JP2014189549A - Accelerator for new generation or hippocampal neuron - Google Patents
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Abstract
Description
本発明は、海馬神経の新生をもたらす剤に関する。 The present invention relates to an agent that causes neogenesis of hippocampal nerves.
成熟した哺乳動物では、一般に神経細胞は***能を持っていない特殊な細胞からなる組織である。そのため一旦傷害を受けると長期にわたって障害が続く。特に脳や脊髄といった中枢神経系では全く再生能がないと言われてきた。
外因性の傷害による脊髄損傷に代表される不随やアルツハイマー病、パーキンソン病といった神経変性疾患に対する治療方法がないことも、中枢神経における再生能が無いことが一つの原因である。一方で、末梢神経は再生能を有しており、一旦切断された後も軸索が再生し機能が回復する。しかしこの場合にも再生に要する期間は数ヶ月から1年以上と長期間を要する。このため、患者にとっての苦痛は大きい。
また神経の再生に長期間を要するために、その間に神経細胞が死滅し機能回復に至らない場合も多い。このように再生能を有する末梢神経の場合も、脳や脊髄といった中枢神経系の環境では全く伸長することはできない。そのため、中枢神経系には神経の伸長を阻止する物質が存在していることが推測されている。この中枢神経系に存在する神経再生阻害物質を抗体などで抑制すると、中枢で一部の神経再生が起こり、機能の回復も見られる。最近、この中枢神経再生阻害因子としてNogoが発見された（非特許文献１：Nature 403,434,2000、非特許文献２：Nature 403,439,2000）。しかし、Nogoを抑制することによって再生する神経線維は一部であり、他の再生阻害物質が存在するのではないかと考えられているが、インビボで神経の再生阻害に働いている因子の一つとしてセフォマリンも推定されている（非特許文献３：Cell 75,217,1993、非特許文献４：Cell 75,1389,1993）が、これまで明らかにされていない。
In mature mammals, nerve cells are generally tissues composed of special cells that do not have division ability. Therefore, once injured, the failure continues for a long time. In particular, it has been said that the central nervous system such as the brain and spinal cord has no regeneration ability.
One of the reasons is that there is no cure for neurodegenerative diseases such as involuntary spinal cord injury due to exogenous injury, Alzheimer's disease, and Parkinson's disease, and the lack of regenerative ability in the central nervous system. On the other hand, the peripheral nerve has a regeneration ability, and even after being cut once, the axon is regenerated and the function is restored. However, even in this case, the period required for the regeneration takes a long period of several months to one year or more. For this reason, the pain for the patient is great.
In addition, since nerve regeneration takes a long time, nerve cells die during that time, and function recovery often does not occur. In the case of the peripheral nerve having the regeneration ability as described above, it cannot be extended at all in the central nervous system environment such as the brain and spinal cord. For this reason, it has been speculated that there exists a substance in the central nervous system that prevents nerve growth. When a nerve regeneration inhibitor that exists in the central nervous system is suppressed with an antibody or the like, partial nerve regeneration occurs in the center, and functional recovery is also seen. Recently, Nogo was discovered as a central nerve regeneration inhibitor (Non-patent document 1: Nature 403,434,2000, Non-patent document 2: Nature 403,439, 2000). However, it is thought that there are some nerve fibers that regenerate by suppressing Nogo and that other regeneration inhibitors exist, but it is one of the factors that work to inhibit nerve regeneration in vivo. Cefofalin has also been estimated (Non-patent Document 3: Cell 75, 217, 1993, Non-Patent Document 4: Cell 75, 1389, 1993), but has not been clarified so far.
近年、アルツハイマー病などの認知症治療薬である塩酸ドネペジル（商品名：アリセプト）が、海馬内のインシュリン様成長因子（IGF）を介して海馬神経を再生させることが発見された（非特許文献５：CLINICIAN、2009、No.583、1077-1085）。このような知見が積み重なることによって再生困難とされてきた中枢神経を新生や再生する薬剤や物質の探索が進められている。特許文献１（特表２０１２−５０８２５４号公報）にはαアゴニストを投与することで中枢神経再生を促進する方法が提案されている。また特許文献２（特表２０１１−５１１６２１号公報）にはCREB経路を活性化させて神経系を活性化させるために、その遺伝子の上流域にあるマイトジェン活性化タンパク質キナーゼ伝達経路及びタンパク質キナーゼを活性化させる技術が提案されている。また特許文献３（特表２００６−５１４６３０号公報）には、神経細胞内のcAMPレベルを上昇させることによって中枢神経系の発生を促進する方法が提案されている。 In recent years, it was discovered that donepezil hydrochloride (trade name: Aricept), a therapeutic agent for dementia such as Alzheimer's disease, regenerates hippocampal nerves via insulin-like growth factor (IGF) in the hippocampus (Non-patent Document 5). : CLINICIAN, 2009, No.583, 1077-1085). The search for drugs and substances that regenerate or regenerate the central nervous system, which has been considered difficult to regenerate due to the accumulation of such knowledge, is in progress. Patent Document 1 (Japanese Patent Publication No. 2012-508254) proposes a method of promoting central nerve regeneration by administering an α agonist. Patent Document 2 (Japanese Patent Publication No. 2011-511621) discloses activating the mitogen-activated protein kinase transmission pathway and protein kinase in the upstream region of the gene in order to activate the CREB pathway and activate the nervous system. Techniques to make it possible have been proposed. Patent Document 3 (Japanese Patent Publication No. 2006-514630) proposes a method of promoting the development of the central nervous system by increasing cAMP levels in nerve cells.
これらの先行技術はいまだ具体化されておらず、中枢神経系特に認知症に係わる海馬神経系の再生や新生を促進する薬剤や物質が希求されている。 These prior arts have not yet been materialized, and there is a need for drugs and substances that promote regeneration and renewal of the central nervous system, particularly the hippocampal nervous system related to dementia.
本発明は、海馬神経の新生を促進する剤を提供することを課題とする。 An object of the present invention is to provide an agent that promotes the development of hippocampal nerves.
本発明者らは、神経系の保護作用と神経伝達に関与することが知られているホスファチジルセリンに着目して試験した結果、ホスファチジルセリンが海馬の神経系を新生することを知見したことに基づき、本発明を提案する。 Based on the fact that phosphatidylserine was born to the nervous system of the hippocampus as a result of testing by paying attention to phosphatidylserine known to be involved in the protective action and neurotransmission of the nervous system, the present inventors The present invention is proposed.
すなわち、本発明は、
（１）ホスファチジルセリンを有効成分とする海馬神経新生促進剤に関する。
That is, the present invention
(1) It relates to a hippocampal neurogenesis promoter containing phosphatidylserine as an active ingredient.
本発明は、新たな海馬神経新生促進剤を提供することができる。さらにまた本発明は海馬の萎縮に伴って発生する知的障害や疾患の予防、治療剤とすることができる。 The present invention can provide a new hippocampal neurogenesis promoting agent. Furthermore, the present invention can be used as a preventive or therapeutic agent for intellectual disabilities and diseases that occur with hippocampal atrophy.
ホスファチジルセリンは細胞膜の構成成分であるグリセロリン脂質の一種で、グリセロール骨格に２つの脂肪酸基とリン酸基、セリン基を持つ両親媒性の物質である。ホスファチジルセリンには天然物由来のものと合成されているものがある。天然物由来のものは、牛脳、骨髄、大豆、乳などから抽出される。合成される場合は、ホスファチジルコリンいわゆるレシチンから塩基交換反応によって作られる。 Phosphatidylserine is a kind of glycerophospholipid that is a component of cell membrane, and is an amphiphilic substance having two fatty acid groups, a phosphate group, and a serine group in the glycerol skeleton. Some phosphatidylserine are synthesized from natural products. Those derived from natural products are extracted from bovine brain, bone marrow, soybean, milk and the like. When synthesized, it is made from phosphatidylcholine so-called lecithin by a base exchange reaction.
本発明に関わるホスファチジルセリンは動植物の高含有組織、例えば脳などから抽出してもよいし、微生物産生によって得ることもできる。また塩基交換反応あるいは他の化学的方法で合成してもよい。 The phosphatidylserine according to the present invention may be extracted from a highly contained tissue of animals and plants, such as the brain, or may be obtained by microbial production. Moreover, you may synthesize | combine by a base exchange reaction or another chemical method.
特に本発明に使用するホスファチジルセリンとしては、大豆レシチンから抽出したものや卵黄由来ホスファチジルセリンが好ましい。卵黄由来のホスファチジルセリンとしては、卵黄に含まれるグリセロリン脂質にセリンを加え、塩基交換により合成し得られたものであってもよい。 In particular, the phosphatidylserine used in the present invention is preferably extracted from soybean lecithin or egg yolk-derived phosphatidylserine. The egg yolk-derived phosphatidylserine may be synthesized by adding serine to glycerophospholipid contained in egg yolk and performing base exchange.
本発明の海馬神経新生又は再生促進剤を製造するには、上記の方法で製造したホスファチジルセリンを用いることができ、常法に従って公知の医薬用無毒性担体と組み合わせて製剤化すればよい。本発明に関する海馬神経新生促進剤は、種々の剤型での投与が可能であり、例えば、経口投与剤としては錠剤、顆粒剤、散剤、カプセル剤、ソフトカプセル剤等の固形剤、溶液剤、懸濁剤、乳剤等の液剤、凍結乾燥製剤等が挙げられ、非経口投与剤としては注射剤のほか、坐剤、噴霧剤、経皮吸収剤等が挙げられ、これらの製剤は製剤上の常套手段により調製することができる。
医薬用担体としては、例えば、グルコース、乳糖、ショ糖、澱粉、マンニトール、デキストリン、脂肪酸グリセリド、ポリエチレングリコール、ヒドロキシエチルデンプン、エチレングリコール、ポリオキシエチレンソルビタン脂肪酸エステル、アミノ酸、アルブミン、水、生理食塩水等が挙げられる。また、必要に応じて、安定化剤、滑剤、湿潤剤、乳化剤、結合剤等の慣用の添加剤を適宜添加することができる。本発明に関わる神経新生促進剤において、卵黄由来ホスファチジルセリンの投与量は、患者の年齢、体重、症状、疾患の程度、投与スケジュール、製剤形態などにより、適宜選択・決定されるが、例えば一日あたり0.01mg/kg〜1000mg/kg体重程度とされ、一日数回に分けて投与してもよい。
In order to produce the hippocampal neurogenesis or regeneration promoter of the present invention, the phosphatidylserine produced by the above method can be used, and it may be formulated in combination with a known non-toxic pharmaceutical carrier in accordance with a conventional method. The hippocampal neurogenesis-promoting agent according to the present invention can be administered in various dosage forms. For example, oral administration includes solid agents such as tablets, granules, powders, capsules, soft capsules, solutions, suspensions, and the like. Examples include suspensions, liquids such as emulsions, freeze-dried preparations, etc. Parenteral preparations include injections, suppositories, sprays, transdermal absorption agents, and the like. It can be prepared by means.
Examples of the pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, albumin, water, and physiological saline. Etc. If necessary, conventional additives such as stabilizers, lubricants, wetting agents, emulsifiers, binders and the like can be appropriately added. In the neurogenesis-promoting agent according to the present invention, the dose of egg yolk-derived phosphatidylserine is appropriately selected and determined depending on the patient's age, weight, symptoms, disease level, administration schedule, formulation form, etc. The dose is about 0.01 mg / kg to 1000 mg / kg body weight per day, and the dose may be divided into several times a day.
また、本発明に関わるホスファチジルセリンは、食経験も豊富なことから安全性が高いと考えられ、海馬神経新生又は再生促進剤としてあるいは認知症やうつ病の予防あるいは治療効果を目的として、機能性食品として摂取することもできる。本発明に関わるホスファチジルセリンを含有することを特徴とする機能性食品は、特定保健用食品、栄養機能性食品、又は健康食品として位置づけることができる。機能性食品としては、例えば、ホスファチジルセリンに適当な助剤を添加した後、慣用の手段を用いて、食用に適した形態、例えば、顆粒状、粒状、錠剤、カプセル剤、ペースト状等に形成したものを用いることができる。この機能性食品は、そのまま食用に供してもよく、また種々の食品（例えばハム、ソーセージ、かまぼこ、ちくわ、パン、バター、粉乳、チョコレートなど）に添加して使用したり、水、酒類、果汁、牛乳、清涼飲料水等の飲料に添加して使用してもよい。かかる食品の形態における本発明の卵黄由来ホスファチジルセリンの摂取量は、対象の年齢、体重、症状、摂取スケジュール、製剤形態などにより、適宜選択・決定されるが、例えば一日あたり0.01mg/kg〜1000mg/kg体重程度とされる。 In addition, phosphatidylserine related to the present invention is considered to be highly safe because it has abundant dietary experience, and is functional as a hippocampal neurogenesis or regeneration promoter, or for the purpose of preventing or treating dementia and depression. It can also be taken as food. A functional food characterized by containing phosphatidylserine according to the present invention can be positioned as a food for specified health use, a nutritional functional food, or a health food. As a functional food, for example, after adding a suitable auxiliary agent to phosphatidylserine, it is formed into an edible form such as granules, granules, tablets, capsules, pastes, etc. using conventional means Can be used. This functional food may be used as it is, or added to various foods (eg ham, sausage, kamaboko, chikuwa, bread, butter, milk powder, chocolate, etc.), water, alcoholic beverages, fruit juice It may be used by adding to beverages such as milk and soft drinks. The intake of egg yolk-derived phosphatidylserine of the present invention in the form of such food is appropriately selected and determined according to the age, weight, symptoms, intake schedule, formulation form, etc. of the subject, for example, 0.01 mg / kg per day About 1000 mg / kg body weight.
以下に実施例、試験例を示し、本発明をさらに説明する。 The following examples and test examples further illustrate the present invention.
＜海馬神経新生試験例＞
１．動物種、系統、及び性別
ラット，Slc：SD、雄
２．週齢
投与開始時：9週齢
<Examples of hippocampal neurogenesis test>
1. Animal species, strain, and sex Rat, Slc: SD, male At the start of administration: 9 weeks of age
３．馴化
入荷時に種、系統、週齢、動物数及び性別を確認し、一般状態および外観を観察するとともに体重を測定した。馴化期間は7日間とした。
４．群分け
投与開始前日に体重を測定し1群6匹に群分けした。
５．個体識別方法
個体識別は尾部に番号を記すことで行った。群分け後の動物は3匹ずつケージに収容し、群、動物番号および個体識別番号を明記したラベルをケージ前面に付けた。
3. Acclimatization Upon arrival, the species, strain, age, number of animals and gender were confirmed, general condition and appearance were observed, and body weight was measured. The acclimatization period was 7 days.
4). Grouping On the day before the start of administration, body weight was measured and divided into 6 groups per group.
5. Individual identification method Individual identification was performed by marking the tail. Three animals after grouping were housed in cages, and a label specifying the group, animal number and individual identification number was attached to the front of the cage.
６．飼育環境
設定温度：23℃（許容範囲21〜25℃）、設定湿度：55%（許容範囲40〜70%）、照明：午前6時点灯、午後6時消灯の12時間、換気回数：17回／時に維持された動物飼育室コンベショナル区域内の飼育室で動物を飼育した。動物はTPX製エコンケージを用いて飼育し、床敷はパルソフト（オリエンタル酵母工業株式会社）を使用して週に1回以上の頻度で交換した。
6). Breeding environment Set temperature: 23 ° C (allowable range 21-25 ° C), set humidity: 55% (allowable range 40-70%), lighting: 12 hours of lighting at 6am, extinguishing at 6pm, ventilation rate: 17 times Animals were reared in a rearing room within a conventional area maintained at / time. The animals were raised using TPX ecocon cages, and the flooring was changed at least once a week using Palsoft (Oriental Yeast Co., Ltd.).
７．飼料
対照群はAIN93M固形飼料（オリエンタル酵母工業株式会社）、試験群はホスファチジルセリンをAIN93Mに混合した固形飼料（αコーンスターチと置換；オリエンタル酵母工業株式会社）を給餌器に入れて自由に摂取させた。
7). Feed The control group was AIN93M solid feed (Oriental Yeast Industry Co., Ltd.) and the test group was freely fed with solid feed mixed with phosphatidylserine in AIN93M (replaced with α corn starch; Oriental Yeast Industry Co., Ltd.). .
８．飲料水
5μカートリッジフィルターを通過させた塩酸水(pH:4〜6)を給水瓶で与えた。
９．投与量
ホスファチジルセリンは、えさ中に0.1質量%を混入した。ラット体重300g、1日摂餌量15gとして投与量を設定した。
8). Drinking water
Hydrochloric acid water (pH: 4-6) passed through a 5μ cartridge filter was given in a water bottle.
9. Dose The phosphatidylserine was mixed in 0.1% by mass in the food. The dose was set with a rat body weight of 300 g and a daily food intake of 15 g.
１０．体重・摂餌量測定
投与期間中、週に2〜3回、体重および摂餌量の測定を行った。
１１．神経新生確認方法
Bromo‐2'‐deoxyuridine (BrdU)の投与によって神経新生の確認を行った。BrdUはDNA合成のときチミジンのアナログとして取り込まれる。BrdUを取り込んだDNA（細胞核）は、DNAに取り込まれたBrdUに特異的な抗体で検出することができ、これを使うと***期（S期）の細胞核をラベルできる。すなわち投与時間を制限してBrdUを投与すれば、その期間にS期にあった細胞を検出できる（pulse labelling）。
次に示す灌流操作の5日前から1日1回5日間，新生細胞（***）マーカーであるBrdU（シグマアルドリッチジャパン株式会社）を50mg/10mL/kg腹腔内投与した。投与は午前9時から12時の間に行った。
10. Body weight and food intake measurement During the administration period, body weight and food intake were measured 2-3 times a week.
11. Neurogenesis confirmation method
Neurogenesis was confirmed by administration of Bromo-2'-deoxyuridine (BrdU). BrdU is incorporated as an analog of thymidine during DNA synthesis. The DNA (cell nucleus) incorporating BrdU can be detected with an antibody specific to BrdU incorporated into the DNA, and this can be used to label cell nucleus in the mitotic phase (S phase). That is, if BrdU is administered while limiting the administration time, cells in S phase during that period can be detected (pulse labeling).
BrdU (Sigma Aldrich Japan Co., Ltd.), a neoplastic cell (division) marker, was administered intraperitoneally 50 mg / 10 mL / kg once a day for 5 days from 5 days before the following perfusion operation. The administration was performed between 9 am and 12 am.
１２．採血、灌流および臓器摘出
PBS(-)を用いて，心臓から全身灌流を行った。その後、4%パラホルムアルデヒド・リン酸緩衝液（4%PFA; 和光純薬工業株式会社）にて灌流固定を行い、脳を摘出した。4%PFAに一晩浸漬後（4℃）、15%スクロース溶液に置き換え、さらに一晩浸漬後（4℃）、30%スクロース溶液に置換え保存した。
１２．凍結切片の作製および切出
スクロース保存した脳を凍結し、クライオミクロトームを用いて凍結切片（30μm）を作製した。切出した脳切片は、0.1%アジ化ナトリウムPBSに浸漬し、4℃で保存した。
12 Blood collection, perfusion and organ removal
Systemic perfusion was performed from the heart using PBS (-). Thereafter, perfusion fixation was performed with 4% paraformaldehyde / phosphate buffer (4% PFA; Wako Pure Chemical Industries, Ltd.), and the brain was removed. After being soaked in 4% PFA overnight (4 ° C.), it was replaced with a 15% sucrose solution, and further soaked overnight (4 ° C.), and then replaced with a 30% sucrose solution and stored.
12 Preparation and excision of frozen sections Sucrose-preserved brains were frozen and frozen sections (30 μm) were prepared using a cryomicrotome. The excised brain section was immersed in 0.1% sodium azide PBS and stored at 4 ° C.
１３．免疫組織染色
切出した脳切片を6枚取出し、免疫組織染色に供した。染色方法は酵素抗体法（DAB染色）を用いた。
13. Immunohistochemical staining Six excised brain sections were taken out and subjected to immunohistochemical staining. As the staining method, the enzyme antibody method (DAB staining) was used.
１４．試薬および試薬の調製
PBST
0.1M Phosphate buffer（37244-35; ナカライテスク） 1000mL
NaCl 90g
10% Triton 300mL
Diluted water 10L
Normal rabbit serum（S-5000, Vector）
200倍の濃度になるようPBSTに希釈した（Blocking）。
Rat anti BrdU（OBT0030S; AbD serotec）
100倍の濃度になるようPBSTに希釈した（一次抗体）。
Biotynylated anti-rat IgG（BA-4001; Vector labolatories）
200倍の濃度になるようPBSTに希釈した（二次抗体）。
AB Reagent VECTASTAIN(R) standard kit（PK-4000, 株式会社フナコシ）
用時調製とし、反応開始の30分前に準備した。
10mL PBSTにA液およびB液を1滴ずつ添加した（AB反応）。
DAB Tris tablet, pH7.6（047-27011; 和光純薬工業株式会社）
用時調製とした。反応開始の1時間前に10mL PBSTに対してDAB tabletを2錠入れ、反応直前にH2O2を0.01%となるよう添加した。
14 Reagents and reagent preparation
PBST
0.1M Phosphate buffer (37244-35; Nacalai Tesque) 1000mL
NaCl 90g
10% Triton 300mL
Diluted water 10L
Normal rabbit serum (S-5000, Vector)
Diluted in PBST to a concentration of 200 times (Blocking).
Rat anti BrdU (OBT0030S; AbD serotec)
Diluted in PBST to a concentration of 100 times (primary antibody).
Biotynylated anti-rat IgG (BA-4001; Vector labolatories)
Diluted in PBST to a 200-fold concentration (secondary antibody).
AB Reagent VECTASTAIN (R) standard kit (PK-4000, Funakoshi Co., Ltd.)
Prepared at the time of use and prepared 30 minutes before the start of the reaction.
Solution A and solution B were added dropwise to 10 mL PBST (AB reaction).
DAB Tris tablet, pH7.6 (047-27011; Wako Pure Chemical Industries, Ltd.)
It was prepared at the time of use. One hour before the start of the reaction, 2 DAB tablets were added to 10 mL PBST, and H2O2 was added to 0.01% immediately before the reaction.
１５．DAB染色
以下の方法でDAB染色を行う。
反応時間 温度
２N HCl 45min 37℃
0.1M Boric acid (pH8.5) 10min RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
Soaking 1% H2O2 in PBST 10min RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
Blocking 30min RT
一次抗体 Overnight 4℃
PBST 10min RT
PBST 10min RT
PBST 10min RT
二次抗体 3h RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
AB反応 1h RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
DAB反応 11min RT
PBST
RT: room temperature
15. DAB staining DAB staining is performed as follows.
Reaction time Temperature 2N HCl 45min 37 ℃
0.1M Boric acid (pH8.5) 10min RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
Soaking 1% H2O2 in PBST 10min RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
Blocking 30min RT
Primary antibody Overnight 4 ℃
PBST 10min RT
PBST 10min RT
PBST 10min RT
Secondary antibody 3h RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
AB reaction 1h RT
PBST 10min RT
PBST 10min RT
PBST 10min RT
DAB reaction 11min RT
PBST
RT: room temperature
１６．マウントおよび封入
MASコートされたスライドガラス（スーパーフロスト，S0944; 松浪硝子株式会社）に脳切片を6枚のせ、一晩置いて乾燥させた。
次いで以下の方法で脱水・脱脂を行い、封入剤を数滴たらしカバーガラスで封入した。
70% EtOH 2min
80% EtOH 2min
90% EtOH 2min
95% EtOH 2min
99. 5% EtOH 5min
99. 5% EtOH 5min
99. 5% EtOH 5min
100% EtOH 10min
100% EtOH 10min
ティッシュクリア 15min
ティッシュクリア 15min
ティッシュクリア 15min
キシレン ∞（封入まで）
16. Mounting and encapsulation
Six brain sections were placed on a MAS-coated slide glass (Super Frost, S0944; Matsunami Glass Co., Ltd.) and allowed to dry overnight.
Next, dehydration and degreasing were performed by the following method, and a few drops of encapsulant were dripped and sealed with a cover glass.
70% EtOH 2min
80% EtOH 2min
90% EtOH 2min
95% EtOH 2min
99. 5% EtOH 5min
99. 5% EtOH 5min
99. 5% EtOH 5min
100% EtOH 10min
100% EtOH 10min
Tissue clear 15min
Tissue clear 15min
Tissue clear 15min
Xylene ∞ (until enclosed)
１７．神経新生促進作用の評価
上記方法で免疫組織染色を行った後、実体顕微鏡下で図１に示す歯状回顆粒層下帯に存在するBrdU陽性細胞の数をそれぞれカウントした。歯状回の面積（図１の 枠内の面積および湾曲部の長さ）をImage J（NIH, Ver.1.63）を用いて算出した。面積から体積を求め、歯状回体積または長さ当たりのBrdU陽性細胞の数（図２の矢印の数）を求めた。
対照群（非投与郡）に比べBrdU陽性細胞数が有意に多い場合を、神経新生促進作用を有するものと評価した。
17. Evaluation of neurogenesis promoting action After immunohistochemical staining by the above method, the number of BrdU positive cells present in the subdentate gyrus granule layer shown in FIG. 1 was counted under a stereomicroscope. The area of the dentate gyrus (the area in the frame of FIG. 1 and the length of the curved portion) was calculated using Image J (NIH, Ver.1.63). The volume was determined from the area, and the number of BrdU positive cells per dentate gyrus volume or length (number of arrows in FIG. 2) was determined.
When the number of BrdU positive cells was significantly higher than that in the control group (non-administration group), it was evaluated as having a neurogenesis promoting effect.
１８．統計解析
結果はすべて平均値±標準誤差で示した。対照群との比較はDunnett’s testを用い、いずれも両側検定で有意水準は5%とした。
18. Statistical analysis All results were expressed as mean ± standard error. Dunnett's test was used for comparison with the control group. In both cases, the significance level was 5% by two-sided test.
＜結果および評価＞
図３に体積換算、図４に長さ換算したBrdU陽性細胞数の結果を示した。いずれの換算結果もホスファチジルセリン投与群は対照群よりも有意なBrdU陽性細胞数の増加が認められた。したがってホスファチジルセリンは海馬神経の新生作用を有することが判明した。
<Results and evaluation>
FIG. 3 shows the result of volume conversion, and FIG. 4 shows the result of length conversion of BrdU positive cells. In any conversion result, a significant increase in the number of BrdU positive cells was observed in the phosphatidylserine-administered group than in the control group. Therefore, phosphatidylserine was found to have a neoplastic effect on hippocampal nerves.
Claims (1)
Hippocampal neurogenesis promoter containing phosphatidylserine as an active ingredient.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019168149A1 (en) * | 2018-03-02 | 2019-09-06 | 一般財団法人糧食研究会 | Peptide capable of improving cognitive function |
| JPWO2019168149A1 (en) * | 2018-03-02 | 2021-02-18 | 一般財団法人糧食研究会 | Peptides that improve cognitive function |
| US11464824B2 (en) | 2018-03-02 | 2022-10-11 | The Food Science Institute Foundation | Peptide capable of improving cognitive function |
| JP7301810B2 (en) | 2018-03-02 | 2023-07-03 | 一般財団法人糧食研究会 | Peptides that improve cognitive function |
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