JP2020147517A - COMPOSITION FOR ENHANCING EXPRESSION OF bFGF - Google Patents
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本発明は、ｂＦＧＦの発現増強作用を有する組成物に関する。 The present invention relates to a composition having an expression enhancing action of bFGF.
ｂＦＧＦ（塩基性線維芽細胞増殖因子）は、線維芽細胞の増殖を著しく促進する蛋白質として見出された。その後、ｉｎ ｖｉｔｒｏにおいて線維芽細胞の増殖を促進するのみならず、血管内皮細胞、血管平滑筋細胞、上皮細胞などの種々の細胞に対してもその増殖や遊走、分化に対する促進作用を有することが明らかにされてきた。また、ｉｎ ｖｉｖｏにおいても、強力な血管新生作用を有することが明らかになってきた。ｂＦＧＦはこのような薬理作用を持つことから、難治性皮膚潰瘍の治療剤として開発が進められ、臨床研究において優れた治療効果と安全性が確認され、現在、市販されている。また、ｂＦＧＦは骨組織に対しても有用な作用を示し、骨折治癒を促進することなどが知られている。 bFGF (Basic Fibroblast Growth Factor) was found as a protein that significantly promotes fibroblast growth. After that, it not only promotes the proliferation of fibroblasts in vitro, but also has an action of promoting the proliferation, migration, and differentiation of various cells such as vascular endothelial cells, vascular smooth muscle cells, and epithelial cells. It has been revealed. In addition, it has been clarified that it has a strong angiogenic effect also in vivo. Since bFGF has such a pharmacological action, it has been developed as a therapeutic agent for intractable skin ulcers, and its excellent therapeutic effect and safety have been confirmed in clinical studies, and it is currently on the market. It is also known that bFGF has a useful action on bone tissue and promotes fracture healing.
一方、近年ｂＦＧＦと視神経系細胞の関係が注目されている。
網膜において視神経細胞は、グリア細胞の一種である多数のミュラー細胞に包まれて存在しており、ミュラー細胞は、神経シナプスを包み込んで保護するとともに、ｂＦＧＦや脳由来神経栄養因子（ＢＤＮＦ）といった網膜中の細胞を保護する物質を産生する。そして、ｂＦＧＦは遺伝性網膜ジストロフィーにおける視細胞障害からの保護、光刺激からの視細胞の保護、網膜色素上皮細胞の貪食促進などの作用を発揮し、視細胞変性を抑制することがいわれている。眼に入った光の受容を主な機能とする視細胞を保護しその機能を維持することは、光の感受性やコントラスト感度、分解能、色覚の認識等の維持・向上に寄与する。また、視細胞の機能障害が発症要因となる加齢黄斑変性や網膜色素変性の予防にも効果が期待される。特許文献１には、網膜神経死の防止及び眼疾患の治療としてｂＦＧＦ又はＦＧＦ−５を投与することが記載されている。
しかしこの治療方法は、投与に当たって外科的な手術と併用しなければならないなど大きな問題がある。
On the other hand, in recent years, attention has been paid to the relationship between bFGF and optic nerve system cells.
In the retina, optic nerve cells exist surrounded by a large number of Muller cells, which are a type of glial cells, and the Muller cells wrap and protect nerve synapses, and the retina such as bFGF and brain-derived neurotrophic factor (BDNF). Produces substances that protect the cells inside. It is said that bFGF exerts actions such as protection from photoreceptor damage in hereditary retinal dystrophy, protection of photoreceptor cells from light stimulation, and promotion of phagocytosis of retinal pigment epithelial cells, and suppresses photoreceptor cell degeneration. .. Protecting and maintaining the function of photoreceptor cells whose main function is the reception of light entering the eye contributes to the maintenance and improvement of light sensitivity, contrast sensitivity, resolution, color vision recognition, and the like. It is also expected to be effective in preventing age-related macular degeneration and retinitis pigmentosa, which are caused by photoreceptor cell dysfunction. Patent Document 1 describes administration of bFGF or FGF-5 for the prevention of retinal nerve death and the treatment of eye diseases.
However, this treatment method has a big problem that it must be administered in combination with surgical operation.
特許文献２には、ミュラー細胞に作用する物質として、ＰＡＣＡＰ（下垂体アデニル酸シクラーゼ活性化ポリペプチド：Ｐｉｔｕｉｔａｒｙ ａｄｅｎｙｌａｔｅ ｃｙｃｌａｓｅ−ａｃｔｉｖａｔｉｎｇ ｐｏｌｙｐｅｐｔｉｄｅ）を投与して、網膜の主要なグリア細胞（神経膠細胞）であるミュラー細胞の内因性生理活性因子ＩＬ−６（インターロイキン−６）の産生を促進し、間接的に、視神経を形成する網膜神経節細胞（ＲＧＣ：Ｒｅｔｉｎａｌ Ｇａｎｇｌｉｏｎ Ｃｅｌｌ）の細胞死を抑制、遅延させることができる治療剤が開示されている。 In Patent Document 2, PACAP (Pituitary adenylate cyclase-activating polypeptide) is administered as a substance acting on Muller cells in major glial cells (glial cells) of the retinal. Promotes the production of the endogenous physiologically active factor IL-6 (interleukin-6) in certain Muller cells, and indirectly suppresses or delays cell death of retinal ganglion cells (RGCs) that form the optic nerve. The therapeutic agents that can be made to do so are disclosed.
一方、フラボノールの一種であるガランギンは、プロポリス中から分離されている（特許文献３〜４）。ガランギンには、抗炎症効果、活性酸素消去酵素の発現促進効果（特許文献５）、肝虚血再灌流障害の緩和効果、アルツハイマーの原因であるβアミロイドタンパク質前躯体（ＡＰＰ）のＢＡＣＥ阻害剤としての用途（特許文献６）などが知られている。
ガランギンの視覚系への作用としては、糖尿病網膜症における血液網膜関門の機能障害の緩和が報告されているが、ガランギンのミュラー細胞や視神経系への作用は知られていない。
On the other hand, galangin, which is a kind of flavonol, is separated from propolis (Patent Documents 3 to 4). Galangin has an anti-inflammatory effect, an effect of promoting the expression of active oxygen scavenging enzyme (Patent Document 5), an effect of alleviating hepatic ischemia-reperfusion injury, and a BACE inhibitor of β-amyloid protein precursor (APP), which is the cause of Alzheimer's disease. (Patent Document 6) and the like are known.
As an effect of galangin on the visual system, alleviation of dysfunction of the blood-retinal barrier in diabetic retinopathy has been reported, but the effect of galangin on Muller cells and the optic nerve system is unknown.
本発明者らは、多数の天然物や化合物のミュラー細胞に対する作用を研究する過程で、網膜中のミュラー細胞に作用してｂＦＧＦの発現を促進する効果を有する物質を見出し、本発明をなした。
すなわち、本発明は、網膜中のミュラー細胞に作用し、ミュラー細胞のｂＦＧＦの発現を促進する組成物を提供することを課題とする。
In the process of studying the effects of many natural products and compounds on Muller cells, the present inventors have found a substance having an effect of acting on Muller cells in the retina to promote the expression of bFGF, and have made the present invention. ..
That is, it is an object of the present invention to provide a composition that acts on Muller cells in the retina and promotes the expression of bFGF in Muller cells.
本発明は、以下の構成からなる。
（１）ガランギンを有効成分として含有する、ｂＦＧＦの発現増強用組成物。
（２）ガランギンを有効成分として含有する、視覚機能改善用の組成物。
The present invention has the following configuration.
(1) A composition for enhancing the expression of bFGF containing galangin as an active ingredient.
(2) A composition for improving visual function, which contains galangin as an active ingredient.
本発明により、ガランギンを有効成分とするミュラー細胞のｂＦＧＦの発現を促進する組成物が提供される。また本発明の組成物はミュラー細胞のｂＦＧＦの発現を促進するため、網膜における視細胞を保護し、その機能を維持することで、光の感受性やコントラスト感度、分解能、色覚の認識、視野、視力（特に夜間視力）等の維持・向上に寄与する。また、視細胞の機能障害が発症要因となる加齢黄斑変性や網膜色素変性の予防にも効果が期待される。さらに、網膜の複合的な機能の障害が要因となって起こるピント調節能の低下や、眼精疲労の症状の改善にも効果をもたらす可能性がある。このため視覚機能改善の目的で使用できる。またガランギンはプロポリス中に含有されており、長期間の摂取経験があり、安全性も高い。ガランギンあるいはガランギンを含有する組成物の経口摂取により、外科的手術よりも非侵襲的かつ安全性の高い方法で網膜中のｂＦＧＦを増加させることができる。 The present invention provides a composition that promotes the expression of bFGF in Muller cells containing galangin as an active ingredient. In addition, since the composition of the present invention promotes the expression of bFGF in Muller cells, it protects photoreceptor cells in the retina and maintains its function, thereby causing light sensitivity, contrast sensitivity, resolution, color vision recognition, visual field, and visual acuity. Contributes to maintenance and improvement of (especially night vision). It is also expected to be effective in preventing age-related macular degeneration and retinitis pigmentosa, which are caused by photoreceptor cell dysfunction. In addition, it may be effective in reducing focus control ability caused by impaired complex function of the retina and improving symptoms of eye strain. Therefore, it can be used for the purpose of improving visual function. In addition, galangin is contained in propolis, has a long-term ingestion experience, and is highly safe. Oral ingestion of galangin or a composition containing galangin can increase bFGF in the retina in a non-invasive and safer way than surgery.
本発明について具体的に説明する。
なお、本発明でいう「組成物」とは、ガランギンを含有し、網膜中のミュラー細胞のｂＦＧＦの発現を促進するものをいう。「組成物」には食品、医薬品、健康食品、動物用飼料を包含する。
また本発明でいう視覚機能改善とは、光の感受性やコントラスト感度、分解能、色覚の認識、視野、視力（特に夜間視力）、ピント調節能の維持・向上、加齢黄斑変性や網膜色素変性、遺伝性網膜疾患の予防および症状緩和、眼精疲労の予防および改善をいう。
The present invention will be specifically described.
The "composition" as used in the present invention refers to a composition containing galangin and promoting the expression of bFGF in Muller cells in the retina. The "composition" includes foods, pharmaceuticals, health foods and animal feeds.
Further, the visual function improvement referred to in the present invention means light sensitivity, contrast sensitivity, resolution, color vision recognition, visual field, visual acuity (particularly night vision), maintenance / improvement of focus adjustment ability, age-related macular degeneration and retinal pigment degeneration. It refers to the prevention and alleviation of hereditary retinal diseases and the prevention and improvement of eye strain.
ガランギン（Ｇａｌａｎｇｉｎ）とは、２−フェニル−３，５，７−トリヒドロキシ−４Ｈ−１−ベンゾピラン−４−オン（２−ｐｈｅｎｙｌ−３，５，７−ｔｒｉｈｙｄｒｏｘｙ−４Ｈ−１−ｂｅｎｚｏｐｙｒａｎ−４−ｏｎｅ）であり、プロポリスや薬用植物エキスに含まれる。ガランギンは下記の化学式１で表すことができる。 Galangin is 2-phenyl-3,5,7-trihydroxy-4H-1-benzopyran-4-one (2-phenyl-3,5,7-trihydroxy-4H-1-benzopyran-4-on). One), which is contained in propolis and medicinal plant extracts. Galangin can be represented by the following chemical formula 1.
ガランギンは、前記のとおり、プロポリスに多く含有されているが、カレーの香辛料の一つとして使用されているガランガル（Ａｌｐｉｎｉａ ｏｆｆｉｃｉｎａｒｕｍ）の根茎に多く含まれている。本発明には、これらの物質から適宜公知の抽出方法を組み合わせて抽出した抽出物を用いることができる。また公知の有機化学合成法（ｄｅ Ｓｏｕｚａ ＲＦ，ｅｔ ａｌ，．Ｓｐｅｃｔｒｏｃｈｉｍ Ａｃｔａ Ａ Ｍｏｌ Ｂｉｏｍｏｌ Ｓｐｅｃｔｒｏｓｃ．２００５ Ｊｕｌ；６１（９）：１９８５−９０．、Ｓａｔｏ Ｓ，ｅｔ ａｌ，．Ｃａｒｂｏｈｙｄｒ Ｒｅｓ． ２００６ Ｊｕｎ １２；３４１（８）：９６４−７０．）により得ることができる。 As mentioned above, galangin is abundantly contained in propolis, but is also abundantly contained in the rhizome of galangal (Alpinia officinarum), which is used as one of the spices for curry. In the present invention, an extract extracted from these substances by appropriately combining known extraction methods can be used. Also known organic chemical synthesis methods (de Souza RF, et al, .Spectrochim Acta A Mol Biomol Spectrosc. 2005 Jul; 61 (9): 1985-90., Sato S, et al, .Carboydr Res. 2006; 341 (8): 964-70.).
さらに得られた合成物や抽出物を公知の分離精製方法を適宜組み合わせてガランギンの純度を高めてもよい。
上記合成や抽出により得られるガランギンは、医薬品上又は食品上許容し得る規格に適合し、本発明の効果を発揮するものであれば、粗精製物であってもよい。
具体的には、抽出としては、水、熱水、アルコール水等の水及び／又は有機溶剤等の極性及び非極性溶剤を用いて行うことが挙げられる。また、分離精製としては、有機溶剤沈殿、遠心分離、限界濾過膜、高速液体クロマトグラフやカラムクロマトグラフ等を用いて行うことが挙げられる。
Further, the obtained synthesis or extract may be appropriately combined with a known separation and purification method to increase the purity of galangin.
The galangin obtained by the above synthesis or extraction may be a crude product as long as it conforms to the standards acceptable for pharmaceuticals or foods and exhibits the effects of the present invention.
Specifically, the extraction may be carried out using water such as water, hot water, alcoholic water and / or polar and non-polar solvents such as an organic solvent. In addition, examples of the separation and purification include organic solvent precipitation, centrifugation, microfiltration membrane, high performance liquid chromatograph, column chromatograph and the like.
ガランギンは、網膜のミュラー細胞に作用し、ｂＦＧＦの発現を促進することができる。従って、ガランギンは、網膜内の視細胞に保護的に作用し、光の感受性やコントラスト感度、分解能、色覚の認識、視野、視力（特に夜間視力）、ピント調節能の維持・向上、加齢黄斑変性や網膜色素変性、遺伝性網膜疾患の予防および症状緩和、眼精疲労の予防および改善などの効果を示す。したがって当該症状若しくは疾患を予防、改善又は治療するための食品・飲料、化粧品、医薬部外品、医薬品等として使用可能である。そして、ガランギンは、これらの作用を示す飲食品、病者用食品、特定保健用食品、機能性表示食品等として利用することができる。 Galangin can act on Muller cells of the retina to promote the expression of bFGF. Therefore, galangin acts protectively on the photoreceptor cells in the retina, and maintains / improves light sensitivity, contrast sensitivity, resolution, color vision recognition, visual field, visual acuity (especially night vision), focus adjustment ability, and age-related macular degeneration. It has effects such as degeneration, retinitis pigmentosa, prevention and alleviation of hereditary retinal diseases, and prevention and improvement of eye strain. Therefore, it can be used as food / beverage, cosmetics, quasi-drugs, pharmaceuticals, etc. for preventing, improving or treating the symptom or disease. Galangin can be used as foods and drinks exhibiting these actions, foods for the sick, foods for specified health use, foods with functional claims, and the like.
ガランギンを食品・飲料として使用する場合、例えば、果汁飲料、炭酸飲料、茶系飲料、乳飲料、アルコール飲料、清涼飲料等の飲料、ゼリー状食品や各種スナック類、焼き菓子、ケーキ類、チョコレート、ガム、飴、スープ類等、あらゆる食品・飲料形態とすることができる。 When galangin is used as a food / beverage, for example, fruit juice beverages, carbonated beverages, tea-based beverages, dairy beverages, alcoholic beverages, soft beverages and other beverages, jelly-like foods and various snacks, baked goods, cakes, chocolates, etc. It can be in any food / beverage form such as gum, candy, soups, etc.
また、ガランギンを含有する組成物をサプリメントや医薬品として使用する場合、例えば、錠剤、顆粒剤等の経口用固形成形剤、内服液剤、シロップ剤等の経口用液体製剤として利用することができる。 When the composition containing galangin is used as a supplement or a pharmaceutical product, it can be used as, for example, an oral solid molding agent such as a tablet or a granule, or an oral liquid preparation such as an oral liquid preparation or a syrup preparation.
ガランギンを含有する製剤は、一般的な製造法により、直接又は製剤上許容し得る担体とともに混合、分散した後、所望の形態に加工することによって得ることができる。この場合、本発明に用いられるこれら植物又はその抽出物の他に、かかる形態に一般的に用いられる植物油、動物油等の油性基剤、鎮痛消炎剤、鎮痛剤、殺菌消毒剤、収斂剤、ホルモン剤、ビタミン類、保湿剤、紫外線吸収剤、アルコール類、キレート剤、ｐＨ調整剤、防腐剤、増粘剤、色素、香料等を本発明の効果を妨害しない範囲で適宜配合することができる。 The galangin-containing preparation can be obtained by mixing and dispersing directly or with a pharmaceutically acceptable carrier by a general production method, and then processing into a desired form. In this case, in addition to these plants or extracts thereof used in the present invention, oily bases such as vegetable oils and animal oils commonly used in such forms, analgesic and anti-inflammatory agents, analgesics, bactericidal disinfectants, astringents, hormones. Agents, vitamins, moisturizers, UV absorbers, alcohols, chelating agents, pH adjusters, preservatives, thickeners, pigments, fragrances and the like can be appropriately blended as long as they do not interfere with the effects of the present invention.
本発明のガランギンを含有する組成物は、特に限定されるものではないが、好ましくはガランギンとして通常全組成の０．００００１〜９９．５質量％の範囲で配合される組成物とする。 The composition containing galangin of the present invention is not particularly limited, but is preferably a composition blended as galangin in the range of 0.00001 to 99.5% by mass of the total composition.
実施例、試験例を示し、本発明についてさらに説明する。
＜実施例１．ｂＦＧＦ発現促進作用＞
（１）初代ミュラー細胞の調製
初代ミュラー細胞は次の方法で調製した。
７日齢のＳＤラット１０匹から網膜を単離した。単離した網膜は、ＰＢＳ（−）で５倍希釈したトリプシン−ＥＤＴＡ（Ｓｉｇｍａ）にＤＮａｓｅＩ（Ｒｏｃｈｅ）を最終濃度が１００ｎｇ／ｍｌになるように添加した溶液１０ｍＬ中で、３７℃で３０分間静置した。
Examples and test examples will be shown, and the present invention will be further described.
<Example 1. bFGF expression promoting action>
(1) Preparation of primary Muller cells The primary Muller cells were prepared by the following method.
Retinas were isolated from 10 7-day-old SD rats. The isolated retina was allowed to stand at 37 ° C. for 30 minutes in 10 mL of a solution of trypsin-EDTA (Sigma) diluted 5-fold with PBS (-) and DNase I (Roche) added to a final concentration of 100 ng / ml. Placed.
次いで、ピペッティング操作により網膜組織を分散させ、２，４００ｒｐｍの回転数、室温で５分間遠心した。遠心後、分散溶液の上清を吸引除去し、１０％ＦＢＳ（ｂｉｏｓｅｒａ）、１％ ペニシリン−ストレプトマイシン（Ｓｉｇｍａ）を含むＤＭＥＭ−Ｆ１２培地（Ｇｉｂｃｏ）２５ｍＬに懸濁した。
懸濁液を７０μＭのセルストレーナー（ＦＡＬＣＯＮ）に通した後、細胞数を計測し、４．２×１０６ｃｅｌｌｓ／ｍｌの濃度になるよう前記の培地に希釈した。
Ｔ−７５細胞培養用フラスコ（住友ベークライト）に１０ｍＬずつ播種し、３７℃、５％ ＣＯ２環境下で培養した（培養０日目）。
培養３日目に、培地を吸引除去し、新しい培地２０ｍＬに交換し、再び３７℃、５％ ＣＯ２環境下でさらに培養した。
The retinal tissue was then dispersed by pipetting and centrifuged at room temperature for 5 minutes at a rotation speed of 2,400 rpm. After centrifugation, the supernatant of the dispersion was removed by suction and suspended in 25 mL of DMEM-F12 medium (Gibco) containing 10% FBS (biosera) and 1% penicillin-streptomycin (Sigma).
After passing the suspension through a 70 μM cell strainer (FALCON), the number of cells was counted and diluted in the above medium to a concentration of 4.2 × 10 6 cells / ml.
10 mL each was seeded in a T-75 cell culture flask (Sumitomo Bakelite) and cultured at 37 ° C. in a 5% CO 2 environment (culture day 0).
On the 3rd day of culturing, the medium was removed by suction, replaced with 20 mL of new medium, and further cultured at 37 ° C. in a 5% CO 2 environment.
培養７日目に、Ｗｅｉ−Ｔａｏ Ｓｏｎｇらの方法（Ｏｐｈｔｈａｌｍｏｌ ２０１３；６（６）：７７８−７８４）に準じて、ミュラー細胞を純化した。
培養８日目に、ミュラー細胞を剥がし、４×１０５ｃｅｌｌｓ／ｍｌの濃度になるよう前記の培地に懸濁し、９６ｗｅｌｌ ｃｅｌｌ ｃｕｌｔｕｒｅ ｐｌａｔｅ（ＦＡＬＣＯＮ）に１００μＬずつ播種し、３７℃、５％ ＣＯ２環境下で培養した。
On the 7th day of culture, Muller cells were purified according to the method of Wei-Tao Song et al. (Ophthermol 2013; 6 (6): 778-784).
On the 8th day of culture, Muller cells were peeled off, suspended in the above medium to a concentration of 4 × 10 5 cells / ml, seeded in 96-well cell culture plate (FALCON) at a rate of 100 μL each, and 37 ° C., 5% CO 2 Cultured in the environment.
（２）ガランギンのｂＦＧＦ発現促進効果の確認
ミュラー細胞のｂＦＧＦ発現促進効果は、上記のミュラー細胞を用いて次のとおり実施した。
ガランギン（Ｇａｌａｎｇｉｎ）は、市販の試薬を用いて、（ＣＡＹＭＡＮ社：純度９８％以上）ＤＭＳＯで溶解し使用した。
(2) Confirmation of bFGF expression promoting effect of galangin The bFGF expression promoting effect of Muller cells was carried out as follows using the above Muller cells.
Galangin was dissolved in DMSO (CAYMAN: purity 98% or more) using a commercially available reagent and used.
培養９日目の初代ミュラー細胞の培地を全量除去し、１％ＦＢＳを含むＤＭＥＭ−Ｆ１２培地１００μＬに交換し、３７℃、５％ ＣＯ２の環境下で９０分間培養し、ミュラー細胞を血清飢餓状態に置いた。ガランギンを１５μＭの濃度になるように添加し、１８０分間培養後に上清を除去し、ＲＮｅａｓｙ ９６ ｋｉｔ（ＱＩＡＧＥＮ）を用い添付の説明書に従ってＲＮＡを調製した。
ＲＮＡ調製の際、ＤＮａｓｅＩＫｉｔ（ＱＩＡＧＥＮ）を用い、ゲノムＤＮＡの除去を行った。
On the 9th day of culture, the medium of the primary Muller cells was completely removed, replaced with 100 μL of DMEM-F12 medium containing 1% FBS, and cultured for 90 minutes in an environment of 37 ° C. and 5% CO 2 , and the Muller cells were serum starved. Put in the state. Galangin was added to a concentration of 15 μM, the supernatant was removed after culturing for 180 minutes, and RNA was prepared using RNeasy 96 kit (QIAGEN) according to the attached instructions.
When preparing RNA, genomic DNA was removed using DNaseIKit (QIAGEN).
約４０ｎｇの調製したＲＮＡは、ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ ｋｉｔ（Ｔａｋａｒａ）を用いて添付の説明書に従いｃＤＮＡを作製した。ｂＦＧＦの発現量はＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ＴａｑＭａｎ Ｇｅｎｅ Ｅｘｐｒｅｓｓｉｏｎ Ａｓｓａｙを用いて、次の方法で定量した。
１μＬのｃＤＮＡとラットｂＦＧＦ Ｔａｑｍａｎ ｐｒｏｂｅ、Ｐｒｅｍｉｘ Ｅｘ Ｔａｑ（タカラバイオ）を所定の割合で混合し、計１０μＬとなるようにＰＣＲ ｇｒａｄｅ ｗａｔｅｒ（Ｉｎｖｉｔｒｏｇｅｎ）を添加し混合したものを反応液とした。
調製した反応液は、Ｑｕａｎｔ Ｓｔｕｄｉｏ ５（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用い、［（９５℃、２０秒）→（９５℃、１秒）→（６０℃、２０秒）］×４５サイクルの反応条件で測定を行った。
About 40 ng of prepared RNA was prepared using PrimeScript RT reagent kit (Takara) according to the attached instructions. The expression level of bFGF was quantified by the following method using Applied Biosystems TaqMan Gene Expression Assay.
1 μL of cDNA, rat bFGF Taqman probe, and Premix Ex Taq (Takarabio) were mixed at a predetermined ratio, and PCR grade water (Invitrogen) was added and mixed so as to have a total of 10 μL, and the mixture was used as a reaction solution.
The prepared reaction solution was measured using Quant Studio 5 (Applied Biosystems) under the reaction conditions of [(95 ° C., 20 seconds) → (95 ° C., 1 second) → (60 ° C., 20 seconds)] × 45 cycles. went.
内在性コントロールにはラットｇａｐＤＨ Ｔａｑｍａｎ ｐｒｏｂｅ（Ｔａｑｍａｎ ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ）を用い、上記と同様の反応条件で測定を行った。測定により得られたＣｔ値からｇａｐＤＨを内部標準としてΔΔＣｔ法により、各サンプルの相対的遺伝子発現量を求めた。 A rat gapDH Taqman probe (Taqman gene expression assay) was used as an endogenous control, and measurement was performed under the same reaction conditions as described above. From the Ct value obtained by the measurement, the relative gene expression level of each sample was determined by the ΔΔCt method using gapDH as an internal standard.
ＰＣＲに使用したＴａｑｍａｎ ｐｒｏｂｅの製品情報、仕様は以下のとおりである。 Ｇａｐｄｈ：Ａｐｐｉｌｉｅｄ ｂｉｏｓｙｓｔｅｍｓ Ａｓｓａｙ ＩＤ：Ｒｎ０１４６２６６２＿ｇ１（Ｄｙｅ：ＶＩＣ，Ｑｕｅｎｃｈｅｒ：ＭＧＢ）
ｂＦＧＦ：Ａｐｐｌｉｅｄ ｂｉｏｓｙｓｔｅｍｓ Ａｓｓａｙ ＩＤ：Ｒｎ００５７０８０９＿ｍ１（Ｄｙｅ：ＦＡＭ，Ｑｕｅｎｃｈｅｒ：ＭＧＢ）
The product information and specifications of Taqman probe used for PCR are as follows. Gapdh: Applied biosystems Assay ID: Rn01462662_g1 (Dye: VIC, Quencher: MGB)
bFGF: Applied biosystems Assay ID: Rn00570809_m1 (Dye: FAM, Quencher: MGB)
（３）結果
図１に示すとおり、ミュラー細胞は、ガランギンの添加により、明らかにｂＦＧＦの発現が促進された。
ミュラー細胞が分泌するｂＦＧＦは、光刺激からの視細胞保護効果が確認されており、さらに視細胞が障害を受けた後に視細胞のｂＦＧＦをはじめとするＦＧＦ受容体の発現が急激に亢進するため、ｂＦＧＦは視細胞保護機構に重要な役割を担っていることが示唆されている。
さらに脱落した視細胞外節を貪食、消化することにより視細胞のターンオーバーに重要な役割を果たしている網膜色素上皮細胞は、ｂＦＧＦにより貪食が促進されるが、視細胞の変性、色素上皮細胞の貪食能の低下は、光の感受性やコントラスト感度、分解能、色覚の認識、視野、視力（特に夜間視力）、ピント調節能の低下、眼精疲労等の症状につながって、加齢黄斑変性、網膜色素変性といった網膜変性疾患の原因にもなる。
したがって、本試験によってガランギンがミュラー細胞のｂＦＧＦの発現促進を行なうことが明らかになったため、これらの障害による視覚機能の維持や改善、網膜疾患の予防や症状緩和にガランギンが有効であることが明らかとなった。
(3) Results As shown in FIG. 1, in Muller cells, the expression of bFGF was clearly promoted by the addition of galangin.
The bFGF secreted by Muller cells has been confirmed to have a protective effect on photoreceptor cells from photostimulation, and the expression of FGF receptors such as bFGF in photoreceptor cells is rapidly increased after the photoreceptor cells are damaged. , BFGF has been suggested to play an important role in the photoreceptor protection mechanism.
Furthermore, retinal pigment epithelial cells, which play an important role in photoreceptor turnover by phagocytosing and digesting the shed photoreceptor segments, are promoted by bFGF, but retinitis pigmentosa and pigment epithelial cells Decreased phagocytosis leads to symptoms such as light sensitivity, contrast sensitivity, resolution, color vision recognition, visual field, visual acuity (especially night vision), decreased focus control, and eye fatigue, leading to age-related macular degeneration and retina. It also causes retinal degeneration diseases such as pigment degeneration.
Therefore, this study revealed that galangin promotes the expression of bFGF in Muller cells, and it is clear that galangin is effective in maintaining and improving visual function due to these disorders, preventing retinal diseases, and alleviating symptoms. It became.
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