カザミノ酸の含量は、０．１〜２．０重量％、好ましくは、０．５〜１．５重量％である。
シリカゲル含有カザミノ水溶液に懸濁した細菌の結膜下への接種菌量は、菌種によって異なるが、１０３〜１０８ＣＦＵ（Ｃｏｌｏｎｙ Ｆｏｒｍａｔｉｏｎ Ｕｎｉｔ）／眼瞼結膜であればよい。
接種菌液の量は、動物、菌量などにより適宜決めればよいが、ウサギにおいては、１０〜１００μＬ、好ましくは、４０〜６０μＬである。
本発明方法によれば、菌接種後、１〜５日で膿点が生じ、菌接種１４日後においても膿点を観察することができる。
【０００８】
【実施例】
以下、実施例により本発明を具体的に説明する。
実施例１
菌株：結膜炎由来黄色ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ）および表皮ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｅｐｉｄｅｒｍｉｄｉｓ）
動物：ウサギ（日本白色在来種）
方法：ミューラー・ヒントン寒天（Ｍｕｅｌｌｅｒ−Ｈｉｎｔｏｎ ａｇａｒ：ＭＨＡ）平板上にて３７℃で一夜培養した菌を、ブレイン・ハート・インフュージョン・ブロス（Ｂｒａｉｎ Ｈｅａｒｔ Ｉｎｆｕｓｉｏｎ Ｂｒｏｔｈ）１０ｍＬに接種して、３７℃で一夜培養後、菌液を室温、３０００ｒｐｍ、１０分間遠心して、培養液を除去した。得られた菌体に、１％シリカゲル（Ｓｉｌｉｃａ ｇｅｌ Ｇ， Ｍｅｒｃｋ Ａｒｔ．７７３１）含有１％カザミノ酸溶液を１ｍＬ加え、良く懸濁したものを公比１０で段階希釈して、各々を接種菌液とした。ペントバルビタールナトリウムの静脈内投与による全身麻酔並びに塩酸オキシブプロカインの点眼による角結膜表面麻酔を施したウサギの上下眼瞼結膜下に、調製した菌液の５０μＬを２５Ｇの注射針をつけた注射筒を用いて接種して、感染を惹起した。感染後、膿点の有無を観察した。また、経日的に、ウサギの全身麻酔並びに角結膜表面麻酔下にて、ダブルレベルスリットナイフで膿点に傷をつけ、膿点内容物を採取して、菌量を測定した。採取した膿点内容物の重量を測定後、滅菌生理食塩液３００μＬを添加して、ホモジナイズした。作製したホモジネートを滅菌生理食塩液で段階希釈し、その５０μＬをＭＨＡ平板に塗布して、３７℃で一夜培養後、菌数を計測して、膿１ｍｇ当たりの菌量を算出した。
結果：黄色ブドウ球菌では、約５×１０３ＣＦＵ／眼瞼結膜以上の菌量で、また、表皮ブドウ球菌では、約２×１０６ＣＦＵ／眼瞼結膜以上の菌量で接種１〜５日後に膿点を生じ、黄色ブドウ球菌では約５×１０７ＣＦＵ／眼瞼結膜以上の菌量で、菌接種１４日後も膿点を認めた。また、両菌株において、接種菌量に依存して膿点内容物中から接種菌が回収された。
【０００９】
【発明の効果】
本発明の方法により、外眼部感染症である麦粒腫の病態モデル動物を作製することができ、作製された麦粒腫病態モデル動物を使用することで抗菌薬の薬効評価をより的確に行うことができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a disease model animal of stye and a disease model animal of stye prepared by the method.
[0002]
[Prior art]
Stye is an acute purulent inflammation due to bacterial infection of the eyelash gland or tarsal gland, and is a disease that requires early cure because of its prominent subjective symptoms and objective findings.
Staphylococcus aureus and Staphylococcus epidermidis make up the majority of streptomycetes, so antibacterial drugs that are susceptible to staphylococci are used for treatment according to the symptoms. Local or systemic administration is performed.
On the other hand, it is important to have an appropriate animal infection model in evaluating the efficacy of extraocular infections such as hordeolum. As a pathological model of extraocular infection, for example, a Pseudomonas aeruginosa corneal-infected rabbit model prepared by instilling a bacterial solution into the cornea after damaging the cornea (New Ophthalmology, Vol. 13, No. 2: 249) -253, 1996), and a conjunctivitis model rabbit (German J. Ophthalomol, Vol. 2: 409-411, 1993), which is prepared by injecting a Staphylococcus aureus solution after damaging the conjunctiva.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for producing a disease model animal for stye and a disease model animal for stye produced by the method.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on the preparation of pathological animal models for extraocular infections, in particular, stye, and as a result, show a disease state similar to that of human stye using staphylococci suspended in an aqueous solution of casamino acid containing silica gel. A rabbit infection model was successfully created, and the present invention was completed.
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for producing a stye infection model of the present invention is characterized by inoculating bacteria suspended in an aqueous solution of casamino acid containing silica gel below the eyelid conjunctiva to induce infection. In addition, the disease model animal of the present invention is a disease model animal of stye showing a disease state similar to human stye caused by infection by inoculation of bacteria suspended in an aqueous solution of casamino acid containing silica gel under the eyelid conjunctiva.
[0006]
The bacterium used in the present invention is not particularly limited as long as it is a causative bacterium of stye, but staphylococcus is preferable, and Staphylococcus aureus or Staphylococcus epidermidis is particularly preferable. The animal used in the present invention is not particularly limited as long as it is a mammal other than a human, and includes monkeys, dogs, rabbits and the like used for experiments, and in particular, rabbit Japanese white native species and rabbit New Zealand white Laboratory rabbits such as species are preferred.
[0007]
Specifically, the production method of the present invention comprises (1) suspending cultured bacterial cells in (2) silica gel-containing casamino acid solution, (3) inoculating under the eyelid conjunctiva of an animal, and (4) infecting the animal. This is a method characterized by causing By this method, a pathological model animal exhibiting symptoms similar to stye can be prepared.
The silica gel particles used in the present invention may have a particle size of 1 to 200 μm, preferably a particle size of 2 to 45 μm.
The content of silica gel in the aqueous solution of casamino acid containing silica gel is 0.1 to 2.0% by weight, preferably 0.5 to 1.5% by weight. The content of casamino acid is 0.1 to 2.0% by weight, preferably 0.5 to 1.5% by weight.
Inoculum of subconjunctival bacteria suspended in silica gel containing casamino aqueous solution varies depending on species, 10 3 to 10 8 CFU may be a (Colony Formation Unit) / palpebral conjunctiva.
The amount of the inoculated bacterial solution may be appropriately determined depending on the animal, the amount of the bacteria, and the like. In rabbits, the amount is 10 to 100 μL, preferably 40 to 60 μL.
According to the method of the present invention, pus spots occur 1 to 5 days after inoculation of the bacterium, and pus spots can be observed even 14 days after the inoculation of the bacterium.
[0008]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples.
Example 1
Bacteria: Staphylococcus aureus and Staphylococcus epidermidis from conjunctivitis
Animal: Rabbit (Japanese native species)
Method: Bacteria cultured overnight at 37 ° C. on Mueller-Hinton agar (MHA) plates were inoculated into 10 mL of Brain Heart Infusion Broth and incubated at 37 ° C. After overnight culture, the bacterial solution was centrifuged at room temperature at 3000 rpm for 10 minutes to remove the culture solution. To the obtained cells, 1 mL of a 1% casamino acid solution containing 1% silica gel (Silica gel G, Merck Art. 7731) was added, and the well-suspended solution was serially diluted at a common ratio of 10 to give each inoculum. And Under the upper and lower eyelid conjunctiva of a rabbit subjected to general anesthesia by intravenous administration of sodium pentobarbital and corneal conjunctival surface anesthesia by instillation of oxybuprocaine hydrochloride, a syringe with 50 μL of the prepared bacterial solution was attached with a 25G needle. Inoculation was used to induce infection. After infection, pus spots were observed. Further, the pus spot was scratched with a double-level slit knife under daily general anesthesia and keratoconjunctival surface anesthesia of the rabbit, and the pus content was collected to measure the bacterial amount. After measuring the weight of the collected pus content, 300 μL of sterile physiological saline was added and homogenized. The prepared homogenate was serially diluted with sterile physiological saline, 50 μL of the diluted homogenate was applied to an MHA plate, cultured at 37 ° C. overnight, the number of bacteria was counted, and the amount of bacteria per 1 mg of pus was calculated.
Results: Staphylococcus aureus at a bacterial mass of about 5 × 10 3 CFU / conjunctiva of the eyelid and conjunctiva, and Staphylococcus epidermidis at a bacterial mass of about 2 × 10 6 CFU / conjunctiva of the eyelid. Spots were produced, and a pus spot was observed 14 days after inoculation of the bacterium with a bacterial amount of about 5 × 10 7 CFU / eyelid conjunctiva or more. In addition, in both strains, the inoculum was recovered from the pus content depending on the amount of the inoculum.
[0009]
【The invention's effect】
According to the method of the present invention, a pathological model animal of stye, which is an extraocular infection, can be produced, and the efficacy of an antimicrobial agent can be more accurately evaluated by using the produced stye pathological model animal. .
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003152420A JP4355172B2 (en) | 2002-05-31 | 2003-05-29 | Stye model animal of stye and production method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002158806 | 2002-05-31 | ||
| JP2003152420A JP4355172B2 (en) | 2002-05-31 | 2003-05-29 | Stye model animal of stye and production method thereof |
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| Publication Number | Publication Date |
|---|---|
| JP2004049228A true JP2004049228A (en) | 2004-02-19 |
| JP4355172B2 JP4355172B2 (en) | 2009-10-28 |
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| JP2003152420A Expired - Fee Related JP4355172B2 (en) | 2002-05-31 | 2003-05-29 | Stye model animal of stye and production method thereof |
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| Country | Link |
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| JP (1) | JP4355172B2 (en) |
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