JP5493149B2 - Intracellular fat globule imaging method, imaging fluorescent agent, and imaging fluorescent agent production method - Google Patents
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- 230000003834 intracellular effect Effects 0.000 title claims description 16
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- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 claims description 58
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Luminescent Compositions (AREA)
Description
本発明は、酸化亜鉛系蛍光技術に関し、特に、細胞内脂肪球のイメージング方法、イメージング用蛍光剤およびイメージング用蛍光剤製造方法に関する。 The present invention relates to a zinc oxide-based fluorescence technique, and more particularly to an intracellular fat globule imaging method, an imaging fluorescent agent, and an imaging fluorescent agent production method.
近年、生活習慣由来の動脈硬化症や肥満（メタボリックシンドローム）に関して脂肪細胞の基礎的な研究が進められつつある。ここで、細胞内の脂肪球（脂肪滴）の染色には、色素染色法であるオイルレッドＯ染色、ズダン染色法などが知られている。また、脂肪球の染色に加えて、細胞核やアクチン等を同時に観察することが求められる場合があり、これを実現するために、蛍光抗体法を取り入れた蛍光イメージング技術が有効とされている。 In recent years, basic research on adipocytes has been promoted regarding lifestyle-related arteriosclerosis and obesity (metabolic syndrome). Here, for the staining of intracellular fat globules (lipid droplets), oil red O staining, Sudan staining, etc., which are pigment staining methods, are known. In addition to staining of fat globules, it may be required to simultaneously observe cell nuclei, actin and the like, and in order to realize this, a fluorescence imaging technique incorporating a fluorescent antibody method is effective.
現在のところ、蛍光イメージングを実現する脂肪球染色キットとしては、プライマリーセル社製による製品が唯一知られており、これによれば、脂肪球部分が蛍光波長５０３ｎｍで発光して観察可能となる。 At present, the only product manufactured by Primary Cell is known as a fat globule staining kit that realizes fluorescence imaging. According to this, the fat globule portion emits light at a fluorescence wavelength of 503 nm and can be observed.
しかしながら、従来の技術では以下の問題点があった。
従来の脂肪球蛍光イメージング技術は、蛍光剤を反応させるために要する時間が３０分程度かかり、迅速な染色ないし蛍光観察ができないという問題点があった。また、安全性については、眼鏡や手袋の保護具が必要であり、人体への接触を避ける必要があり操作性に劣るという問題点があった。また、価格も高額であるという問題点があった。
However, the conventional technique has the following problems.
The conventional fat globule fluorescence imaging technique has a problem that it takes about 30 minutes to react with the fluorescent agent, and rapid staining or fluorescence observation cannot be performed. As for safety, glasses and gloves must be protected, and contact with the human body must be avoided, resulting in poor operability. In addition, there is a problem that the price is high.
すなわち、解決しようとする問題点は、安価に供給でき、染色時間も短く、安全性の高い脂肪球イメージング技術を提供する点である。 That is, the problem to be solved is to provide a fat globule imaging technique that can be supplied at low cost, has a short staining time, and is highly safe.
請求項１に記載の発明は、蛍光発光する酸化亜鉛粒子を濃度６０％〜７０％のイソプロピルアルコール水溶液に分散させて得ることを特徴とする、細胞内脂肪球のイメージング用蛍光剤製造方法である。
The invention according to claim 1, the zinc oxide particles to fluoresce and wherein the capable dispersed in isopropyl alcohol aqueous solution having a concentration of 60% to 70%, by imaging a fluorescent agent manufacturing method of intracellular fat globules is there.
請求項２に記載の発明は、請求項１に記載のイメージング用蛍光剤製造方法において、酸化亜鉛粒子の粒径が１ｎｍ〜５μｍであることを特徴とする。
The invention according to claim 2 is the method for producing an imaging fluorescent agent according to claim 1, wherein the particle diameter of the zinc oxide particles is 1 nm to 5 μm .
請求項３に記載の発明は、蛍光発光する酸化亜鉛粒子を濃度６０％〜７０％のイソプロピルアルコール水溶液に分散させた液を用いて細胞内脂肪球をイメージングすることを特徴とする細胞内脂肪球イメージング方法である。
According to a third aspect of the invention, intracellular fat, characterized by imaging the intracellular fat globules by using a liquid prepared by dispersing zinc oxide particles fluoresce isopropyl alcohol aqueous solution having a concentration of 60% to 70% A sphere imaging method.
請求項４に記載の発明は、請求項３に記載の細胞内脂肪球イメージング方法において、酸化亜鉛粒子の粒径が１ｎｍ〜５μｍであることを特徴とする。
The invention according to claim 4 is the intracellular fat globule imaging method according to claim 3, wherein the zinc oxide particles have a particle size of 1 nm to 5 μm .
請求項５に記載の発明は、蛍光発光する酸化亜鉛粒子を濃度６０％〜７０％のイソプロピルアルコール水溶液に分散させたことを特徴とする細胞内脂肪球のイメージング用蛍光剤である。
The invention of claim 5 is the imaging fluorescence agent of intracellular fat globules, characterized in that the zinc oxide particles to fluoresce dispersed in isopropyl alcohol aqueous solution having a concentration of 60% to 70%.
請求項６に記載の発明は、請求項５に記載のイメージング用蛍光剤において、酸化亜鉛粒子の粒径が１ｎｍ〜５μｍであることを特徴とする。
A sixth aspect of the present invention is the imaging fluorescent agent according to the fifth aspect, wherein the particle size of the zinc oxide particles is 1 nm to 5 μm .
以上の発明において、酸化亜鉛粒子は、脂肪球を良好にイメージングすべく１ｎｍ〜５μｍ程度の粒径が好適であるが、蛍光発光をするものであれば特に限定されない。このような酸化亜鉛粒子は、例えば、本出願人が権利者である特許文献１にかかる公報に開示するように、酸素ガスと窒素ガスとを含む混合ガスを雰囲気ガスとし、その中でアーク放電を用いて亜鉛を加熱して蒸発させ、亜鉛蒸発量が過多となり酸素欠損の存在する粒子とならないように亜鉛蒸発量を抑制することにより得ることができる。
In the above invention, the zinc oxide particles, but a particle size of about 1nm~5μm order to satisfactorily image the fat肪球is suitable is not particularly limited as long as the fluorescent emission. Such zinc oxide particles are, for example, a mixed gas containing oxygen gas and nitrogen gas as an atmospheric gas as disclosed in the gazette according to Patent Document 1 to which the present applicant is the right holder, and arc discharge therein. It can be obtained by heating and evaporating zinc to suppress the amount of zinc evaporation so that the amount of zinc evaporation is excessive and particles with oxygen vacancies are not formed.
この方法により得られる酸化亜鉛粒子は、条件を最適化することにより室温で３７５ｎｍ付近の強い蛍光を示す。また、亜鉛の蒸発量の制御により可視光で発光する酸化亜鉛粒子の生成も可能である。また、この方法によれば、亜鉛純度が純度９９．９９％以下の亜鉛を用いても製造できるため、本発明の蛍光剤を安価に提供することも可能となる。 The zinc oxide particles obtained by this method exhibit strong fluorescence around 375 nm at room temperature by optimizing the conditions. In addition, it is possible to generate zinc oxide particles that emit light with visible light by controlling the evaporation amount of zinc. Moreover, according to this method, since it can manufacture even if it uses zinc whose purity is 99.99% or less, the fluorescent agent of the present invention can be provided at low cost.
なお、蛍光剤は、さらに他の成分を含むことを妨げない。すなわち、ここでは必須成分ないし主成分を規定したものであり、脂肪球の蛍光イメージングを妨げないのであれば、適宜必要に応じて他の成分を含んでいても良いものとする。 The fluorescent agent does not prevent further including other components. In other words, essential components or main components are defined here, and other components may be included as necessary as long as they do not interfere with fluorescence imaging of fat globules.
また、本発明は、イソプロピルアルコール水溶液濃度が６０％〜７０％であると特に脂肪球の蛍光イメージングに適していることを発見したことによる発明である。なお、濃度が９０％を超えると脂肪球がほぼ溶出してしまい、濃度が４０％未満であると蛍光強度が相対的に小さくなり効率的な蛍光観測ができなくなるので、４０％以上９０％以下、好ましくは５０％以上８０％以下であって、特に好ましい濃度が６０％以上７０％以下の範囲である。 In addition, the present invention is based on the discovery that the concentration of the isopropyl alcohol aqueous solution is 60% to 70%, which is particularly suitable for fluorescent imaging of fat globules. If the concentration exceeds 90%, fat globules are almost eluted, and if the concentration is less than 40%, the fluorescence intensity becomes relatively small and efficient fluorescence observation becomes impossible. Preferably, it is 50% or more and 80% or less, and a particularly preferable concentration is in the range of 60% or more and 70% or less.
本発明によれば、安価な酸化亜鉛粒子を用いることができるため、蛍光剤等を安価に供給でき、染色時間も短く、また、原料そのものの安全性が高いため蛍光剤等としても安全性の高い脂肪球イメージング技術を提供することができる。 According to the present invention, since inexpensive zinc oxide particles can be used, a fluorescent agent or the like can be supplied at a low cost, the dyeing time is short, and the safety of the raw material itself is high, so that the fluorescent agent is also safe. High fat globule imaging technology can be provided.
以下、本発明の実施の形態を図面を参照しながら詳細に説明する。
＜イソプロピルアルコール中の酸化亜鉛の蛍光特性＞
まず、イソプロピルアルコール１００％液に、特許文献１に示す本願発明者により先に技術開発された酸化亜鉛粒子（約１００ｎｍの単分散粒子）の分散液を作成し、これの蛍光特性を調べた。
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
<Fluorescence characteristics of zinc oxide in isopropyl alcohol>
First, a dispersion of zinc oxide particles (monodispersed particles of about 100 nm), which was previously developed by the inventors of the present application shown in Patent Document 1, was prepared in a 100% isopropyl alcohol solution, and the fluorescence characteristics thereof were examined.
図１（ａ）は、蛍光顕微鏡の波長３６５ｎｍの励起光を用いた発光スペクトルである。ここで、励起フィルタＢＰ３６０−３７０ｎｍ，吸収フィルタＢＡ４２０−４６０ｎｍ，ダイクロックミラー４００ｎｍとした。図１（ｂ）は、共焦点レーザー用の波長４０５ｎｍの励起光を用いた発光スペクトルである。何れも、可視光でも観察可能であることが確認できる。 FIG. 1A shows an emission spectrum using excitation light having a wavelength of 365 nm of a fluorescence microscope. Here, the excitation filter BP360-370 nm, the absorption filter BA420-460 nm, and the dichroic mirror 400 nm were used. FIG. 1B is an emission spectrum using excitation light with a wavelength of 405 nm for a confocal laser. It can be confirmed that both can be observed with visible light.
＜予備実験１＞
まず、アカミミガメの肝臓を使用して、肝細胞内に脂肪球があることを確認した。具体的には、門脈より４％パラホルムアルデヒドを環流して化学固定し、マイクロスライサーにて３０μｍの切片を作成した。これを、一般的な中性脂質の脂溶性染料であるオイルレッドＯによる染色後、光学顕微鏡によって肝細胞内の脂肪球の局在を確認した。
<Preliminary experiment 1>
First, we confirmed the presence of fat globules in hepatocytes using the red sea turtle liver. Specifically, 4% paraformaldehyde was refluxed and chemically fixed from the portal vein, and a 30 μm section was prepared with a micro slicer. This was stained with oil red O, which is a general lipid-soluble dye for neutral lipids, and the localization of fat globules in hepatocytes was confirmed by an optical microscope.
図２は、肝細胞内の脂肪球の染色結果の様子を示した光学顕微鏡写真である。図中のＨは、肝細胞の核であって、その周囲に赤色に染色された脂肪球が多数確認できる。 FIG. 2 is an optical micrograph showing the result of staining of fat globules in hepatocytes. H in the figure is the nucleus of the hepatocyte, and a large number of fat globules stained in red can be confirmed around it.
＜実験１＞
アカミミガメの肝細胞の脂肪球の存在および分布の様子がわかったので、次に、本発明の蛍光剤により蛍光イメージングが可能であるかを検討した。まず、イソプロピルアルコール１００％液に、本願発明者により先に技術開発された酸化亜鉛粒子（特許文献１）の飽和溶液を作成した。これに水を添加して６０％溶液の蛍光剤を作成した。
<Experiment 1>
Since the presence and distribution of fat globules in red sea turtle hepatocytes was found, it was next examined whether fluorescence imaging was possible with the fluorescent agent of the present invention. First, a saturated solution of zinc oxide particles (Patent Document 1), which was previously developed by the inventors of the present application, was prepared in a 100% isopropyl alcohol solution. Water was added thereto to prepare a 60% solution fluorescent agent.
予備実験１と同様に準備した肝臓の切片を６０％イソプロピルアルコール水溶液に１分間浸し、その後、３７度の蛍光剤に５分間浸漬して脂肪球に酸化亜鉛粒子を定着させた。次に６０％イソプロピルアルコール水溶液で切片を２分間洗浄し、さらに蒸留水で１分間洗浄した。 A slice of the liver prepared in the same manner as in Preliminary Experiment 1 was immersed in a 60% isopropyl alcohol aqueous solution for 1 minute, and then immersed in a 37-degree fluorescent agent for 5 minutes to fix the zinc oxide particles on the fat globules. Next, the section was washed with 60% isopropyl alcohol aqueous solution for 2 minutes and further washed with distilled water for 1 minute.
これをスライドグラスに載せ、グリセリンを滴下後、カバーガラスをかけて封入した。これを蛍光顕微鏡で観測した。このとき、励起波長３６５ｎｍの紫外光を照射して、３９０ｎｍ〜４２０ｎｍを透過するフィルタを用いて蛍光を観測した。図３は、蛍光イメージング結果を表した蛍光顕微鏡写真である。図示したように脂肪球部分が蛍光を発しており、その様相は予備実験１で染色された結果と同じであることが確認できる。以上により、本発明の蛍光剤によれば、従来の３０分程度かかる反応ステップを５分程度と著しく短縮でき、簡便に脂肪球を蛍光イメージングできることが確認できた。 This was placed on a slide glass, and glycerin was dropped, followed by covering with a cover glass. This was observed with a fluorescence microscope. At this time, ultraviolet light having an excitation wavelength of 365 nm was irradiated, and fluorescence was observed using a filter that transmitted from 390 nm to 420 nm. FIG. 3 is a fluorescence micrograph showing the result of fluorescence imaging. As shown in the figure, the fat globule portion fluoresces, and it can be confirmed that the appearance is the same as the result stained in the preliminary experiment 1. From the above, according to the fluorescent agent of the present invention, it was confirmed that the conventional reaction step of about 30 minutes can be remarkably shortened to about 5 minutes, and fat globules can be easily fluorescently imaged.
＜予備実験２＞
次に、異なる分布を示す脂肪球であってもイメージングが可能であるかを検討するため、トラフグの肝臓の脂肪球の染色をおこなうこととした。具体的には、トラフグの肝臓の門脈より４％パラホルムアルデヒドを環流して化学固定し、マイクロスライサーにて３０μｍの切片を作成した。これを、一般的な中性脂質の脂溶性染料であるオイルレッドＯによる染色後、光学顕微鏡によって肝細胞内の脂肪球の局在を確認した。
<Preliminary experiment 2>
Next, in order to investigate whether or not fat globules with different distributions can be imaged, it was decided to stain the fat globules in the trough puffer liver. Specifically, 4% paraformaldehyde was refluxed from the portal vein of the trough puffer liver and chemically fixed, and a 30 μm section was prepared with a micro slicer. This was stained with oil red O, which is a general lipid-soluble dye for neutral lipids, and the localization of fat globules in hepatocytes was confirmed by an optical microscope.
図４は、肝細胞内の脂肪球の染色結果の様子を示した光学顕微鏡写真である。図から明らかなように、アカミミガメの様相とは異なり大脂肪球（脂肪球）が肝小葉内にびまん性に多数存在し、これが赤色に染色されていることが確認できる。 FIG. 4 is an optical micrograph showing the result of staining of fat globules in hepatocytes. As is apparent from the figure, it can be confirmed that many large fat globules (fat globules) are diffusely present in the liver lobule and are stained red, unlike the appearance of red turtles.
＜実験２＞
トラフグの肝細胞の脂肪球の存在および分布の様子がわかったので、次に、本発明の蛍光剤により、このようなびまん性の脂肪球でも蛍光イメージングが可能であるかを検討することとした。実験１と同様に酸化亜鉛粒子を分散させた６０％イソプロピルアルコール水溶液の蛍光剤を作成した。
<Experiment 2>
Now that we know the presence and distribution of fat globules in trough liver cells, we decided to investigate whether fluorescent imaging of these diffuse fat globules is possible with the fluorescent agent of the present invention. . As in Experiment 1, a fluorescent agent of 60% isopropyl alcohol aqueous solution in which zinc oxide particles were dispersed was prepared.
実験１と同様に蛍光剤を反応させた後洗浄等をおこない、同じく同様に蛍光観察した。図５は、蛍光イメージング結果を表した蛍光顕微鏡写真である。なお、実験２では白黒写真による撮影とした。図から明らかなように、脂肪球部分の発光の様子は予備実験２の光学顕微鏡の観測結果と同様の様相であり適正に蛍光イメージングされていることが確認できた。
以上から、本発明の蛍光剤は、脂肪球であれば、その粗密や分布に依存せず好適にイメージングできるものであることがわかった。
In the same manner as in Experiment 1, the fluorescent agent was reacted and then washed, and the fluorescence was similarly observed. FIG. 5 is a fluorescence micrograph showing fluorescence imaging results. In Experiment 2, black and white photography was used. As is clear from the figure, the state of light emission from the fat globule portion was the same as the observation result of the optical microscope in Preliminary Experiment 2, and it was confirmed that fluorescence imaging was appropriately performed.
From the above, it was found that the fluorescent agent of the present invention can be suitably imaged if it is a fat globule, regardless of its density or distribution.
次に、他の溶媒による蛍光イメージングの可能性を検討した。試験片としては、予備実験２および実験２と同様のトラフグの肝臓細胞を用いた。
＜実験３＞
実験１，２と同じ酸化亜鉛粒子を、ＤＭＳＯ（ジメチルスルホキシド）６０％水溶液に溶解したものを蛍光剤とし、実験２と同様の手順で肝臓切片に反応させた。これを蛍光顕微鏡で観察したが蛍光は観測されなかった。なお、結果は黒一色の写真であるので図示を省略する。
Next, the possibility of fluorescence imaging with other solvents was examined. As test pieces, the same trough puffer liver cells as those in Preliminary Experiment 2 and Experiment 2 were used.
<Experiment 3>
The same zinc oxide particles as those in Experiments 1 and 2 dissolved in a 60% aqueous solution of DMSO (dimethyl sulfoxide) were used as a fluorescent agent, and the liver slices were reacted in the same procedure as in Experiment 2. This was observed with a fluorescence microscope, but no fluorescence was observed. In addition, since a result is a black-colored photograph, illustration is abbreviate | omitted.
＜実験４＞
実験１，２と同じ酸化亜鉛粒子を、１００％イソプロピルアルコールに飽和分散させたもの（希釈なし）を蛍光剤とし、実験２と同様の手順で肝臓切片に反応させた。これを蛍光顕微鏡で観察したが蛍光は観測されなかった。なお、結果は黒一色の写真であるので図示を省略する。これは、アルコール濃度が高いため、脂質が溶出してしまったものと考えられた。
<Experiment 4>
The same zinc oxide particles as those in Experiments 1 and 2 were dispersed in 100% isopropyl alcohol in a saturated manner (no dilution) as a fluorescent agent, and reacted with the liver sections in the same manner as in Experiment 2. This was observed with a fluorescence microscope, but no fluorescence was observed. In addition, since a result is a black-colored photograph, illustration is abbreviate | omitted. This was thought to be due to the elution of lipid due to the high alcohol concentration.
＜実験５＞
他のアルコールを溶媒とすることとし、エチルアルコール、Ｔブチルアルコールを用いた酸化亜鉛粒子分散液を作成し、実験２と同様の手順で肝臓切片に反応させた。これを蛍光顕微鏡で観察したが蛍光は観測されなかった。なお、結果は黒一色の写真であるので図示を省略する。
<Experiment 5>
Using other alcohol as a solvent, a zinc oxide particle dispersion using ethyl alcohol and T-butyl alcohol was prepared, and the liver slice was reacted in the same procedure as in Experiment 2. This was observed with a fluorescence microscope, but no fluorescence was observed. In addition, since a result is a black-colored photograph, illustration is abbreviate | omitted.
以上の結果から、酸化亜鉛粒子は、驚くべきことに、イソプロピルアルコールを媒介とした場合に、特異的に細胞内脂肪球への結合能力が発揮されることがわかった。 From the above results, it was found that the zinc oxide particles surprisingly exhibit the ability to specifically bind to intracellular fat globules when isopropyl alcohol is mediated.
＜実験６＞
次に、イソプロピルアルコール水溶液の好適な濃度を検討することとした。酸化亜鉛粒子を１００％イソプロピルアルコールに飽和分散させたものを希釈して、５０％希釈液、５５％希釈液、７５％希釈液、８０％希釈液を調整した。これを上述の実験と同様な手法により、アカミミガメの肝臓切片を用いた実験をおこなった。蛍光観察をおこなったところ５５％液と７５％液では、微弱ながら観察が可能であったが、細胞内の十分な脂肪球の情報が得られないレベルであった。なお、５０％液と８０％液は、今回の励起波長では蛍光観察されなかった。以上から、濃度は６０〜７０％とすることとした。
<Experiment 6>
Next, a suitable concentration of the isopropyl alcohol aqueous solution was examined. A solution obtained by saturatedly dispersing zinc oxide particles in 100% isopropyl alcohol was diluted to prepare a 50% diluted solution, a 55% diluted solution, a 75% diluted solution, and an 80% diluted solution. An experiment using a slice of red turtle liver was performed by the same method as that described above. When fluorescence observation was performed, the 55% solution and the 75% solution were able to be observed although they were weak, but the level of information on sufficient fat globules in the cells could not be obtained. In the 50% solution and the 80% solution, fluorescence was not observed at this excitation wavelength. From the above, the concentration was determined to be 60 to 70%.
本発明は、生物・医学的な研究のみならず食品加工分野にも応用が可能であり、食品の品質管理および食品中の脂質の分布域を観察する利用法を挙げることができる。具体的には、乳製品、食肉、魚肉、加工食品などの工場では安全性が特に重要視される背景のもと、脂質の測定にはＦＴ−ＩＲなどの高価な装置が導入されている。しかしながら、脂質の分布の測定にはＭＲＩを使用すれば、短時間で測定可能であるが、非常に高価な装置で管理が難しいなどの問題があり、工場では用いられてこなかった。本発明によれば簡便に分布や割合などを蛍光観察できるという利点を有する。
The present invention can be applied not only to biological / medical research but also to the field of food processing, and examples include the use of food quality control and observation of lipid distribution areas in food. Specifically, expensive factories such as FT-IR have been introduced for lipid measurement under the background that safety is particularly important in factories such as dairy products, meat, fish and processed foods. However, if MRI is used for the measurement of lipid distribution, it can be measured in a short time, but it has been difficult to manage with a very expensive apparatus and has not been used in factories. According to the present invention, there is an advantage that the distribution and ratio can be easily observed with fluorescence.
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