JPH09206096A - Assessment of differentiation and maturity of t-cell-based oncocyte - Google Patents
Assessment of differentiation and maturity of t-cell-based oncocyteInfo
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- JPH09206096A JPH09206096A JP8039124A JP3912496A JPH09206096A JP H09206096 A JPH09206096 A JP H09206096A JP 8039124 A JP8039124 A JP 8039124A JP 3912496 A JP3912496 A JP 3912496A JP H09206096 A JPH09206096 A JP H09206096A
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- Prior art keywords
- agglutination
- abrin
- cells
- cell
- maturity
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、生きているＴ細胞
を用いてその分化、成熟度を判定する方法に関し、例え
ば白血病やガンの進行状態を推測するデータを得るため
に有用なものである。The present invention relates to a method for judging differentiation and maturity using living T cells, and is useful, for example, for obtaining data for estimating the progress of leukemia or cancer. .
【０００２】[0002]
【従来の技術】アブリンはトウアズキ（Ａｂｒｕｓ ｐ
ｒｅｃａｔｉｏｒｉｕｓ）の種子中に含まれる分子量約
６３，０００のレクチンで、セファロース４Ｂに対する
親和性の相違によりアブリン‐ａとアブリン‐ｂに分け
られている。これらのアブリンは、Ａ鎖及びＢ鎖から成
り、Ｂ鎖で糖類と結合したのち、Ａ鎖が細胞内に取り込
まれタンパク合成を阻害するため、強い毒性を示す。2. Description of the Related Art Abrin is a kind of azuki bean (Abrus p.
lectin having a molecular weight of about 63,000 contained in the seeds of S. recatiorius), and is divided into abrin-a and abrin-b by differences in affinity for Sepharose 4B. These abrins are composed of an A chain and a B chain, and after binding to a saccharide with the B chain, the A chain is taken into cells and inhibits protein synthesis, so that it exhibits strong toxicity.
【０００３】このものは、細胞凝集活性を有することか
ら、これを利用して種々の生理作用の評価を行うことが
できることが知られている。従来、このような凝集活性
を測定する方法としては、単にこの溶液を所定の細胞に
混合し、凝集の有無を観察する手法がとられてきたが、
これは一定濃度ごとに凝集反応を判断するため、凝集の
強度や速度の解析が不可能であり、利用範囲が制限され
るのを免れなかった。[0003] It is known that since it has a cell aggregation activity, it can be used to evaluate various physiological actions. Conventionally, as a method for measuring such agglutinating activity, a method of simply mixing this solution with predetermined cells and observing the presence or absence of agglutination has been adopted.
In this method, since the agglutination reaction is determined for each constant concentration, it is impossible to analyze the intensity and speed of the agglutination, and the use range was inevitably limited.
【０００４】[0004]
【発明が解決しようとする課題】本発明の課題は、アブ
リンの細胞凝集反応を分光的手法により測定し、その結
果を数値的に解析して、簡単に凝集速度及び凝集強度等
の凝集パラメータを求め、かつそれを利用して、Ｔ細胞
腫瘍の分化、成熟度を判定する方法を提供することであ
る。SUMMARY OF THE INVENTION An object of the present invention is to measure the agglutination reaction of abrin by a spectroscopic method, numerically analyze the results, and easily determine the agglutination parameters such as agglutination rate and agglutination strength. It is an object of the present invention to provide a method for determining and utilizing the T cell tumor differentiation and maturity.
【０００５】[0005]
【課題を解決するための手段】本発明者らは、細胞の懸
濁液にアブリンを添加すると、時間が経過するに従っ
て、細胞の凝集反応が進行し、凝集生成物が沈殿する結
果、懸濁液の濁度が次第に減少する傾向を示すが、この
状態をグラフとして表わした凝集曲線から求められる凝
集速度及び凝集強度は、細胞数及びアブリン濃度の対数
に依存して直線的に増加すること、及びこれらの数値は
Ｔ細胞の分化、成熟の程度が進むとともに高くなること
を見出し、この知見に基づいて本発明をなすに至った。Means for Solving the Problems The present inventors have found that when abrin is added to a suspension of cells, as the time elapses, the agglutination reaction of the cells progresses, and as a result, the agglutination product precipitates. Although the turbidity of the liquid tends to gradually decrease, the aggregation rate and the aggregation strength obtained from the aggregation curve representing this state as a graph are linearly increased depending on the logarithm of the cell number and the abrin concentration, The inventors have found that these values increase as the degree of differentiation and maturation of T cells increases, and based on this finding, the present invention has been accomplished.
【０００６】すなわち、本発明によれば、Ｔ細胞系腫瘍
細胞懸濁液にアブリンを添加し、経時的に濁度の減少度
を測定し、それに基づいて作成した凝集曲線から凝集速
度及び凝集強度を求め、その数値からＴ細胞腫瘍の分
化、成熟度を判定することができる。That is, according to the present invention, abrin is added to a T cell tumor cell suspension, the degree of turbidity decrease is measured over time, and the aggregation rate and the aggregation strength are determined from an aggregation curve prepared based on the turbidity. , And the differentiation and maturity of the T cell tumor can be determined from the numerical values.
【０００７】[0007]
【発明の実施の形態】本発明方法により、分化、成熟度
を判定しうるＴ細胞としては、ヒト由来のもの例えばジ
ャーカット（Ｊｕｒｋａｔ）株、ハット（Ｈｕｔ）‐１
０２株、モルト（Ｍｏｌｔ）４株、ケー・オー・ピー・
テー（ＫＯＰＴ）４株、アール・ピー・エム・アイ（Ｒ
ＰＭＩ）８４０２株のようなヒト急性白血病及び菌状息
肉症由来のものやその他のガン由来のものを用いること
ができる。そして、このような各種病因細胞由来のＴ細
胞の分化、成熟度を判定することにより、間接的に病状
の進行度を判断することができる。BEST MODE FOR CARRYING OUT THE INVENTION The T cells whose differentiation and maturity can be determined by the method of the present invention include those derived from humans, such as the Jurkat strain and the Hut-1.
02 shares, 4 Molt shares, KOP
KOP 4 shares, RPM I (R
Those derived from human acute leukemia and mycosis fungoides such as PMI) 8402 strain and those derived from other cancers can be used. Then, the degree of progression of the disease state can be indirectly determined by determining the degree of differentiation and maturation of the T cells derived from such various pathogenic cells.
【０００８】次に本発明方法において用いられるアブリ
ンは、アブリン‐ａとアブリン‐ｂのいずれでもよい
が、前者の方が後者よりも高い凝集能力を示す。Next, the abrin used in the method of the present invention may be either abrin-a or abrin-b, but the former has a higher aggregation ability than the latter.
【０００９】本発明方法は、例えば適当な条件下で培養
した各株Ｔ細胞の対数増殖期にあるものを、生理的塩類
溶液でリンスし、この中に所定のアブリン溶液を加え、
３７℃に維持して、波長６００ｎｍの吸光度として経時
的な濁度変化を測定する。得られた結果から図１に示す
ような凝集曲線が描かれる。In the method of the present invention, for example, the T cells in the logarithmic growth phase of each strain cultured under appropriate conditions are rinsed with a physiological salt solution, and a predetermined abrin solution is added thereto.
While maintaining the temperature at 37 ° C., the change in turbidity over time is measured as the absorbance at a wavelength of 600 nm. An aggregation curve as shown in FIG. 1 is drawn from the obtained results.
【００１０】この凝集曲線に対する接線の傾きとして、
凝集速度が、また凝集開始時の吸光度と、凝集終了後の
吸光度の差として、凝集強度がそれぞれ得られる。この
ようにして得られる凝集速度及び凝集強度の、アブリン
濃度の対数値に対する関係の１例をグラフに示すと図２
のようになり、また細胞数に対する関係をグラフに示す
と図３のようになる。The slope of the tangent to this aggregation curve is:
The aggregation strength is obtained as the aggregation rate and the difference between the absorbance at the start of aggregation and the absorbance at the end of aggregation. FIG. 2 is a graph showing an example of the relationship between the aggregation rate and the aggregation strength obtained as described above and the logarithmic value of the abrin concentration.
FIG. 3 shows the relationship with the number of cells in a graph.
【００１１】図２から分るように、凝集強度、凝集速度
ともに、アブリン濃度の対数に依存して直線的に増加す
るがアブリン濃度が０．８μＭ以上になると増加が認め
られなくなるため、アブリン濃度としては０．４μＭ以
下にするのが好ましい。As can be seen from FIG. 2, both the cohesive strength and the coagulation rate increase linearly depending on the logarithm of the abrin concentration, but when the abrin concentration exceeds 0.8 μM, the increase is not observed. Is preferably set to 0.4 μM or less.
【００１２】また、図３から分るように凝集速度につい
ては、細胞数が４×１０6個／ｍｌ以上になると相関性
が低下するので細胞数としては３×１０6個／ｍｌ以下
の範囲とするのが好ましい。As can be seen from FIG. 3, the agglutination rate decreases when the number of cells is 4 × 10 6 cells / ml or more, so that the number of cells is in the range of 3 × 10 6 cells / ml or less. It is preferred that
【００１３】このようにして測定された凝集速度及び凝
集強度は、Ｔ細胞系腫瘍の成熟した株であるジャーカッ
ト（Ｊｕｒｋａｔ）で大きく、幼若株のピー・アール・
エム・アイ（ＰＲＭＩ）８４０２ではほとんど０にな
る。また、両者の中間の成熟度をもつ株であるモルト
（Ｍｏｌｔ）４及びハット（Ｈｕｔ）１０２の場合は、
凝集速度と凝集強度もその中間の値になる。The agglutination rate and agglutination strength measured in this manner are large in Jurkat, a mature T cell tumor, and the young strain P.R.
It is almost 0 in the MI (PRMI) 8402. In the case of Malt 4 and Hut 102, which are strains having an intermediate maturity between the two,
The agglomeration rate and the agglomeration strength also have intermediate values.
【００１４】[0014]
【実施例】次に実施例により本発明をさらに詳細に説明
する。EXAMPLES The present invention will be described in more detail with reference to examples.
【００１５】なお、各例で用いたアブリン‐ａ及びアブ
リン‐ｂは、「ネイチュア（Ｎａｔｕｒｅ）」，第７
巻，第２９２ページ（１９７０年）記載のリン（Ｌｉ
ｎ）の方法に従い、タイ国産トウアズキ種子から調製し
た。また、Ｔ細胞の各株は、牛胎児血清１０重量％、グ
ルタミン１重量％、ストレプトマイシン５ｍｇ／リット
ル、ペニシリン５０，０００ＩＵを含むＰＲＭＩ‐１６
４０培地中、５％ＣＯ2雰囲気中で培養したものであ
る。The abrin-a and abrin-b used in each example are described in "Nature", No. 7
Vol., P. 292 (1970).
According to the method of n), it was prepared from the seeds of the spruce toads produced in Thailand. In addition, each T cell line is a PRMI-16 containing 10% by weight of fetal bovine serum, 1% by weight of glutamine, 5 mg / liter of streptomycin, and 50,000 IU of penicillin.
Cultured in a 40 medium and 5% CO 2 atmosphere.
【００１６】実施例１ 対数増殖期にあるジャーカット（Ｊｕｒｋａｔ）株Ｔ細
胞を生理的塩類溶液ＰＢＳ（−）で３回リンスし、細胞
数を３×１０6個／ｍｌに調整した。次にこのＴ細胞を
含む細胞懸濁液１．７ｍｌをかきまぜながら３７℃で３
分間予備培養したのち、０．４μＭのアブリン‐ｂ溶液
８５μｌを添加し、６００ｎｍの波長における吸光度を
測定して凝集曲線を作成した。この凝集曲線から求めた
凝集速度（ΔＡ／分）は０．２３、凝集強度（ΔＡ）は
０．９７であった。Example 1 Jurkat strain T cells in the logarithmic growth phase were rinsed three times with physiological saline PBS (-) to adjust the cell number to 3 × 10 6 cells / ml. Next, 1.7 ml of the cell suspension containing the T cells was stirred at 37 ° C for 3 hours.
After pre-incubation for minutes, 85 μl of a 0.4 μM Abrin-b solution was added, and the absorbance at a wavelength of 600 nm was measured to prepare an aggregation curve. The aggregation rate (ΔA / min) determined from the aggregation curve was 0.23, and the aggregation strength (ΔA) was 0.97.
【００１７】幼若期のアール・ピー・エム・アイ（ＲＰ
ＭＩ）８４０２株Ｔ細胞について、同様の操作を繰り返
し、凝集曲線を作成した。この凝集曲線から求めた凝集
速度及び凝集強度はいずれも０であった。[0017] R.P.M.I.
MI) The same operation was repeated for the 8402 strain T cells to prepare an aggregation curve. The coagulation rate and cohesion strength obtained from this coagulation curve were both 0.
【００１８】実施例２ アブリン‐ｂの代りにアブリン‐ａを用い、全く同様に
してジャーカット（Ｊｕｒｋａｔ）株Ｔ細胞の凝集速度
（ΔＡ／分）及び凝集強度（ΔＡ）を求めたところ、そ
れぞれ１．４２及び０．８３であった。また、アール・
ピー・エム・アイ（ＲＰＭＩ）８４０２株Ｔ細胞につい
ての凝集速度（ΔＡ／分）及び凝集強度（ΔＡ）は、そ
れぞれ０．２９及び０．５であった。Example 2 Agrin-a was used in place of Abrin-b, and the aggregation rate (ΔA / min) and aggregation strength (ΔA) of Jurkat strain T cells were determined in exactly the same manner. 1.42 and 0.83. Also,
Aggregation rate (ΔA / min) and aggregation strength (ΔA) for the PMI (RPMI) 8402 strain T cells were 0.29 and 0.5, respectively.
【００１９】[0019]
【発明の効果】本発明によると、アブリンを用いること
により、簡単な分光的手法でＴ細胞系の腫瘍細胞の分
化、成熟度を判定することができるので、ガンや白血病
の進行状況を間接的に知ることができる上に、生きてい
る細胞を直接アッセイできるという利点もある。According to the present invention, the use of abrin allows the differentiation and maturity of T cell tumor cells to be determined by a simple spectroscopic method, so that the progress of cancer or leukemia can be indirectly determined. And the ability to assay live cells directly.
【図１】 Ｔ細胞の凝集曲線の例を示すグラフ。FIG. 1 is a graph showing an example of an aggregation curve of T cells.
【図２】 アブリン濃度と凝集速度及び凝集強度との関
係を示すグラフ。FIG. 2 is a graph showing the relationship between the abrin concentration and the aggregation rate and the aggregation strength.
【図３】 細胞数と凝集速度及び凝集強度との関係を示
すグラフ。FIG. 3 is a graph showing the relationship between the number of cells and the aggregation rate and aggregation intensity.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 星野 友昭 福岡県久留米市旭町67 久留米大学内 (72)発明者 伊藤 恭悟 福岡県久留米市旭町67 久留米大学内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomoaki Hoshino 67 Asahimachi, Kurume City, Fukuoka Prefecture Kurume University (72) Inventor Kyogo Ito 67 Asahimachi, Kurume City Fukuoka Prefecture In Kurume University
Claims (4)
加し、経時的に濁度の減少度を測定し、それに基づいて
作成した凝集曲線から凝集速度及び凝集強度を求め、そ
の数値からＴ細胞の分化、成熟度を判定する方法。1. An abrin is added to a T cell tumor cell suspension, the degree of turbidity decrease is measured over time, and the aggregation rate and the aggregation intensity are determined from an aggregation curve created based on the turbidity. A method for determining T cell differentiation and maturity.
る請求項１記載の方法。2. The method according to claim 1, wherein the T cells are derived from human acute leukemia.
ｍｌ以下の細胞数とする請求項１又は２記載の方法。3. The concentration of T cell suspension is 3 × 10 6 cells /
3. The method according to claim 1, wherein the number of cells is not more than ml.
請求項１ないし３のいずれかに記載の方法。4. The method according to claim 1, wherein the concentration of abrin is 0.4 μM or less.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8039124A JP2802364B2 (en) | 1996-02-02 | 1996-02-02 | Method for determining differentiation and maturity of T cell tumor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8039124A JP2802364B2 (en) | 1996-02-02 | 1996-02-02 | Method for determining differentiation and maturity of T cell tumor cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09206096A true JPH09206096A (en) | 1997-08-12 |
| JP2802364B2 JP2802364B2 (en) | 1998-09-24 |
Family
ID=12544360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8039124A Expired - Lifetime JP2802364B2 (en) | 1996-02-02 | 1996-02-02 | Method for determining differentiation and maturity of T cell tumor cells |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2802364B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6420171B1 (en) | 1999-11-30 | 2002-07-16 | Japan As Represented By Secretary Of Agency Of Industrial Science And Technology | Leukemic cell-adsorbing material containing lectin protein from Agrocybe cylindracea or jequirity plant seed |
| US6987022B2 (en) * | 1999-11-30 | 2006-01-17 | National Institute Of Advanced Industrial Science And Technology | Leukemic cell-adsorbing material containing lectin protein from Dolichos beans or soybeans |
-
1996
- 1996-02-02 JP JP8039124A patent/JP2802364B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6420171B1 (en) | 1999-11-30 | 2002-07-16 | Japan As Represented By Secretary Of Agency Of Industrial Science And Technology | Leukemic cell-adsorbing material containing lectin protein from Agrocybe cylindracea or jequirity plant seed |
| US6987022B2 (en) * | 1999-11-30 | 2006-01-17 | National Institute Of Advanced Industrial Science And Technology | Leukemic cell-adsorbing material containing lectin protein from Dolichos beans or soybeans |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2802364B2 (en) | 1998-09-24 |
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