JP2003075437A - Detection method of fragmentation of dna by cell apoptosis derivation due to near-infrared spectrochemical analysis method - Google Patents
Detection method of fragmentation of dna by cell apoptosis derivation due to near-infrared spectrochemical analysis methodInfo
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】この発明は、近赤外分光分析
法による細胞アポトーシス誘導によるＤＮＡの断片化の
検出方法に関する。TECHNICAL FIELD The present invention relates to a method for detecting DNA fragmentation due to cell apoptosis induction by near infrared spectroscopy.
【０００２】[0002]
【従来の技術】可視域に隣接する近赤外領域の光を用い
て物質の定量、定性分析を行う近赤外分光分析法は、近
年、農業分野をはじめ医療分野等の様々な分野で利用さ
れている（岩元睦夫、河野澄夫、魚住純「近赤外分光法
入門」幸書房、１９９４年９月１０日初版発行）。近赤
外分光分析法は、
１）エネルギーの低い電磁波を用いるので試料を損傷す
ることがない。
２）固体、粉体、繊維、液体、気体など様々な状態の試
料に適用することができる。
３）赤外にくらべ近赤外では水の吸収強度が弱くなるの
で、水溶液あるいは含水系での分析ができる。
４）紫外光や可視光にくらべ近赤外光は、厚い組織をも
透過することができる。などの利点を有する。2. Description of the Related Art Near-infrared spectroscopy, which performs quantitative and qualitative analysis of substances using light in the near-infrared region adjacent to the visible region, has recently been used in various fields such as agriculture and medical fields. (Mutio Iwamoto, Sumio Kono, Jun Uozumi "Introduction to Near Infrared Spectroscopy" Koshobo, published on September 10, 1994 first edition). The near-infrared spectroscopic method 1) does not damage the sample because it uses electromagnetic waves with low energy. 2) It can be applied to samples in various states such as solid, powder, fiber, liquid and gas. 3) The absorption intensity of water in the near infrared is weaker than that in the infrared, so that analysis in an aqueous solution or a water-containing system can be performed. 4) Compared to ultraviolet light and visible light, near-infrared light can also penetrate thick tissues. It has advantages such as
【０００３】近赤外分光分析はその定量、定性分析を行
うためにいわゆるケモメトリクスと呼ばれる手法を用い
る。これは多変量解析手法や統計解析手法を用いて化学
分析を行う手法で、近年、パーソナルコンピュータの発
達とともに発展してきた。最近の近赤外分光分析では回
帰分析あるいはＰＬＳ回帰分析といった多変量解析手法
を用いて行われている。また、ニューラルネットワーク
やカオス理論等の解析への応用も試みられている。The near-infrared spectroscopic analysis uses a so-called chemometrics technique to perform its quantitative and qualitative analysis. This is a method of performing chemical analysis using a multivariate analysis method or a statistical analysis method, and has been developed in recent years with the development of personal computers. In recent near infrared spectroscopy, multivariate analysis methods such as regression analysis or PLS regression analysis are used. In addition, application to analysis of neural networks and chaos theory has also been attempted.
【０００４】定量分析を行う場合、たとえば、ＰＬＳ(P
anial Least Square）回帰分析と呼ばれる手法を用い
る。前記回帰分析は、予め本従来例の分析装置を用いて
既知濃度の試料の吸収スペクトルを測定する実験より検
量線を作成することにより行われる。前記検量線は、濃
度既知の複数試科から測定した吸光スペクトルを説明変
量とし、試料中成分の濃度を目的変数として回帰分析す
ることにより求める。When performing a quantitative analysis, for example, PLS (P
anial Least Square) A method called regression analysis is used. The regression analysis is performed by preparing a calibration curve in advance by an experiment in which the absorption spectrum of a sample having a known concentration is measured using the analyzer of the conventional example. The calibration curve is obtained by performing a regression analysis using the absorption spectra measured from a plurality of trial departments of known concentrations as explanatory variables and the concentrations of the components in the sample as objective variables.
【０００５】上記のような優れた特性を有する近赤外分
光分析法は、臨床医学の分野で注目を集め、細胞の分
析、検出やモニタリング等に適用されている。The near-infrared spectroscopic method having the above-mentioned excellent characteristics has been attracting attention in the field of clinical medicine and is applied to cell analysis, detection, monitoring and the like.
【０００６】[0006]
【発明が解決しようとする課題】本願発明の目的は、近
赤外分光分析法（ＮＩＲ法）を用いてＤＮＡの挙動解析
を行った例がないことに着目して、形態学的に変化した
細胞や病的な状態であるアポトーシス細胞の検出、特に
近赤外分光分析法による細胞アポトーシス誘導によるＤ
ＮＡの断片化の検出方法を提供することである。The object of the present invention has been changed morphologically, focusing on the fact that there is no example of behavior analysis of DNA using near infrared spectroscopy (NIR method). Detection of cells or apoptotic cells in a pathological state, especially by induction of cell apoptosis by near infrared spectroscopy
It is to provide a method for detecting NA fragmentation.
【０００７】[0007]
【課題を解決するための手段】本願発明は、ＤＮＡを含
む試料に８００〜２５００ｎｍ波長の近赤外光を照射す
る工程、前記試料からの反射光又は透過光を受光して近
赤外光領域に含まれる波長に対する強度をセンサよって
検出し、吸光度スぺクトルを計測する工程、前記吸光度
スぺクトルのうち１乃至複数の特定波長の吸光度スぺク
トルを解析して前記試料中の細胞アポトーシス誘導によ
るＤＮＡの断片化を検出する工程、とからなることを特
徴とする近赤外分光分析法による細胞アポトーシス誘導
によるＤＮＡの断片化の検出方法である。前記特定波長
は、１４０５〜１４２５、１５１０〜１５３５、１５４
５〜１５７０、１６００〜１６２５、１８４５〜１８６
０、１８６５〜１８９０ｎｍである。According to the present invention, a step of irradiating a sample containing DNA with near infrared light having a wavelength of 800 to 2500 nm, a reflected light or a transmitted light from the sample is received, and a near infrared light region is received. Detecting the intensity for the wavelength contained in the sample with a sensor to measure the absorbance spectrum, and inducing cell apoptosis in the sample by analyzing the absorbance spectrum of one or more specific wavelengths of the absorbance spectrum And a step of detecting the fragmentation of the DNA by the method, and the method for detecting the fragmentation of the DNA by inducing cell apoptosis by the near-infrared spectroscopy. The specific wavelengths are 1405 to 1425, 1510 to 1535, 154.
5-1570, 1600-1625, 1845-186
0, 1865 to 1890 nm.
【０００８】[0008]
【発明の実施の形態】図１は、本発明において使用する
近赤外分光分析装置の模式図を示すもので、１１００〜
２５００ｎｍの波長の近赤外光を光源１１からの光を順
次フィルタ１２を通して、特定波長のみを通すように制
御装置１５によりフィルタを切り替えて、２ｎｍの分解
能で分光する。この分光された光を試料台１３中の測定
すべき試料に照射する。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a schematic view of a near-infrared spectroscopic analyzer used in the present invention.
The near-infrared light having a wavelength of 2500 nm is sequentially passed through the filter 12 by the light from the light source 11, and the control device 15 switches the filters so that only specific wavelengths are allowed to pass therethrough, and the light is dispersed at a resolution of 2 nm. The sample to be measured in the sample table 13 is irradiated with this dispersed light.
【０００９】試料から反射または透過してくる反射光又
は透過光をセンサ１４（透過光センサ１４ａ、反射光セ
ンサ１４ｂからなる。）によって検出し、波長毎に吸光
度スぺクトルを計測する。The sensor 14 (comprising a transmitted light sensor 14a and a reflected light sensor 14b) detects reflected light or transmitted light reflected or transmitted from the sample, and measures the absorbance spectrum for each wavelength.
【００１０】試料の近赤外域における吸収バンドを調べ
る。近赤外分光装置は、ＢＲＡＮ＋ＬＵＥＢＢＲ社製Ｉ
ｎｆｒａＡｌｙｚｅｒ５００を用い、近赤外吸収スぺク
トルの測定波長範囲は１１００−２５００ｎｍ、測定温
度２５℃とし、この範囲を２ｎｍ間隔で走査測定する。The absorption band of the sample in the near infrared region is examined. The near-infrared spectroscopic device is I manufactured by BRAN + LUEBBR
Using nfraAlyzer 500, the measurement wavelength range of the near infrared absorption spectrum is 1100-2500 nm, the measurement temperature is 25 ° C., and this range is scanned and measured at 2 nm intervals.
【００１１】以下に、細胞アポトーシスの検出方法の詳
細を説明する。The details of the method for detecting cell apoptosis will be described below.
【００１２】先ず、細胞アポトーシスの誘導は、ＦＢＳ
（Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ ウシ胎児血
清）を１０％、ペニシリンを１％添加したＲＰＭＩ Ｍ
ｅｄｉｕｍを用いて、ヒト乳ガン由来乳腺上皮細胞（Ｍ
ＣＦ−７）１×１０6個を３７℃の温度条件下で、５％
ＣＯ2インキュベーター内で培養する。次いで、スタウ
ロスポリン（非特異的リン酸化阻害剤）の濃度が１μＭ
となるように処理し、６時間培養して細胞のアポトーシ
スを起こさせる。未処理のものをコントロールとした。[0012] First, the induction of cell apoptosis is performed by using FBS.
RPMI M with 10% (Fetal Bovine Serum fetal bovine serum) and 1% penicillin added
human breast cancer-derived mammary epithelial cells (M
5% of CF-7) 1 × 10 6 under the temperature condition of 37 ° C.
Culture in a CO 2 incubator. Next, the concentration of staurosporine (non-specific phosphorylation inhibitor) was 1 μM
And the cells are cultured for 6 hours to induce cell apoptosis. The untreated one was used as a control.
【００１３】細胞のＤＮＡの抽出には、和光純薬製「Ｗ
ＡＫＯ ＤＮＡ Ｅｘｔｒａｃｔｏｒ ＷＢ Ｋｉｔ」
（ヨウ化ナトリウム法）を使用した。For extracting DNA of cells, "W" manufactured by Wako Pure Chemical Industries, Ltd.
AKO DNA Extractor WB Kit "
(Sodium iodide method) was used.
【００１４】先ず、細胞をＰＢＳ（Ｐｈｏｓｐｈａｔｅ
Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎｅリン酸緩衝液）で洗
浄後、マイクロチューブに回収する。マイクロチューブ
によって回収した細胞を３０００Ｇ、５分間遠心し、上
清を除去する。First, the cells are put into PBS (Phosphate).
After washing with Buffered Saline phosphate buffer), it is collected in a microtube. The cells collected by the microtube are centrifuged at 3000 G for 5 minutes, and the supernatant is removed.
【００１５】分離された細胞に溶解液１ｍｌを加えて、
ＶＯＲＴＥＸによって攪拌する。更に、１００００Ｇ、
１分間遠心し、上清を除去する。この操作を再度繰り返
す。1 ml of lysate was added to the separated cells,
Stir with VORTEX. Furthermore, 10000G,
Centrifuge for 1 minute and remove the supernatant. Repeat this operation again.
【００１６】上記操作で得られたものを、酵素反応液２
００μｌとタンパク分解酵素１０μｌを添加し、混合
し、３７℃の温度で１時間保温し、ヨウ化ナトリウム溶
液を３００μｌ加えて混合し、イソプロピルアルコール
を０．５μｌ加えて混合し、ＤＮＡを沈殿させる。The product obtained by the above operation is used as the enzyme reaction solution 2
00 μl and 10 μl of proteolytic enzyme are added, mixed and kept at 37 ° C. for 1 hour, 300 μl of sodium iodide solution is added and mixed, and 0.5 μl of isopropyl alcohol is added and mixed to precipitate DNA.
【００１７】上記操作で得られたＤＮＡを含む液を１０
０００Ｇ、１０分間遠心し、上清を除去し、洗浄液を１
ｍｌ加えて混合し、さらに１００００Ｇ、５分間遠心
し、上清を除去し、洗浄液を１ｍｌ加えて混合した後、
更に、１００００Ｇ、５分間遠心し、上清を除去し、乾
燥させて、ＤＮＡを５μｇ抽出する。A solution containing the DNA obtained by the above operation was added to 10
Centrifuge at 000G for 10 minutes, remove the supernatant, and wash with 1
After adding 1 ml and mixing, further centrifugation at 10,000 G for 5 minutes, removing the supernatant, and adding 1 ml of the washing solution and mixing,
Further, the mixture is centrifuged at 10000 G for 5 minutes, the supernatant is removed, and dried to extract 5 μg of DNA.
【００１８】抽出されたＤＮＡの濃度の調整は、分光光
度計（ＢＥＣＫＭＡＮ社製 ＤＵ６００）を用いて測定
し、ＤＮＡ濃度をそれぞれ１．０μｇ／ｍｌ、０．１μ
ｇ／ｍｌ、０．０１μｇ／ｍｌに調整し、それぞれの試
料について、検出を行った。本発明の近赤外分光法によ
れば、０．０１μｇ／ｍｌ低濃度の濃度でも検出でき
た。The concentration of the extracted DNA was adjusted by using a spectrophotometer (DU600 manufactured by BECKMAN), and the DNA concentrations were 1.0 μg / ml and 0.1 μm, respectively.
It was adjusted to g / ml and 0.01 μg / ml, and detection was performed for each sample. According to the near-infrared spectroscopy of the present invention, detection was possible even at a low concentration of 0.01 μg / ml.
【００１９】上記した細胞アポトーシスが誘導された試
料と、未処理の細胞であるコントロールとを赤外分光装
置にかけて、角波長毎の吸収スぺクトルを収集し、１０
回ずつ測定した。また、両試料とも、ＤＮＡ電気泳動法
によって、ＤＮＡの断片化を確認した。The sample in which the above-mentioned cell apoptosis was induced and a control which was an untreated cell were applied to an infrared spectroscope to collect absorption spectra for each angular wavelength.
Measured each time. In both samples, DNA fragmentation was confirmed by DNA electrophoresis.
【００２０】図２は、２次微分スぺクトルの主成分分析
（ＰＣＡ）の線図を示している。FIG. 2 shows a principal component analysis (PCA) diagram of the second derivative spectrum.
【００２１】図３、図４は、その２次微分スぺクトルを
示す線図である。図３の下段の図３（ｂ）は、波長範囲
１４００ｎｍ〜１４３０ｎｍの部分拡大図であって、特
に波長１４１０ｎｍの領域では、アポトーシスを起こし
たＤＮＡとコントロールＤＮＡとの判別が有効であるこ
とを示している。図４の下段の図４（ｂ）は、波長範囲
１８７０ｎｍ〜１８９０ｎｍの部分拡大図であって、特
に波長１８８２ｎｍの領域では、アポトーシスを起こし
たＤＮＡとコントロールＤＮＡとの判別が有効であるこ
とを示している。FIG. 3 and FIG. 4 are diagrams showing the second derivative spectrum. FIG. 3B in the lower part of FIG. 3 is a partially enlarged view in the wavelength range of 1400 nm to 1430 nm, and shows that the discrimination between apoptotic DNA and control DNA is effective particularly in the wavelength range of 1410 nm. ing. FIG. 4B in the lower part of FIG. 4 is a partially enlarged view of the wavelength range 1870 nm to 1890 nm, and shows that the discrimination between apoptotic DNA and control DNA is effective particularly in the wavelength 1882 nm region. ing.
【００２２】収集した吸光度スぺクトル２次微分変換し
たものについて、図２に示されているように、２次微分
による主成分分析をおこなった。いずれも、第１主成分
（Ｆａｃｔｏｒ１）、第２主成分（Ｆａｃｔｏｒ２）に
よってアポトーシスを起こしたＤＮＡを検出することが
できた。As shown in FIG. 2, principal component analysis based on the second derivative was performed on the collected second derivative of the absorbance spectrum. In both cases, it was possible to detect DNA that had undergone apoptosis by the first main component (Factor 1) and the second main component (Factor 2).
【００２３】２次微分変換したスぺクトルは、第１主成
分によって、検出することができた。第１主成分は、図
２（ｂ）の相関スぺクトル線図から相関のある特徴的な
波長領域、１４０５〜１４２５、１５１０〜１５３５、
１５４５〜１５７０、１６００〜１６２５、１８４５〜
１８６０、１８６５〜１８９０ｎｍを抽出することがで
きた。これらの波長を重回帰分析を行うことによって、
アポトーシスを起こしたＤＮＡとコントロールＤＮＡと
を明確に検出することができる。The second-differential-transformed spectrum could be detected by the first principal component. The first principal component is a characteristic wavelength region having correlation from the correlation spectrum diagram of FIG. 2B, 1405 to 1425, 1510 to 1535,
1545 ~ 1570, 1600 ~ 1625, 1845 ~
It was possible to extract 1860, 1865-1890 nm. By performing multiple regression analysis on these wavelengths,
Apoptotic DNA and control DNA can be clearly detected.
【００２４】図８は、２次微分スぺクトルに基づいてク
ラスタ分析をした樹状図である。細胞アポトーシスを起
こしたＤＮＡサンプルＡ．１〜Ａ．１０と、コントロー
ルＤＮＡサンプルＣ．１〜Ｃ．１０は、類似値０．４に
着目すると、前者と後者とが二つのグループに明確に分
類されていることが分り、細胞アポトーシスを起こした
ＤＮＡサンプルを検出できることが分る。FIG. 8 is a dendrogram obtained by cluster analysis based on the second derivative spectrum. DNA sample with cell apoptosis 1-A. 10 and the control DNA sample C.I. 1-C. Regarding No. 10, focusing on the similarity value of 0.4, it is found that the former and the latter are clearly classified into two groups, and it is found that a DNA sample in which cell apoptosis has occurred can be detected.
【００２５】[0025]
【発明の効果】本発明によれば、 （１）抗ガン剤による細胞アポトーシスの測定 （２）環境汚染によるＤＮＡ損傷の測定 （３）遺伝子組み換え作物の検出 が可能となるという効果を有する。According to the present invention, (1) Measurement of cell apoptosis by anti-cancer drug (2) Measurement of DNA damage due to environmental pollution (3) Detection of genetically modified crops Has the effect that it becomes possible.
【図１】本発明の、近赤外分光分析装置の模式図。FIG. 1 is a schematic diagram of a near-infrared spectroscopic analyzer according to the present invention.
【図２】近赤外分光分析法により得られた２次微分スぺ
クトルの主成分分析の線図。FIG. 2 is a diagram of principal component analysis of a second derivative spectrum obtained by near infrared spectroscopy.
【図３】近赤外分光分析法により得られた細胞アポとシ
ース誘導したＤＮＡとコントロールＤＮＡののそれぞれ
の吸光度スぺクトルの２次微分スぺクトルと、その一部
を拡大した線図。FIG. 3 is a second-order differential spectrum of the respective absorbance spectra of the cell apo, the sheath-induced DNA, and the control DNA obtained by near-infrared spectroscopy, and an enlarged diagram of a part thereof.
【図４】近赤外分光分析法により得られた細胞アポとシ
ース誘導したＤＮＡとコントロールＤＮＡののそれぞれ
の吸光度スぺクトルの２次微分スぺクトルと、その一部
を拡大した線図。FIG. 4 is a second-order differential spectrum of the respective absorbance spectra of the cell apo, the sheath-induced DNA, and the control DNA obtained by the near-infrared spectroscopy, and an enlarged diagram of a part thereof.
【図５】近赤外分光分析法により得られた細胞アポとシ
ース誘導したＤＮＡとコントロールＤＮＡののそれぞれ
の吸光度スぺクトルの２次微分スぺクトルをクラスタ分
析から得られた樹状図。FIG. 5 is a dendrogram obtained by cluster analysis of the second-order differential spectra of the absorbance spectra of the cell apo, the sheath-induced DNA, and the control DNA obtained by near-infrared spectroscopy.
１１ 光源、１２ フィルタ、１３ 試料台、１４ セ
ンサ、１５ 制御装置11 light source, 12 filter, 13 sample stage, 14 sensor, 15 control device
【手続補正書】[Procedure amendment]
【提出日】平成１３年９月４日（２００１．９．４）[Submission date] September 4, 2001 (2001.9.4)
【手続補正１】[Procedure Amendment 1]
【補正対象書類名】図面[Document name to be corrected] Drawing
【補正対象項目名】全図[Correction target item name] All drawings
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【図１】 [Figure 1]
【図２】 [Fig. 2]
【図３】 [Figure 3]
【図４】 [Figure 4]
【図５】 [Figure 5]
───────────────────────────────────────────────────── フロントページの続き Ｆターム(参考） 2G045 DA12 DA13 FA11 FA25 2G059 AA05 BB12 DD16 EE01 EE02 EE12 FF12 GG10 HH01 HH06 JJ01 JJ03 MM01 MM02 PP04 4B063 QA01 QQ42 QX10 ─────────────────────────────────────────────────── ─── Continued front page F-term (reference) 2G045 DA12 DA13 FA11 FA25 2G059 AA05 BB12 DD16 EE01 EE02 EE12 FF12 GG10 HH01 HH06 JJ01 JJ03 MM01 MM02 PP04 4B063 QA01 QQ42 QX10
Claims (2)
ｎｍ波長の近赤外光を照射する工程、 前記試料からの反射光又は透過光を受光して近赤外光領
域に含まれる波長に対する強度をセンサよって検出し、
吸光度スぺクトルを計測する工程、 前記吸光度スぺクトルのうち１乃至複数の特定波長の吸
光度スぺクトルを解析して前記試料中の細胞アポトーシ
ス誘導によるＤＮＡの断片化を検出する工程、 とからなることを特徴とする近赤外分光分析法による細
胞アポトーシス誘導によるＤＮＡの断片化の検出方法。1. A sample comprising DNA is 800 to 2500.
irradiating near-infrared light of nm wavelength, the reflected light or transmitted light from the sample is received and the intensity for the wavelength contained in the near-infrared light region is detected by a sensor,
Measuring the absorbance spectrum; analyzing the absorbance spectrum of one or more specific wavelengths of the absorbance spectrum to detect the fragmentation of DNA in the sample due to the induction of cell apoptosis. A method for detecting fragmentation of DNA by inducing cell apoptosis by near-infrared spectroscopy.
１５１０〜１５３５、１５４５〜１５７０、１６００〜
１６２５、１８４５〜１８６０、１８６５〜１８９０ｎ
ｍであることを特徴とする請求項１の近赤外分光分析法
による細胞アポトーシス誘導によるＤＮＡの断片化の検
出方法。2. The specific wavelength is 1405 to 1425,
1510-1535, 1545-1570, 1600
1625, 1845-1860, 1865-1890n
The method for detecting DNA fragmentation by inducing cell apoptosis by near infrared spectroscopy according to claim 1, wherein m is m.
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| JP (1) | JP2003075437A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1313624C (en) * | 2004-06-15 | 2007-05-02 | 中国科学院大连化学物理研究所 | Detecting miniflow controlled chip from single-cell wilting DNA fragmentation |
| CN100425976C (en) * | 2005-03-21 | 2008-10-15 | 江苏省人民医院 | Method for quick-speed in-situ authentication of peripheral nerve tracts |
| CN111471589A (en) * | 2020-04-23 | 2020-07-31 | 华中科技大学 | Infrared quantitative PCR nucleic acid detection device |
-
2001
- 2001-09-04 JP JP2001266741A patent/JP2003075437A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1313624C (en) * | 2004-06-15 | 2007-05-02 | 中国科学院大连化学物理研究所 | Detecting miniflow controlled chip from single-cell wilting DNA fragmentation |
| CN100425976C (en) * | 2005-03-21 | 2008-10-15 | 江苏省人民医院 | Method for quick-speed in-situ authentication of peripheral nerve tracts |
| CN111471589A (en) * | 2020-04-23 | 2020-07-31 | 华中科技大学 | Infrared quantitative PCR nucleic acid detection device |
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