JP2012029675A - Method for testing cytotoxicity - Google Patents
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Abstract
Description
本発明は、細胞に被検査物を曝して、細胞の変化を観察する細胞毒性検査方法であって、被検査物に曝した細胞をライブ観察することで、細胞がどのように変化するか、特に分裂時間および分裂から分裂までの間を示す分裂間時間を調べる事で、被検査物の毒性を検査する方法に関する。 The present invention is a cytotoxicity test method for observing changes in cells by exposing the test object to the cells, and how the cells change by observing the cells exposed to the test object live, In particular, the present invention relates to a method for inspecting the toxicity of an object to be inspected by examining the division time and the inter-division time indicating the period between division.
体内組織や血液に直接触れる薬剤や医療機器は、安全性が求められる。そのため、体内組織や血液に対してどれほどの影響があるかは、使用される前に検査される必要がある。一方、ほとんどの物質は体組織にとって毒にも薬にもなりえる。そこで、問題はどれほどの量が体に入れば、毒としての効果を示すかという点である。このような検査では、被検査物を体組織に曝す若しくは摂取させる量を変えて、結果を観察する必要があるので、膨大な検体が必要となる。 Drugs and medical devices that come into direct contact with body tissues and blood are required to be safe. Therefore, it is necessary to examine how much it has an influence on body tissues and blood before being used. On the other hand, most substances can be poisons or drugs for body tissues. Therefore, the problem is how much amount enters the body and shows the effect as a poison. In such a test, it is necessary to observe the result by changing the amount of the object to be inspected or ingested to the body tissue, so that a huge number of specimens are required.
動物を使った検査は信頼性は高いが１次サーチ用としては、費用がかかりすぎるという問題も発生する。そこで、確立された細胞株を用いたスクリーニングが行われている。これらは、細胞毒性試験と呼ばれる試験方法であり、例えば、培地抽出法、細胞増殖抑制試験、直接接触法等が挙げられる。 Although testing using animals is highly reliable, there is a problem that it is too expensive for a primary search. Therefore, screening using established cell lines is performed. These are test methods called cytotoxicity tests, and examples include a medium extraction method, a cell growth inhibition test, and a direct contact method.
これらの試験の原理は多数の培養細胞を用いて、細胞の生死で毒性を評価することにある。有害物質の影響を受けた細胞は、ネクローシス若しくはアポトーシスによって死に至るため、多くの培養細胞に被検査物を与えて、その後の細胞の死亡率若しくは生存率を調べる。つまり、細胞個々には有害物質に対する感受性に違いがあったとしても、多くの細胞について同一の被検査物の影響を調べる事で、統計的には毒性が評価できるという技術思想に基づくものである。 The principle of these tests is to use a large number of cultured cells to evaluate the viability of the cells by viability. Since cells affected by harmful substances are killed by necrosis or apoptosis, a test object is given to many cultured cells, and the mortality or survival rate of the subsequent cells is examined. In other words, even if there is a difference in sensitivity to harmful substances in each cell, it is based on the technical idea that toxicity can be statistically evaluated by examining the influence of the same test object on many cells. .
このような方法に関する技術はすでに開示されているものがあり、特許文献１（特開平０５−３３６９９６号）には、コラーゲンマトリックスに表皮細胞と繊維芽細胞とを一緒に培養する場合は、生存細胞の数をカウントするのが極めて困難という課題に対して、生存細胞の数のカウントをより容易にできる方法が開示されている。 Techniques relating to such a method have already been disclosed, and in Patent Document 1 (Japanese Patent Laid-Open No. 05-336996), when epidermal cells and fibroblasts are cultured together in a collagen matrix, viable cells are disclosed. In response to the problem that it is extremely difficult to count the number of living cells, a method that can more easily count the number of viable cells is disclosed.
また、特許文献２（特開平１０−３２７８５４号）にも、培養細胞の生死細胞を直接計数するのは不可能であるとして、接着性細胞を基板上で培養した後、被検査物に曝して、染色した後計数する方法が開示されている。この方法では、１個１個の細胞の生死を正確にカウントすることができるとされている。さらに、特許文献２では、観察に自動ステージとカメラを用いることで接着性細胞の生存率の決定を自動化できることが開示されている。 Further, in Patent Document 2 (Japanese Patent Laid-Open No. 10-327854), it is impossible to directly count the viable and dead cells of the cultured cells, and after culturing the adherent cells on the substrate, the cells are exposed to the test object. A method of counting after staining is disclosed. According to this method, it is said that the life and death of each cell can be accurately counted. Furthermore, Patent Document 2 discloses that the determination of the survival rate of adherent cells can be automated by using an automatic stage and a camera for observation.
一方、細胞の一つ一つに着目し、より詳細に薬剤が細胞に及ぼす影響を視覚的に観察する方法も提案されている。特許文献３（特表２００５−５３５３４８号）では、単一の蛍光タンパク質を導入した細胞に対して発光強度を時間関数として観察することで、増殖速度や薬剤感受性を生体外でリアルタイムに判定する技術が開示されている。 On the other hand, a method of visually observing the influence of a drug on cells in detail has been proposed by paying attention to each cell. In patent document 3 (Japanese translations of PCT publication No. 2005-535348), the technique which determines the proliferation rate and a drug sensitivity in real time in vitro by observing the light emission intensity as a time function with respect to the cell which introduce | transduced the single fluorescence protein. Is disclosed.
細胞を使った毒性試験は、細胞の生死によって被検査物の毒性を評価する方法であり、比較的容易で多くのロットの試験ができるため、一次スクリーンの目的には合致したものである。しかし、評価が細胞の生死であるため、細胞がどのような過程で死んでしまったのかは明確でない。また、細胞は被検査物が作用した直後に死んだのか、ある程度細胞分裂をしてから死んだのかという点についても判断できない。 The toxicity test using cells is a method for evaluating the toxicity of an object to be inspected based on the life or death of cells, and it is relatively easy and can test many lots. Therefore, it is suitable for the purpose of the primary screen. However, since the evaluation is the survival of the cell, it is not clear how the cell died. In addition, it cannot be judged whether the cell died immediately after the test object acted or whether it died after some cell division.
さらに、例えば、被検査物によって、細胞の分裂過程が停止してしまい、実際には死んでいると判断すべき場合、つまり、アポトーシスでもなくネクローシスでもない状態で、あたかも生きているように見えるような状態は、誤って生きていると判断してしまうという課題があった。 In addition, for example, if an object to be examined stops the cell division process and it should be judged that it is actually dead, that is, it appears to be alive in a state that is neither apoptotic nor necrotic. However, there was a problem that it was judged that it was alive by mistake.
特許文献３の実施例９や実施例１０には、蛍光タンパク質を導入した細胞の分裂過程やアポトーシスの状態を時間毎に撮影して、認識できる点が開示されているが、ある細胞の分裂過程若しくはアポトーシスの場面を蛍光タンパク質を用いて観察した点が開示されているのみであり、試験若しくは検査にどのように使えるかについてはなんら開示されていない。 In Example 9 and Example 10 of Patent Document 3, it is disclosed that a cell division process and a state of apoptosis introduced with a fluorescent protein can be taken and recognized every time, but a cell division process is disclosed. Or only the point which observed the scene of apoptosis using fluorescent protein is disclosed, and it is not disclosed at all how it can be used for a test or a test | inspection.
すなわち、同じ細胞株とはいえ、個々の細胞の被検査物に対する感受性の違いをどのように判断するかという点がなんら考慮されていない。 That is, no consideration is given to how to determine the difference in sensitivity of each cell to the test object even though they are the same cell line.
本発明は、上記の課題に鑑みて想到されたものであり、細胞の内部で生じる変化まで追跡することで、被検査物の細胞に与える効果を評価することを目的とする。より具体的には、本発明は、
分裂しても染色体の数が変化しない細胞に蛍光タンパク質を導入する工程と、
前記蛍光タンパク質が導入された細胞を被検査物に曝す工程と、
前記被検査物に曝された細胞をライブ観察し、少なくとも分裂時間を測定する工程を有す
る細胞毒性検査方法を提供するものである。
The present invention has been conceived in view of the above problems, and an object of the present invention is to evaluate the effect of a test object on cells by tracing changes occurring inside the cells. More specifically, the present invention provides:
Introducing a fluorescent protein into a cell whose number of chromosomes does not change when it divides;
Exposing the cells into which the fluorescent protein has been introduced to a test object;
The present invention provides a cytotoxicity testing method comprising a step of performing live observation of cells exposed to the test object and measuring at least the division time.
本発明では、分裂しても染色体の数が変化しない細胞を用いるので、集団の中の任意の細胞がほぼ集団全体の細胞の性質を反映する。つまり、細胞分裂時間や細胞分裂に至るまでの時間（細胞分裂間時間）が、全ての細胞においてほぼ同じである。したがって、被検査物に曝した細胞集団の中から選んだ数個の細胞の変化を調べることで、集団全体の結果を判断できる。これによって、被検査物による細胞の変化をより詳細に追跡することができる。 In the present invention, a cell whose number of chromosomes does not change even when it divides is used, so that any cell in the population reflects the properties of the cells of the entire population. That is, the cell division time and the time until cell division (inter-cell division time) are almost the same in all cells. Therefore, by examining changes in several cells selected from the cell population exposed to the test object, the result of the entire population can be determined. Thereby, the change of the cell by the test object can be traced in more detail.
また、ライブ観察によって、細胞分裂の時間や細胞分裂間時間を測定し、同じ集団の細胞で被検査物に曝されなかった細胞の場合と比較することで、詳細な検査であるにも関わらず、被検査物の毒性を評価することができる。 In addition, by measuring the time of cell division and the time between cell divisions by live observation, and comparing with the case of cells that were not exposed to the test object in the same group of cells, it is a detailed test despite being a detailed test. The toxicity of the test object can be evaluated.
さらに、細胞分裂の停止によって、あたかも生きているように見える状態も、同じ性質
を有して被検査物に曝されなかった細胞と比較することで、確認することができ、そのよ
うな被検査物の毒性を評価することができる。
Furthermore, the state that appears to be alive due to the cessation of cell division can be confirmed by comparing with cells that have the same properties and have not been exposed to the test object. The toxicity of the product can be evaluated.
（実施の形態１）
本発明の細胞毒性検査方法で用いる細胞は、分裂によって染色体の本数が変化せず、不死の細胞である。さらに、癌化していない細胞であるのが好ましい。このような細胞の候補の１つは、幹細胞であるが、すでに株化された公知の細胞も使用できる可能性がある。例えば、ＮＩＨ３Ｔ３は、マウスの胎児皮膚から分離したものであり、正常細胞の性質と無限に分裂する性質を併せ持つ。Ｖｅｒｏ細胞は、アフリカミドリザル由来の細胞で、不死の性質を持つが、癌化していない。
(Embodiment 1)
The cells used in the cytotoxicity test method of the present invention are immortal cells whose chromosome numbers do not change by division. Furthermore, cells that are not cancerous are preferred. One such cell candidate is a stem cell, although known cells already established may be used. For example, NIH3T3 is isolated from mouse fetal skin and has both the properties of normal cells and the ability to divide indefinitely. Vero cells are derived from African green monkeys and have immortal properties, but are not cancerous.
本明細書ではｍ５ｓという京都大学放射線生物研究センター佐々木正夫教授による細胞（大阪府立大学先端科学イノベーションセンター児玉靖司教授より分与）を用いた。この細胞は、染色体構成が安定的に維持されていることがよく知られている。なお、分裂によって染色体数が変化しないというのは、細胞分裂によって染色体が異なる細胞の発生率が４０％以下をいうものとする。 In the present specification, m5s cells by Professor Masao Sasaki of Kyoto University's Radiation Biology Research Center (distributed by Professor Shinji Kodama, Advanced Science Innovation Center, Osaka Prefecture University) were used. It is well known that the chromosome structure of this cell is stably maintained. Note that the fact that the number of chromosomes does not change due to division means that the incidence of cells having different chromosomes due to cell division is 40% or less.
また、本発明に用いる細胞は、接着性の細胞であるのがよい。本発明では、数回の細胞分裂を追う必要があるので、浮遊細胞では追跡できない場合もあるからである。 The cells used in the present invention are preferably adhesive cells. This is because in the present invention, it is necessary to follow several cell divisions.
本発明の検査方法では、このような細胞に蛍光タンパク質を導入する。導入の方法は特に制限されず、公知の方法を用いることができる。導入できる蛍光タンパク質も特に制限されない。より具体的には、ＧＦＰ（Ｇｒｅｅｎ Ｆｌｕｏｒｅｓｃｅｎｃｅ Ｐｒｏｔｅｉｎ）、やＹＦＰ（Ｙｅｌｌｏｗ Ｆｌｏｕｒｅｓｃｅｎｃｅ Ｐｒｏｔｅｉｎ）、ＣＦＰ（Ｃｙａｎ Ｆｌｕｏｒｅｓｃｅｎｃｅ Ｐｒｏｔｅｉｎ）、ＲＦＰ（Ｒｅｄ Ｆｌｏｕｒｅｓｃｅｎｃｅ Ｐｒｏｔｅｉｎ）などが好適に利用できる。これらのタンパク質は、コードする塩基配列が確立されているので、プラスミドに組み込みやすい。 In the inspection method of the present invention, a fluorescent protein is introduced into such cells. The introduction method is not particularly limited, and a known method can be used. The fluorescent protein that can be introduced is not particularly limited. More specifically, GFP (Green Fluorescence Protein), YFP (Yellow Fluorescence Protein), CFP (Cyan Fluorescence Protein), RFP (Red Fluorescence Protein), etc. can be used suitably. These proteins are easily incorporated into plasmids since the encoded nucleotide sequences have been established.
また、これらの蛍光タンパク質は、細胞中で目標とするタンパク質をコードする塩基配列に続けてプラスミド中に組み込み、細胞に導入する。目標とするタンパク質が生成された際に観察できるようにするためである。 In addition, these fluorescent proteins are incorporated into a plasmid following the base sequence encoding the target protein in the cell and introduced into the cell. This is so that the target protein can be observed when it is generated.
また、本発明の検査方法に用いる細胞には、少なくとももう１箇所に蛍光タンパク質を導入してもよい。この蛍光タンパク質は、細胞の分裂を観察するためである。したがって、この蛍光タンパク質は、細胞核や、細胞壁といった細胞の外観に関わる部分のタンパク質に対して発光させる。また、この蛍光タンパク質は、先程の蛍光タンパク質とは発光の波長が異なるものが良い。これらを区別できるようにするためである。 Moreover, you may introduce | transduce fluorescent protein into the cell used for the test | inspection method of this invention at least at another place. This fluorescent protein is for observing cell division. Therefore, this fluorescent protein emits light to the protein in the part related to the appearance of the cell such as the cell nucleus and the cell wall. In addition, the fluorescent protein is preferably different from the previous fluorescent protein in the emission wavelength. This is so that these can be distinguished.
蛍光タンパク質を導入した細胞は、適量を検査用とコントロール用に分け、検査用の細胞は被検査物に曝す。被検査物は、検査用の細胞を培養している容器中に投入したまま、検査を行ってもよいし、検査用の細胞を、被検査物に所定の期間曝してから再び培地中で培養してもよい。 The cells into which the fluorescent protein is introduced are divided into appropriate amounts for testing and control, and the testing cells are exposed to the test object. The object to be inspected may be inspected while being put in the container in which the cells for inspection are cultured, or the cells for inspection are exposed to the object to be inspected for a predetermined period and then cultured again in the medium. May be.
被検査物に曝された細胞は、ライブ観察によって状態を観察する。ライブ観察とは、細胞を生きたまま観察し記録することをいう。この際、細胞には励起光をあてて、導入された蛍光タンパク質が発光する光を顕微鏡を介して観察・記録する。 The state of the cells exposed to the test object is observed by live observation. Live observation refers to observing and recording cells alive. At this time, the cells are irradiated with excitation light, and the light emitted from the introduced fluorescent protein is observed and recorded through a microscope.
使用する細胞が接着性の細胞であれば、この観察の際に、移動することが少ないので、長期間に渡り同一の細胞を観察することができる。 If the cells to be used are adherent cells, they are less likely to move during this observation, so that the same cells can be observed over a long period of time.
観察は、染色体が凝縮を始めてから分離するまでの時間である細胞分裂時間と、細胞の分裂と分裂の間を表す、細胞分裂間時間の２種類の時間を少なくとも観察・記録する。細胞は被検査物から影響を受けると、細胞分裂時間が変化するからである。したがって、同じ集団から培養された細胞を比較のために観察しておくことが必要である。 Observation observes and records at least two types of time, ie, a cell division time, which is a time from the start of condensation to separation, and a time between cell divisions. This is because cell division time changes when cells are affected by the test object. It is therefore necessary to observe cells cultured from the same population for comparison.
また、本発明では、被検査物の影響が少なくて、細胞分裂を複数回行った後に被検査物の影響がでる場合も観察・記録することができる。 Further, in the present invention, it is possible to observe and record even when the influence of the inspection object is small and the influence of the inspection object appears after a plurality of cell divisions.
本発明では、分裂しても染色体の数が変化しない細胞を用いる。以下にそのような細胞について説明する。 In the present invention, a cell whose number of chromosomes does not change even when it divides is used. Such cells are described below.
以下のように、ヒストンＨ３と共に発現する蛍光タンパク質用プラスミドの例として、蛍光タンパク質ｍＣｈｅｒｒｙとＶｅｎｕｓについて作製した。 As described below, fluorescent proteins mCherry and Venus were prepared as examples of fluorescent protein plasmids expressed with histone H3.
＜＜実施例の可視化細胞の作製と観察＞＞
＜（１）プラスミドＤＮＡの作製＞
（ａ）ｐｍＣｈｅｒｒｙ−Ｈ３
ヒストンＨ３−赤色蛍光蛋白質発現プラスミド（ｍＣｈｅｒｒｙ−Ｈ３）は、ｐＲＳＥＴ−ｍＣｈｅｒｒｙ（カリフォルニア大学 Ｔｓｉｅｎ博士より寄贈）を鋳型とし、プライマーＣとＨを用いてＰＣＲ法を利用して増幅させ、ｍｃｈｅｒｒｙ断片を得た。それぞれのプライマーは表１に示す。なお、プライマーＣは配列番号１、プライマーＨは配列番号２である。ｐＥＣＦＰ−Ｈ３をＮｈｅＩ、ＢｇｌＩＩで切断しＥＣＦＰ部分を取り除いた部位に、ＮｈｅＩ、ＢｇｌＩＩで切断したｍＣｈｅｒｒｙ断片を挿入することによりｐｍＣｈｅｒｒｙ−Ｈ３を作製した。
<< Preparation and Observation of Visualized Cells of Examples >>
<(1) Preparation of plasmid DNA>
(A) pmCherry-H3
The histone H3-red fluorescent protein expression plasmid (mCherry-H3) is amplified using the PCR method with primers C and H using pRSET-mCherry (contributed by Dr. Tsien, University of California) to obtain an mcherry fragment. It was. Each primer is shown in Table 1. Primer C is SEQ ID NO: 1 and primer H is SEQ ID NO: 2. pmCherry-H3 was prepared by inserting the mCherry fragment cleaved with NheI and BglII into the site where pECFP-H3 was cleaved with NheI and BglII and the ECFP part was removed.
（ｂ）ｐＶｅｎｕｓ−Ｌ２０１Ａ−Ｈ３
黄緑色蛍光タンパク質融合タンパク質の発現ベクターとして、黄緑色蛍光タンパク質（Ｖｅｎｕｓ−Ｌ２０１Ａ）の遺伝子を含むベクターである哺乳動物細胞用発現ベクターｐＶｅｎｕｓ−Ｌ２０１Ａ−Ｃ１を使用した。なお、Ｖｅｎｕｓ−Ｌ２０１Ａは、黄緑色蛍光タンパク質ＶｅｎｕｓのプラスミドであるｐＣＳ２−Ｖｅｎｕｓ（理化学研究所脳科学総合研究センター 宮脇敦史博士より寄贈）を鋳型にメガプライマー法を用いて、そのＮ末端から２０１番目のロイシンをアラニンに置き換えたものである。
(B) pVenus-L201A-H3
As an expression vector for the yellow-green fluorescent protein fusion protein, mammalian cell expression vector pVenus-L201A-C1, which is a vector containing the gene for yellow-green fluorescent protein (Venus-L201A), was used. Venus-L201A is the 201st plasmid from the N-terminus using the megaprimer method using pCS2-Venus (contributed by Dr. Atsushi Miyawaki, RIKEN Brain Science Institute), which is a plasmid for the yellow-green fluorescent protein Venus, as a template. The leucine was replaced with alanine.
具体的には、１回目のＰＣＲでは、Ｃｈｅｒｒｙ−Ｆｗ（Ｐｒｉｍｅｒ−１）とＶｅｎｕｓ−Ｃ−Ｌ２０１Ａ（Ｐｒｉｍｅｒ−２）の間を増幅した。Ｐｒｉｍｅｒ−１により、Ｖｅｎｕｓのコーディング領域の上流に制限酵素Ｎｈｅ Ｉの認識部位を作製した。また、Ｐｒｉｍｅｒ−２により、アミノ酸残基２０１番目のロイシンをアラニンに変異（Ｌ２０１Ａ）させた。この変異導入時に、アミノ酸残基２０２番目セリンのアミノ酸配列は変えずに、塩基配列を変えることにより、制限酵素Ｍｌｕ Ｉの認識部位を作製した。 Specifically, in the first PCR, amplification was performed between Cherry-Fw (Primer-1) and Venus-C-L201A (Primer-2). A recognition site for the restriction enzyme Nhe I was created upstream of the coding region of Venus by Primer-1. In addition, leucine at amino acid residue 201 was mutated to alanine (L201A) by Primer-2. At the time of this mutagenesis, the recognition site for the restriction enzyme Mlu I was prepared by changing the base sequence without changing the amino acid sequence of the amino acid residue 202th serine.
２回目のＰＣＲでは、１回目のＰＣＲ産物をメガプライマーとして、Ｖｅｎｕｓ−Ｃ−Ｒｅｖ−２（Ｐｒｉｍｅｒ−３）との間を増幅した。Ｐｒｉｍｅｒ−３により、ストップコドンを取り除き、さらに、Ｖｅｎｕｓのコーディング領域の下流に制限酵素ＢｇｌＩＩの認識部位を作製した。プライマーの塩基配列を表１に示す。塩基配列は左から５´末端で、右端は３´末端である。また、プライマー１は配列番号３、プライマー２は配列番号４、プライマー３は配列番号５である。 In the second PCR, the first PCR product was used as a megaprimer to amplify between Venus-C-Rev-2 (Primer-3). The stop codon was removed by Primer-3, and a recognition site for the restriction enzyme BglII was created downstream of the coding region of Venus. Table 1 shows the primer base sequences. The base sequence is the 5 ′ end from the left and the right end is the 3 ′ end. Primer 1 is SEQ ID NO: 3, primer 2 is SEQ ID NO: 4, and primer 3 is SEQ ID NO: 5.
そして、Ｖｅｎｕｓのコーディング領域の上流と下流にそれぞれ作成した制限酵素認識部位を、ＮｈｅＩとＢｇｌＩＩで制限酵素処理し、インサートを作製した。 Then, the restriction enzyme recognition sites prepared respectively upstream and downstream of the coding region of Venus were subjected to restriction enzyme treatment with NheI and BglII to prepare inserts.
また、ｐＥＧＦＰ−Ｃ１をＮｈｅＩとＢｇｌＩＩで制限酵素処理し、ＥＧＦＰのコーディング領域を取り除いて、ベクターを作製した。そして、インサートとベクターのライゲーションを行い、ｐＶｅｎｕｓ−Ｌ２０１Ａ−Ｃ１を作製した。これらの過程を図４に示す。 Also, pEGFP-C1 was treated with restriction enzymes NheI and BglII, and the EGFP coding region was removed to prepare a vector. Then, the insert and vector were ligated to prepare pVenus-L201A-C1. These processes are shown in FIG.
次に上記ｐＶｅｎｕｓ−Ｌ２０１Ａ−Ｃ１をＭＣＳ内にある制限酵素ＢａｍＨＩ及びＥｃｏＲＩで切断し、該切断部位に上記のプラスミドＤＮＡであるｍＣｈｅｒｒｙ−Ｈ３を同じ制限酵素で切断することで得られたヒストンＨ３のｃＤＮＡ断片を導入することにより、黄緑色蛍光タンパク質融合タンパク質を発現させるためのプラスミドＤＮＡであるｐＶｅｎｕｓ−Ｌ２０１Ａ−Ｈ３を作製した。プラスミドＤＮＡの精製には、「Ｑｕａｎｔｕｍ Ｐｒｅｐ Ｐｌａｓｍｉｄ Ｍｉｎｉｐｒｅｐ Ｋｉｔ」（ＢＩＯ−ＲＡＤ社製）を使用した。 Next, the above pVenus-L201A-C1 was cleaved with restriction enzymes BamHI and EcoRI in MCS, and the above-mentioned plasmid DNA mCherry-H3 was cleaved with the same restriction enzymes. By introducing the cDNA fragment, pVenus-L201A-H3, which is a plasmid DNA for expressing the yellow-green fluorescent protein fusion protein, was prepared. For the purification of plasmid DNA, “Quantum Prep Plasmid Miniprep Kit” (manufactured by BIO-RAD) was used.
＜（２）プラスミドＤＮＡの導入および安定形質転換細胞の取得＞
次に、上記のプラスミドＤＮＡを細胞に導入して、安定形質転換細胞を得た。上記プラスミドＤＮＡを導入する細胞として、マウス胎児線維芽細胞株ｍ５ｓ細胞を用いた。１５％ＦＢＳ含有Ｄ−ＭＥＭ培地（日水製薬製）にて上記ｍ５ｓ細胞を３７℃、５％炭酸ガス空気下で培養した。対数増殖期にある細胞をトリプシン溶液ではがし、ファルコンチューブに回収した。遠心分離（１０００ｒｐｍ／５分）し１×ＰＢＳ（−）で洗浄し、細胞数が１．２×１０７ｃｅｌｌｓ／ ｍｌになるよう１×Ｋ−ＰＢＳ（３０ｍＭ ＮａＣｌ， １２０ｍＭ ＫＣｌ， ８ｍＭ Ｎａ２ＨＰＯ４， １．５ｍＭ ＫＨ２ＰＯ４， ５ｍＭ ＭｇＣｌ２）に懸濁した。
<(2) Introduction of plasmid DNA and acquisition of stably transformed cells>
Next, the above plasmid DNA was introduced into the cells to obtain stably transformed cells. The mouse embryo fibroblast cell line m5s cell was used as a cell into which the plasmid DNA was introduced. The m5s cells were cultured in a 15% FBS-containing D-MEM medium (manufactured by Nissui Pharmaceutical) at 37 ° C. under 5% carbon dioxide air. Cells in the logarithmic growth phase were peeled off with trypsin solution and collected in a falcon tube. Centrifugation (1000 rpm / 5 minutes), washing with 1 × PBS (−), and 1 × K-PBS (30 mM NaCl, 120 mM KCl, 8 mM Na 2 HPO) so that the number of cells becomes 1.2 × 10 7 cells / ml. 4 , 1.5 mM KH 2 PO 4 , 5 mM MgCl 2 ).
この細胞懸濁液４８０μｌをマイクロチューブに移し氷中で５分静置後、上記プラスミドＤＮＡ２０μｌを添加し、氷中で１０分静置した。その後懸濁液を冷却した４ｍｍ幅シングルキュベット（ＢＩＯ−ＲＡＤ社製）に移し、パルス発生装置「Ｇｅｎｅ Ｐｕｌｓｅｒ Ｘｃｅｌｌ」（ＢＩＯ−ＲＡＤ社製）を用いて２７０Ｖ、１０５０μＦの条件で、上記プラスミドＤＮＡをｍ５ｓ細胞に導入し直ちに１０分間氷冷した。氷冷後無血清Ｄ−ＭＥＭを０．５ｍｌ加えて室温で１０分静置し、パスツールピペットで回収した細胞を９０ｍｍシャーレ数枚に播いた。 480 μl of this cell suspension was transferred to a microtube and allowed to stand in ice for 5 minutes, and then 20 μl of the plasmid DNA was added and allowed to stand in ice for 10 minutes. Thereafter, the suspension was transferred to a cooled 4 mm single cuvette (manufactured by BIO-RAD), and the above plasmid DNA was transferred using a pulse generator “Gene Pulser Xcell” (manufactured by BIO-RAD) at 270 V and 1050 μF. The cells were introduced into m5s cells and immediately cooled on ice for 10 minutes. After ice-cooling, 0.5 ml of serum-free D-MEM was added and allowed to stand at room temperature for 10 minutes, and the cells collected with a Pasteur pipette were seeded on several 90 mm dishes.
細胞を播いたシャーレに選択培地Ｇ４１８（ナカライ社製）を２００〜６００μｇ／ｍｌとなるように添加し、３〜５日毎に培地交換をして培養を続けた。約２週間経過後、形成されたコロニーをクローニングリングを用いて個々に回収し、カバーグラスを入れた１２穴マイクロプレートに移して培養した。次の操作（形質転換細胞での目的とする構造の可視化の確認）により、各蛍光タンパク質が核で発現し、かつ大核など異常な核が少ないと確認された細胞を安定形質転換細胞株とし、９０ｍｍシャーレにて培養し、液体窒素中にて保存した。安定形質転換細胞株として得る事ができたのは、ｍＣｈｅｒｒｙ−Ｈ３導入株が５クローンと、Ｖｅｎｕｓ−Ｌ２０１Ａ−Ｈ３導入株が４クローンであった。取得した蛍光細胞を表２に示す。 A selective medium G418 (manufactured by Nacalai) was added to the petri dish seeded with the cells so as to be 200 to 600 μg / ml, and the medium was changed every 3 to 5 days to continue the culture. After about 2 weeks, the formed colonies were individually collected using a cloning ring, transferred to a 12-well microplate with a cover glass, and cultured. By the next operation (confirmation of visualization of the target structure in the transformed cells), cells that have been confirmed that each fluorescent protein is expressed in the nucleus and that there are few abnormal nuclei such as macronuclei are defined as stable transformed cell lines. The cells were cultured in a 90 mm petri dish and stored in liquid nitrogen. As a stably transformed cell line, 5 clones with mCherry-H3 and 4 clones with Venus-L201A-H3 were obtained. The obtained fluorescent cells are shown in Table 2.
＜（３）形質転換細胞での目的とする構造の可視化の確認＞
上記のカバーグラス上で培養された細胞を４％パラホルムアルデヒドで２０分間固定し、０．１％トリトンＸ−１００で５分間処理した。細胞核をＤＡＰＩ溶液（１μｇ／ｍｌ）で染色し、対物レンズ「ＰｌａｎＡｐｏ ６０ｘ」（ＮＡ１．４０、ニコン社製）を備えた蛍光顕微鏡「Ｅｃｌｉｐｓｅ Ｅ６００」（ニコン社製）を用いて観察した。ＤＡＰＩ、ｍＣｈｅｒｒｙ、Ｖｅｎｕｓはそれぞれの蛍光を観察するにあたってＵＶ−１Ａ（ＥＸ ３６５ ／ １０ ＤＭ４００ ＢＡ４００）、Ｔｅｘａｓ Ｒｅｄ（ＥＸ ５４０−５８０ ＤＭ５９５ ＢＡ６００−６６０）、Ｙｅｌｌｏｗ ＧＦＰ（ＥＸ ４６５−４９５ ＤＭ５０５ ＢＡ５１５−５５５）を使用した。目的とする細胞構造（核）がそれぞれの蛍光を放っていることを確認した。
<(3) Confirmation of visualization of target structure in transformed cells>
The cells cultured on the above cover glass were fixed with 4% paraformaldehyde for 20 minutes and treated with 0.1% Triton X-100 for 5 minutes. Cell nuclei were stained with a DAPI solution (1 μg / ml), and observed using a fluorescence microscope “Eclipse E600” (Nikon) equipped with an objective lens “PlanApo 60x” (NA 1.40, Nikon). DAPI, mCherry, and Venus have UV-1A (EX 365/10 DM400 BA400), Texas Red (EX 540-580 DM595 BA600-660), Yellow GFP (EX 465-495 DM505 BA515-555) It was used. It was confirmed that the target cell structure (nucleus) emitted each fluorescence.
＜染色体数の確認＞
次に上記の操作で得る事のできた安定形質転換細胞株の染色体の数を確認した。
<Confirmation of the number of chromosomes>
Next, the number of chromosomes of the stably transformed cell line obtained by the above operation was confirmed.
＜（１）染色体展開標本の作製＞
対数増殖期にあるｍ５ｓ蛍光細胞にコルセミドを終濃度０．０６μｇ／ｍｌになるよう添加し、２時間３７℃、５％炭酸ガス空気下で培養した。その後トリプシン溶液で細胞をはがしてファルコンチューブに回収し、遠心分離（１０００ｒｐｍ／５分）した。その上澄みを捨て、０．０７５Ｍ塩化カリウム水溶液２ｍｌを加えて穏やかに懸濁し、２５分間室温において低張処理を行った。処理後、氷冷したカルノア液（酢酸：メタノール＝１：３）２ｍｌを重層し、穏やかに撹拌した。遠心分離（１０００ｒｐｍ／５分）し上澄みを捨てた後、カルノア液４ｍｌを加え穏やかに撹拌し氷中に５分間静置した。その後遠心分離（１０００ｒｐｍ／５分）し上澄みを捨て、カルノア液４ｍｌで再度撹拌、氷冷を行った。
<(1) Preparation of chromosome expansion specimen>
Colcemid was added to m5s fluorescent cells in the logarithmic growth phase to a final concentration of 0.06 μg / ml, and the cells were cultured for 2 hours at 37 ° C. in 5% carbon dioxide air. Thereafter, the cells were peeled off with a trypsin solution, collected in a falcon tube, and centrifuged (1000 rpm / 5 minutes). The supernatant was discarded, and 2 ml of 0.075 M potassium chloride aqueous solution was added to suspend the mixture gently, and hypotonic treatment was performed at room temperature for 25 minutes. After the treatment, 2 ml of ice-cooled Carnoy's solution (acetic acid: methanol = 1: 3) was layered and gently stirred. After centrifugation (1000 rpm / 5 minutes) and discarding the supernatant, 4 ml of Carnoy's solution was added, gently stirred and allowed to stand in ice for 5 minutes. Thereafter, the mixture was centrifuged (1000 rpm / 5 minutes), the supernatant was discarded, and the mixture was stirred again with 4 ml of Carnoy's solution and cooled with ice.
カルノア液１ｍｌを加えた細胞懸濁液を２０μｌとり、染色体メタフェーズ標本作製用装置ＨＡＮＡＢＩ（ＡＤＳＴＥＣ社製）を用いて、スライドグラス上に滴下した。標本の乾燥後、ＤＡＰＩ溶液（１μｇ／ｍｌ）で核・染色体を染色し、グリセロールで標本を封入した。 20 μl of cell suspension added with 1 ml of Carnoy's solution was taken and dropped onto a slide glass using a chromosome metaphase specimen preparation apparatus HANABI (manufactured by ADSTEC). After the specimen was dried, nuclei and chromosomes were stained with a DAPI solution (1 μg / ml), and the specimen was encapsulated with glycerol.
＜（２）染色体数の測定＞
上記の標本を、対物レンズ「ＰｌａｎＡｐｏ ６０ｘ」（ＮＡ１．４０、ニコン社製）を備えた蛍光顕微鏡「Ｅｃｌｉｐｓｅ Ｅ６００」（ニコン社製）を用いて観察した。ＤＡＰＩが観察可能なフィルターを用いて、展開された染色体について観察した。染色体が展開しているもののうち、明らかに染色体数が形質転換前と異なる２クローン、即ち、４割が６０本以上の染色体をもつクローン（ｍＣｈｅｒｒｙ−Ｈ３ Ｃ１８）と、８割が７０〜８０本の染色体をもつクローン（ｍＣｈｅｒｒｙ−Ｈ３ Ａ２０）、を除き、各クローンの細胞５０個分についてその染色体数をカウントした。
<(2) Measurement of the number of chromosomes>
The specimen was observed using a fluorescence microscope “Eclipse E600” (Nikon Corp.) equipped with an objective lens “PlanApo 60x” (NA 1.40, Nikon Corp.). The developed chromosomes were observed using a DAPI observable filter. Among clones that have developed chromosomes, two clones that clearly differ in number of chromosomes from before transformation, that is, 40% have 60 or more chromosomes (mCherry-H3 C18) and 80% have 70-80 The number of chromosomes was counted for 50 cells of each clone, except for the clone (mCherry-H3 A20).
得られた蛍光細胞には、染色体数が形質転換前のｍ５ｓ細胞の染色体数に近いクローン（ｍＣｈｅｒｒｙ−Ｈ３ Ａ１４， Ｖｅｎｕｓ−Ｈ３ Ａ１４など）もあれば、染色体数がばらついているクローン（ｍＣｈｅｒｒｙ−Ｈ３ Ｂ２−１２）もあった。図１でｍＣｈｅｒｒｙ−Ｈ３ Ａ１４， Ｂ２−１２、Ｖｅｎｕｓ−Ｈ３ Ａ１４の染色体数の分布図を示す。 Among the obtained fluorescent cells, there are clones (mCherry-H3A14, Venus-H3A14, etc.) whose number of chromosomes is close to that of m5s cells before transformation, and clones (mCherry-H3) whose number of chromosomes varies. B2-12) was also present. FIG. 1 shows the distribution of chromosome numbers of mCherry-H3 A14, B2-12, and Venus-H3 A14.
図１は、縦軸が細胞数を表し、横軸が染色体数を表す。図１（ａ）、（ｂ）、（ｃ）がそれぞれｍＣｈｅｒｒｙ−Ｈ３ Ａ１４， Ｂ２−１２、Ｖｅｎｕｓ−Ｈ３ Ａ１４の結果である。 In FIG. 1, the vertical axis represents the number of cells, and the horizontal axis represents the number of chromosomes. 1A, 1B, and 1C show the results of mCherry-H3 A14, B2-12, and Venus-H3 A14, respectively.
本発明の検査方法には、分裂後の染色体数が変わらない細胞を使用するので、例えば、クローン（ｍＣｈｅｒｒｙ−Ｈ３ Ｂ２−１２）は、利用することができない。以下の実施例では、クローン（ｍＣｈｅｒｒｙ−Ｈ３ Ａ１４）を用いた。もちろん、クローン（Ｖｅｎｕｓ−Ｈ３ Ａ１４）を用いることもできる。 In the test method of the present invention, since the number of chromosomes after division does not change, for example, a clone (mCherry-H3 B2-12) cannot be used. In the following examples, a clone (mCherry-H3 A14) was used. Of course, a clone (Venus-H3 A14) can also be used.
この細胞を３５ｍｍガラスボトムデイッシュ（イワキ社製）へ接種し、ＩＮＵ−ＮＩ−ＦＩ顕微鏡保温装置「ステージトップインキュベーター」（東海ヒット社製）で培養した。 The cells were inoculated into a 35 mm glass bottom dish (manufactured by Iwaki Co., Ltd.) and cultured with an INU-NI-FI microscope heat retaining device “Stage Top Incubator” (manufactured by Tokai Hit Co., Ltd.).
次に比較例として用いた細胞について説明する。 Next, a cell used as a comparative example will be described.
＜＜比較例の可視化細胞の作製と観察＞＞
＜（１）プラスミドＤＮＡの作製＞
（ａ）ｍＰｌｕｍ−Ｈ３
ｐＢＡＤ−ｍＰｌｕｍ（カリフォルニア大学 Ｔｓｉｅｎ博士より寄贈）を鋳型とし、プライマーＣおよびＭを用いてＰＣＲ法を利用して増幅させ、蛍光タンパク質ｍＰｌｕｍ遺伝子断片を得た。なお、プライマーＭは配列番号６であるｐＥＧＦＰ−Ｃ１（クロンテック社）をＮｈｅＩ、ＢｇｌＩＩで切断し、ＥＧＦＰ遺伝子を取いた部位に、あらかじめＮｈｅＩ、ＢｇｌＩＩで処理したｍＰｌｕｍ遺伝子断片を挿入することにより、ｐｍＰｌｕｍ−Ｃ１を作製した。
<< Preparation and Observation of Visualized Cell of Comparative Example >>
<(1) Preparation of plasmid DNA>
(A) mPlum-H3
pBAD-mPlum (contributed by Dr. Tsien, University of California) was used as a template, and amplification was performed using the primers C and M using the PCR method to obtain a fluorescent protein mPlum gene fragment. Primer M was prepared by cleaving pEGFP-C1 (Clontech) of SEQ ID NO: 6 with NheI and BglII, and inserting the mPlum gene fragment previously treated with NheI and BglII into the site where the EGFP gene was removed. -C1 was produced.
ヒストンＨ３−赤色蛍光蛋白質発現プラスミド（ｍＰｌｕｍ−Ｈ３）は、ｐｍＰｌｕｍ−Ｃ１をＢａｍＨＩ、ＥｃｏＲＩで切断した部位に、ｐＥＣＦＰ−Ｈ３をＢａｍＨＩ、ＥｃｏＲＩで切断して得られたヒストンＨ３遺伝子断片を挿入することにより作製した。 In the histone H3-red fluorescent protein expression plasmid (mPlum-H3), the histone H3 gene fragment obtained by cleaving pECFP-H3 with BamHI and EcoRI is inserted into the site obtained by cleaving pmPlum-C1 with BamHI and EcoRI. It was produced by.
＜（２）プラスミドＤＮＡの導入および安定形質転換細胞の取得＞
上記プラスミドＤＮＡを導入する細胞として、ヒト乳癌細胞株（ＭＤＡ４３５）由来の４色可視化細胞株ＭＤＡ−Ａｕｒｏ／ｉｍｐ／Ｈ３／ＡＦ（Ｓｕｇｉｍｏｔｏ ｅｔ ａｌ． Ｍｕｔａｔｉｏｎ Ｒｅｓｅａｒｃｈ ６５７， ５６−６２， ２００８、）を用いた。１０％ＦＢＳ含有Ｄ−ＭＥＭ培地（日水製薬製）にて上記細胞株を３７℃、５％炭酸ガス空気下で培養した。対数増殖期にある細胞をトリプシン溶液ではがし、ファルコンチューブに回収した。遠心分離（１０００ｒｐｍ／５分）し１×ＰＢＳ（−）で洗浄し、細胞数が１．２×１０７ｃｅｌｌｓ／ｍｌになるよう１×Ｋ−ＰＢＳに懸濁した。
<(2) Introduction of plasmid DNA and acquisition of stably transformed cells>
As a cell into which the plasmid DNA is introduced, a 4-color visualization cell line MDA-Auro / imp / H3 / AF derived from a human breast cancer cell line (MDA435) (Sugimoto et al. Mutation Research 657, 56-62, 2008) is used. It was. The cell line was cultured in 10% FBS-containing D-MEM medium (manufactured by Nissui Pharmaceutical) at 37 ° C. under 5% carbon dioxide air. Cells in the logarithmic growth phase were peeled off with trypsin solution and collected in a falcon tube. Centrifugation (1000 rpm / 5 minutes), washing with 1 × PBS (−), and suspending in 1 × K-PBS so that the number of cells was 1.2 × 10 7 cells / ml.
この細胞懸濁液４８０μｌをマイクロチューブに移し氷中で５分静置後、上記プラスミドＤＮＡ２０μｌを添加し、氷中で１０分静置した。その後懸濁液を冷却した４ｍｍ幅キュベット（ＢＩＯ−ＲＡＤ社製）に移し、パルス発生装置「Ｇｅｎｅ Ｐｕｌｓｅｒ」（ＢＩＯ−ＲＡＤ社製）を用いて２００Ｖ、９５０μＦの条件で、上記プラスミドＤＮＡを薬剤耐性遺伝子（ｂｌｅ）発現プラスミドＤＮＡとともにＭＤＡ細胞に導入し直ちに１０分間氷冷した。氷冷後無血清Ｄ−ＭＥＭを０．５ｍｌ加えて室温で１０分静置し、パスツールピペットで回収した細胞を９０ｍｍシャーレ数枚に播いた。 480 μl of this cell suspension was transferred to a microtube and allowed to stand in ice for 5 minutes, and then 20 μl of the plasmid DNA was added and allowed to stand in ice for 10 minutes. Thereafter, the suspension was transferred to a cooled 4 mm width cuvette (manufactured by BIO-RAD), and the plasmid DNA was drug resistant under the conditions of 200 V and 950 μF using a pulse generator “Gene Pulser” (manufactured by BIO-RAD). The gene (ble) expression plasmid DNA was introduced into MDA cells and immediately cooled on ice for 10 minutes. After ice-cooling, 0.5 ml of serum-free D-MEM was added and allowed to stand at room temperature for 10 minutes, and the cells collected with a Pasteur pipette were seeded on several 90 mm dishes.
細胞を播いたシャーレに選択培地Ｚｅｏｃｉｎ（インビトロゲン社製）を１５μｇ／ｍｌとなるように添加し、３〜５日毎に培地交換をして培養を続けた。約２週間経過後、形成されたコロニーをクローニングリングを用いて個々に回収し、カバーグラスを入れた１２穴マイクロプレートに移して培養した。上記で説明した形質転換細胞での目的とする構造の可視化の確認の操作により、赤色の蛍光を放つｍＰｌｕｍ蛍光タンパク質が核に局在する細胞株７株（Ｎｏ．３，６，７，９，１３，３０，３１）をｍＰｌｕｍ−ヒストンＨ３安定形質転換細胞株として取得し、９０ｍｍシャーレにて培養し、液体窒素中にて保存した。 The selection medium Zeocin (manufactured by Invitrogen) was added to the petri dish seeded with the cells so that the concentration was 15 μg / ml, and the culture was continued after changing the medium every 3 to 5 days. After about 2 weeks, the formed colonies were individually collected using a cloning ring, transferred to a 12-well microplate with a cover glass, and cultured. Seven cell lines (No. 3, 6, 7, 9, 9) in which mPlum fluorescent protein emitting red fluorescence is localized in the nucleus by the above-described operation for confirming the visualization of the target structure in the transformed cells. 13, 30, 31) was obtained as an mPlum-histone H3 stable transformed cell line, cultured in a 90 mm petri dish, and stored in liquid nitrogen.
比較例として用いた細胞は得られたｍＰｌｕｍ−ヒストンＨ３安定形質転換細胞株（Ｎｏ．９）を用いた。細胞を３５ｍｍガラスボトムデイッシュ（イワキ社製）へ接種し、ＩＮＵ−ＮＩ−ＦＩ顕微鏡保温装置「ステージトップインキュベーター」（東海ヒット社製）で培養した。 The obtained mPlum-histone H3 stable transformed cell line (No. 9) was used as a cell used as a comparative example. The cells were inoculated into a 35 mm glass bottom dish (manufactured by Iwaki Co., Ltd.) and cultured with an INU-NI-FI microscope heat retaining device “Stage Top Incubator” (manufactured by Tokai Hit Co., Ltd.).
次に可視化細胞のライブ観察について説明する。 Next, live observation of visualized cells will be described.
＜＜可視化細胞のライブ観察＞＞
ライブ観察には以下の装置を用いた。ＥＣＬＩＰＳＥ ＴＥ３００（ニコン社製）へ、ＰｌａｎＡｐｏ ＶＣ ６０Ｘ対物レンズ（ニコン社製）とＯＲＣＡ−ＥＲ ＣＣＤカメラ（浜松ホトニクス社製）、さらに、励起、吸収フィルターホイールとＺ軸モータ（Ｌｕｄｌ Ｅｌｅｃｔｒｏｎｉｃ Ｐｒｏｄｕｃｔｓ社製）を備えたＭＡＣ５０００コントローラーを取り付けた。
<< Live observation of visualized cells >>
The following apparatus was used for live observation. ECLIPSE TE300 (made by Nikon), PlanApo VC 60X objective lens (made by Nikon), ORCA-ER CCD camera (made by Hamamatsu Photonics), excitation, absorption filter wheel and Z-axis motor (made by Ludl Electronic Products) A MAC5000 controller equipped with was attached.
１００Ｗのハロゲンランプを光源にし、励起、吸収フィルターの波長特性は、それぞれ、５７２をピークに３５ｎｍ幅のものと６３２をピークに６０ｎｍ幅のもので、ダイクロイックミラーは８９００６ｂｓ （８９００６ ＣＦＰ／ＹＦＰ／ｍＣｈｅｒｒｙ−ＥＴ、Ｃｈｒｏｍａ Ｔｅｃｈｎｏｌｏｇｙ社製）を用いた。 Using a 100 W halogen lamp as the light source, the wavelength characteristics of the excitation and absorption filters are 35 nm wide at 572 and 60 nm wide at 632, respectively, and the dichroic mirror is 89006bs (89006 CFP / YFP / mCherry- ET, manufactured by Chroma Technology).
生細胞のタイムラプス画像は、ＭａｃＯＳ Ｘ（三谷コーポレーション社製）用ルミナビジョンソフトウェアを用いて、焦点面を２μｍずつ移動させつつ、２〜４分のタイムラプスで取得した。 The time-lapse image of the living cells was acquired with a time lapse of 2 to 4 minutes using Luminovision software for MacOS X (manufactured by Mitani Corporation) while moving the focal plane by 2 μm.
得られたＺ軸画像スタックは、ルミナビジョンソフトウェアを用いて最大値投影法にて変換し、画像解析した。 The obtained Z-axis image stack was converted by the maximum value projection method using Lumina Vision software, and image analysis was performed.
上記の観察装置を用いて、実施例クローン（ｍＣｈｅｒｒｙ−Ｈ３ Ａ１４）と比較例クローン（ｍＰｌｕｍ−ヒストンＨ３−Ｎｏ３）のライブ観察を行った。観察はそれぞれ複数個の細胞についてライブ観察し、所定時間毎に写真撮影した。得られた写真から細胞の分裂時間（染色体が凝縮してから２極に分かれるまでの時間）を判定した。実施例クローン（ｍＣｈｅｒｒｙ−Ｈ３ Ａ１４）の分割時間を図２（ａ）に、また比較例クローン（ｍＰｌｕｍ−ヒストンＨ３−Ｎｏ３）の分割時間を図２（ｂ）に示す。 Using the above-described observation apparatus, live observation of the example clone (mCherry-H3 A14) and the comparative example clone (mPlum-histone H3-No3) was performed. For observation, a plurality of cells were observed live, and photographs were taken every predetermined time. From the obtained photograph, the cell division time (the time from when the chromosomes were condensed to when they were separated into two poles) was determined. FIG. 2 (a) shows the division time of the example clone (mCherry-H3 A14), and FIG. 2 (b) shows the division time of the comparative clone (mPlum-histone H3-No3).
図２（ａ）のグラフを参照して、実施例の細胞は、平均的な分裂時間は３６．５分であり、観察した１６個の細胞すべてが、ほぼ±４分以内（±３．８３分）と細胞分裂時間がよくそろっていた。一方、図２（ｂ）を参照して、比較例クローン（ｍＰｌｕｍ−ヒストンＨ３−Ｎｏ３）では、観察した３２個の細胞の分裂時間はそれぞれまちまちであった。 Referring to the graph of FIG. 2 (a), the cells of the examples had an average division time of 36.5 minutes, and all the 16 cells observed were within approximately ± 4 minutes (± 3.83). Min) and cell division time were well aligned. On the other hand, with reference to FIG. 2 (b), in the comparative clone (mPlum-histone H3-No3), the observed division times of the 32 cells varied.
実施例クローンの分裂時間がそろっているのは、分裂した後の染色体の数が変化しないことに起因すると考えた。すなわち、実施例クローンのような細胞株の細胞は、分裂時間がよくそろっている。その特徴の一つが分裂しても染色体に変化が少ないということであると考えた。一方、比較例クローンの場合のように、分裂後の染色体の数が細胞毎にバラバラであると、細胞の分裂時間もまちまちとなってしまう。逆に言うと、分裂しても染色体の数に大きな変化のない細胞は、その株に属する全ての細胞について、ほぼ同じ分裂時間を有すると考えられる。本発明は、このような性質を利用し、被検査物に曝す細胞を分裂しても染色体の数に変化のない細胞として、そのうち、数個の細胞を詳細に観察し、分裂時間等を観察することで、細胞毒性を検査するものである。 It was thought that the fact that the division time of the Example clones was the same was caused by the fact that the number of chromosomes after division did not change. That is, the cells of the cell line such as the example clone have a good division time. We thought that one of the characteristics was that there was little change in the chromosome even if it divided. On the other hand, if the number of chromosomes after division varies from cell to cell, as in the case of the comparative clone, the cell division time also varies. Conversely, cells that do not change significantly in the number of chromosomes when they divide are considered to have approximately the same division time for all cells belonging to the strain. The present invention makes use of such properties, and as a cell whose number of chromosomes does not change even when the cell exposed to the test object is divided, several cells are observed in detail, and the division time is observed. By doing so, the cytotoxicity is examined.
＜＜分裂時間の変化＞＞
ｍＣｈｅｒｒｙ−Ｈ３−Ａ１４−６株を３５ｍｍガラスボトムディッシュ（イワキ社製）に播き、一日培養後、薬剤マイトマイシンＣ（以後ＭＭＣと表記する）を終濃度３００ｎＭ（ＩＣ５０濃度の約３倍で、投与後２４時間後に小核発生頻度が約３倍になる濃度）になるよう投与した。投与後４時間経過した後、培地を新しいものに交換し薬剤を除去し、細胞を記述のライブ観察装置に設置し観察を行った。結果を図３に示す。縦軸は細胞数を表し、横軸は分裂時間（分）である。
<< Changes in division time >>
The mCherry-H3-A14-6 strain is seeded in a 35 mm glass bottom dish (manufactured by Iwaki Co., Ltd.) and cultured for one day. The concentration was such that the micronucleus frequency was approximately 3 times later 24 hours later. After 4 hours from the administration, the medium was replaced with a new one, the drug was removed, and the cells were placed in the described live observation apparatus for observation. The results are shown in FIG. The vertical axis represents the number of cells, and the horizontal axis represents the division time (minutes).
分裂時間は、２４分から１０８分まで広がった。ライブ観察した６８細胞での分裂時間の平均は４０分であり、ＳＤは±１８分であった。これは、薬剤に曝されなかったコントロールが平均３６．５分±４分であったことを考えると、ばらつきが大きくなったと判断できる。また、分裂時間が２４分から４８分までのグループと、６０分から１０８分までのグループに分かれたという見方もできる。 The division time extended from 24 minutes to 108 minutes. The average mitotic time of 68 cells observed live was 40 minutes and the SD was ± 18 minutes. This can be judged to have increased variability, considering that the average of controls that were not exposed to the drug was 36.5 minutes ± 4 minutes. It can also be said that the division time was divided into a group of 24 to 48 minutes and a group of 60 to 108 minutes.
しかも、この細胞は、集団として特性がそろっているので、分裂時間が長くなり、その分散も長くなったのは、個々の細胞自体の特性ではなく、薬の影響であったと判断できる。このことから、影響のわからない薬剤に対して、数十個単位の細胞をライブ観察し、コントロールの細胞と比較することで、薬剤の影響を分裂時間の変化として捉えることができる。 Moreover, since these cells have characteristics as a group, it can be determined that the division time is long and the dispersion is long because of the influence of drugs, not the characteristics of individual cells themselves. From this, it is possible to grasp the influence of the drug as a change in mitotic time by observing several tens of cells live for a drug whose effect is not known and comparing it with a control cell.
また、本発明の毒性検査では、細胞の分裂状態を直接観察するので、細胞の生き死にだけでなく、細胞に生じた変化も確認することができる。例えば、図３のグラフにおいて、黒の部分は、細胞に小核が生じたものである。 Further, in the toxicity test of the present invention, since the cell division state is directly observed, it is possible to confirm not only the survival of the cell but also the change occurring in the cell. For example, in the graph of FIG. 3, the black part is a cell in which micronuclei are generated.
（実施の形態２）
＜細胞系譜図＞
観察には得られた蛍光細胞のうち、染色体数が形質転換前のｍ５ｓ細胞の染色体数に近いクローンｍＣｈｅｒｒｙ−Ｈ３ Ａ１４−６株を用いた。細胞を３５ｍｍガラスボトムデイッシュ（イワキ社製）へ接種し、ＩＭＦ−Ｉ−Ｗ 蒸留水供給装置（東海ヒット社製） を取り付けた、ＩＮＵ Ｇ２−ＷＤＳ−Ｐ顕微鏡保温装置（東海ヒット社製）で培養した。
(Embodiment 2)
<Cell lineage chart>
Among the obtained fluorescent cells, a clone mCherry-H3 A14-6 strain having a chromosome number close to that of m5s cells before transformation was used for the observation. Cells were inoculated into a 35 mm glass bottom dish (manufactured by Iwaki) and an INU-I-W distilled water supply device (manufactured by Tokai Hit) was attached. In culture.
ＥＣＬＩＰＳＥ Ｔｉ顕微鏡（ニコン社製）へ、Ｐｌａｎ Ａｐｏ ４０Ｘ対物レンズ（ニコン社製）とＩｍａｇＥＭ ＥＭ−ＣＣＤカメラ（浜松ホトニクス社製）とさらに、ＰｒｏＳｃａｎ ＩＩ ステージ・Ｚ軸コントローラー（ＰＲＩＯＲ社製）を取り付けた。 A Plan Apo 40X objective lens (Nikon), ImagEM EM-CCD camera (Hamamatsu Photonics), and ProScan II stage / Z-axis controller (PRIOR) were attached to an ECLIPSE Ti microscope (Nikon). .
１００Ｗのハロゲンランプを光源にし、適切な波長の光を得るために、波長特性が５７２をピークに３５ｎｍ幅と６３２をピークに６０ｎｍ幅の、ダイクロイックミラー（Ｓｅｍｒｏｋ社製）を用いた。 In order to obtain light having an appropriate wavelength using a 100 W halogen lamp as a light source, a dichroic mirror (manufactured by Semlok Co., Ltd.) having a wavelength characteristic of 35 nm width with a peak of 572 and a width of 60 nm with a peak of 632 was used.
生細胞のタイムラプス画像は、Ｗｉｎｄｏｗｓ（登録商標）用Ｖｏｌｏｃｉｔｙソフトウェア（Ｉｍｐｒｏｖｉｓｉｏｎ社製）を用いて、焦点面を２μｍずつ移動させつつ、６分のタイムラプスで取得した。 A time-lapse image of the living cells was acquired with a time lapse of 6 minutes while moving the focal plane by 2 μm by using Volocity software for Windows (registered trademark) (manufactured by Improvision).
この細胞の分裂時間と分裂間時間がどの程度安定しているかを表３に示す。表３でＭＤＡは５色可視化したヒト由来の癌細胞である。このＭＤＡ細胞とＡ１４−６株を６５時間まで調べた際の分裂時間と分裂間時間を比較したものである。ライブ撮影は上記のように６分間隔で撮影しているので、例えばＡ１４−６株は、６５時間後であっても分裂時間が写真２コマ程度の差しかない。すなわち、５１個の細胞について、６５時間経過した後であっても、ほぼ同じ分裂時間で分裂が行われていることを示している。 Table 3 shows how stable the cell division time and the time between divisions are. In Table 3, MDA is a human-derived cancer cell visualized in five colors. This is a comparison of the division time and the time between divisions when the MDA cells and the A14-6 strain were examined up to 65 hours. Since live shooting is taken at intervals of 6 minutes as described above, for example, the A14-6 strain has a split time of about two frames even after 65 hours. That is, it shows that about 51 cells are divided at almost the same division time even after 65 hours.
一方、ＭＤＡの場合は、分裂時間の平均からの上下の広がり幅が１４０分近くあり、平均の８５．７分よりも大きくなる。これらの細胞でこれだけ分裂時間に差ができると、任意に選んだ細胞が全体を代表しているとは言い難い。 On the other hand, in the case of MDA, the vertical spread from the average splitting time is nearly 140 minutes, which is larger than the average of 85.7 minutes. If these cells have such a difference in division time, it is difficult to say that the cells selected arbitrarily represent the whole.
細胞分裂時に染色体が分離する画像が取得できた。取得画像より、染色体が凝縮してから２極に分かれるまでの時間を調べたところ、染色体の分裂時間のばらつきが少なく、ほぼそろっていることが分かった。さらに長時間（約６５時間）の撮影で、最高で５回の連続する分裂像が取得でき、染色体が分裂してから再び分裂するまでの分裂間時間も、ばらつきが少なかった。 Images of chromosome separation during cell division were obtained. From the acquired images, when the time from when the chromosomes were condensed until they were separated into two poles was examined, it was found that there was little variation in the chromosome division time and almost all of them. Furthermore, by taking a long time (about 65 hours), it was possible to obtain a maximum of 5 consecutive division images, and there was little variation in the time between divisions after the chromosomes were divided again.
図５は、上記のｍＣｈｅｒｒｙ−Ｈ３ Ａ１４−６の特定した細胞についての、分裂の世代を示す系譜図である。それぞれの代の細胞には、最初の親をＣ１とし、その後Ｃ２、Ｃ３・・、と番号を付与した。同じ世代の細胞には、Ｃ３ａ、Ｃ３ｂなどのように数字の後にアルファベットを付与して区別する。 FIG. 5 is a lineage diagram showing the generation of division for the identified cells of mCherry-H3 A14-6 described above. The cells of each generation were given the first parent as C1, and then numbered C2, C3,. Cells of the same generation are distinguished by adding an alphabet after the number, such as C3a, C3b, etc.
図中丸で囲んだ数字は、これらの細胞の分裂の回数を表す。また、世代間をつなぐ線の長さは、大まかに分裂間時間の長さを反映させている。この系譜図を見ると、最初の細胞Ｃ１が分裂して２つのＣ２（Ｃ２ａ、Ｃ２ｂ）ができ、それぞれの細胞からさらに分裂してＣ３（Ｃ３ａ、Ｃ３ｂ、Ｃ３ｃ、Ｃ３ｄ）を生んでいることを順次観測できていることが分かる。 The numbers circled in the figure represent the number of divisions of these cells. In addition, the length of the line connecting the generations roughly reflects the length of the time between divisions. Looking at this genealogy, it can be seen that the first cell C1 has divided to form two C2 (C2a, C2b), and further divided from each cell to give C3 (C3a, C3b, C3c, C3d). It can be seen that it can be observed sequentially.
図中で、「ＯＵＴ」は顕微鏡の視野から外れ、観測出来なくなったことを示す。本発明の細胞は壁面に固着しやすいので、顕微鏡を視野を動かすことで、ある程度追跡することができる。しかし、分裂の際には、大きく動くことがあり、すると、追跡できる視野範囲からはずれてしまう。 In the figure, “OUT” is out of the field of view of the microscope and cannot be observed. Since the cell of the present invention tends to adhere to the wall surface, it can be traced to some extent by moving the field of view of the microscope. However, there may be large movements at the time of splitting, which will deviate from the field of view that can be tracked.
なお、特定の細胞は複数個を選んで観測している。５回の分裂まで観測し、観測した全ての細胞の分裂時間と分裂間時間の平均を求めると、分裂時間は平均２３．９±４．８８分（ｎ＝６３）であり、分裂間時間の平均は１３．２±２．５３時間（ｎ＝４０）であった。このように、分裂しても染色体の数が変わらない細胞は、培地中で順調に分裂し、その分裂の系譜図を追跡することができる。 A specific cell is selected and observed. Observing up to 5 divisions, and finding the average of the division time and the time between divisions of all the observed cells, the average division time is 23.9 ± 4.88 minutes (n = 63). The average was 13.2 ± 2.53 hours (n = 40). Thus, a cell whose number of chromosomes does not change even when it divides can divide smoothly in the medium, and the lineage diagram of the division can be traced.
なお、分裂時間と分裂間時間のｎ数に違いがあるのは、分裂回数が多くなると、視野の外に出て観測できなくなる細胞が増えるからである。 Note that the difference in n number between the division time and the time between divisions is that as the number of divisions increases, the number of cells that go out of the field of view and cannot be observed increases.
＜ＭＭＣ処理細胞のライブ観察＞
ｍＣｈｅｒｒｙ−Ｈ３ Ａ１４−６株を３５ｍｍガラスボトムディッシュ（イワキ社製）に播き、一日培養後、薬剤マイトマイシンＣ （以後ＭＭＣと表記する）を終濃度３００ｎＭ （ＩＣ５０濃度の約３倍）になるよう投与した。投与後４時間経過した後、培地を新しいものに交換し薬剤を除去し、細胞を記述のライブ観察装置に設置し観察を行った。その結果、小核が形成される様子が観察でき、連続する分裂において小核が続けてできるもの、２回目以降の分裂で小核ができるもの、小核をもつ細胞のその後の様子を追うことができた。
<Live observation of MMC-treated cells>
The mCherry-H3 A14-6 strain is seeded on a 35 mm glass bottom dish (manufactured by Iwaki), and after culturing for one day, the drug mitomycin C (hereinafter referred to as MMC) reaches a final concentration of 300 nM (about three times the IC 50 concentration) Was administered. After 4 hours from the administration, the medium was replaced with a new one, the drug was removed, and the cells were placed in the described live observation apparatus for observation. As a result, you can observe the formation of micronuclei, follow the subsequent state of cells that have micronuclei, what can be made in the second and subsequent divisions I was able to.
図６には、ＭＭＣで処理した細胞の系譜図を示す。図中丸で囲んだ数字は図５と同じく分裂の回数である。同じ世代の分裂には丸で囲んだ数字の後にハイフンをつけアルファベットで区別した。また、細胞を示す丸の左肩に小さな丸がついているものは、小核が発生した事を表す。図５のＭＭＣにさらされていない細胞の場合と比較すると、分裂間時間が延びているのがわかる。また、丸５で示す細胞は、初代から４代目で初めて小核が発生しており、初代の細胞が蒙った影響が、２代目、３代目で発現しなくても、４代目以降で発現することが観察された。 FIG. 6 shows a genealogy diagram of cells treated with MMC. The numbers in circles in the figure are the number of divisions as in FIG. The division of the same generation is distinguished by an alphabet with a hyphen after the circled number. A cell with a small circle on the left shoulder of a cell indicates that a micronucleus has occurred. Compared to the case of the cells not exposed to the MMC in FIG. In addition, the cells indicated by circle 5 have micronuclei for the first time from the first generation to the fourth generation, and the effects of the first generation cells are expressed after the fourth generation even if they are not expressed by the second generation and the third generation. It was observed.
一度小核ができた細胞は、さまざまな変化を起こすが、例えば図７に示すようにある親からの子が２つとも小核を発生し、その後分裂しきれないまま繋がってしまうといったケース（図７（ｂ）参照）であったり、分裂した細胞の片方にだけ小核ができて、小核ができた細胞はその後死んでしまったり（図７（ｃ）参照）といったケースがある。もちろん、図６に示したように、小核を発生したまま分裂を続ける細胞もあった。なお、図７（ａ）は、細胞Ｃ２ｂが２つに分裂し（Ｃ３ｃおよびＣ３ｄ）、その後小核部分同士で結合する（Ｃ４ｃ、Ｃ４ｄ）ところを撮影した連続写真である。左上から右、左下から右下と時間は過ぎてゆく。 Once a cell has formed a micronucleus, it undergoes various changes. For example, as shown in Fig. 7, two children from a parent generate both micronuclei and then connect without being able to divide ( 7 (see FIG. 7 (b)), or micronuclei are formed only in one of the divided cells, and the cells in which the micronuclei are formed then die (see FIG. 7 (c)). Of course, as shown in FIG. 6, some cells continued to divide while generating micronuclei. FIG. 7 (a) is a series of photographs taken of the cell C2b dividing into two (C3c and C3d) and then joining at the micronucleus parts (C4c, C4d). Time passes from upper left to right and from lower left to lower right.
このように、本発明で用いた細胞を薬剤に曝し、特定の細胞についてその系譜図を薬剤に曝さなかった細胞と比較することで、薬剤の細胞に対する世代を通じた影響をアッセイすることができる。特に、薬剤の濃度が薄い場合において、どの程度の影響があるのかは、この系譜図を比較することで、これまでにないアッセイが可能となる。 Thus, by exposing the cells used in the present invention to a drug and comparing the lineage diagram of a particular cell with cells that have not been exposed to the drug, the effects of the drug on the cells over generations can be assayed. In particular, when the concentration of the drug is low, it is possible to perform an unprecedented assay by comparing this genealogy diagram to the extent of the effect.
本発明は、培養細胞を用いた薬剤の細胞に対する影響について、詳細な確認が可能である。 In the present invention, it is possible to confirm in detail the influence of a drug using cultured cells on cells.
Claims (2)
前記蛍光タンパク質が導入された細胞を被検査物に曝す工程と、
前記被検査物に曝された細胞をライブ観察し、少なくとも分裂時間を測定する工程を有する細胞毒性検査方法。 Introducing a fluorescent protein into a cell whose number of chromosomes does not change when it divides;
Exposing the cells into which the fluorescent protein has been introduced to a test object;
A cytotoxicity testing method comprising a step of observing a cell exposed to the test object live and measuring at least a division time.
前記蛍光タンパク質が導入された細胞を被検査物に曝す工程と、
前記被検査物に曝された細胞をライブ観察し、前記細胞が少なくとも２回以上分裂する間の分裂時間および分裂間時間を測定する工程を有する細胞毒性検査方法。
Introducing a fluorescent protein into a cell whose number of chromosomes does not change when it divides;
Exposing the cells into which the fluorescent protein has been introduced to a test object;
A cytotoxicity testing method comprising a step of observing a cell exposed to the test object live and measuring a division time and a division time during which the cell divides at least twice.
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