JPH07289288A - Method for evaluating effect of antirheumatic medicine - Google Patents
Method for evaluating effect of antirheumatic medicineInfo
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- JPH07289288A JPH07289288A JP11016794A JP11016794A JPH07289288A JP H07289288 A JPH07289288 A JP H07289288A JP 11016794 A JP11016794 A JP 11016794A JP 11016794 A JP11016794 A JP 11016794A JP H07289288 A JPH07289288 A JP H07289288A
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、抗リウマチ薬の効果評
価方法に係り、詳細には、リウマチ疾患のモデルを使用
したイムノモニタリング法に基づく、抗リウマチ薬の効
果評価方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating the effects of antirheumatic drugs, and more particularly to a method for evaluating the effects of antirheumatic drugs based on an immunomonitoring method using a model of rheumatic diseases.
【０００２】[0002]
【従来の技術】従来より薬剤の薬効評価については、臨
床評価、病理学的評価に加え、免疫学的評価を詳細に行
なうのが一般的である。そのなかでも、関節炎治療にお
ける薬効評価については、ｉｎ ｖｉｖｏにおける疾患
モデルを用いて行なうことが最近の薬剤開発研究の中で
行なわれている。特に慢性関節リウマチに対する新しい
抗リウマチ薬を開発するに際しては、その薬効評価を行
なうために、ｉｎ ｖｉｖｏにおける病態をｉｎ ｖｉ
ｔｒｏで効率よく再現するより簡便な疾患モデルの確立
が必要である。これまでにも、かかる目的に使用されて
きた関節炎モデルは数多く存在する。例えば、誘発モデ
ルとして、小動物に誘発させるアジュバント関節炎、Ｉ
Ｉ型コラーゲン関節炎、プリスタン関節炎、あるいは関
節炎自然発症モデルとしてのＭＲＬ／ｌｐｒ、その他の
突然変異マウスなどが、疾患モデルとして用いられてき
た。しかしながらラットやマウスなどの小動物に発症す
るこれら関節炎は、慢性関節リウマチの病態と部分的に
類似してはいるが、ヒトの慢性関節リウマチを必ずしも
正確には再現していないものである。特に慢性関節リウ
マチの治療目標は、増殖性滑膜炎（パンヌス）による関
節破壊を抑制することであるが、この滑膜炎を再現する
ｉｎ ｖｉｔｒｏのヒト由来細胞モデルがないのが現状
である。最近に至り、Ｈｏｌｏｓｈｉｔｚらは、慢性関
節リウマチ患者の滑膜由来単核細胞からパンヌス様組織
を形成する細胞モデルを報告している（Ａｒｔｈｒｉｓ
ｔｉｓ Ｒｈｅｕｍ，３４：６７９−６８６，１９９
１）。しかしながら、この系は特異的抗原である抗酸菌
由来蛋白とＴ細胞増殖因子ＩＬ−２を人為的に加えて作
成したものであり、慢性関節リウマチに普遍的な病態モ
デルとはいえないものであり、かかる細胞モデルを使用
して抗リウマチ薬の薬効評価をしたとしても、実際の病
態との比較のうえでの正確な薬効評価とはなり得ないも
のである。2. Description of the Related Art Conventionally, in order to evaluate the efficacy of drugs, it is general to carry out detailed immunological evaluation in addition to clinical evaluation and pathological evaluation. Among them, the evaluation of drug efficacy in the treatment of arthritis has been carried out in recent drug development studies using an in vivo disease model. In particular, when developing a new antirheumatic drug for rheumatoid arthritis, in order to evaluate the drug efficacy, the in vivo pathological condition is examined in vivo.
It is necessary to establish a simpler disease model that can be efficiently reproduced in tro. There are many arthritis models that have been used for this purpose. For example, as an induction model, adjuvant arthritis induced by small animals, I
Type I collagen arthritis, pristane arthritis, or MRL / lpr as a spontaneous arthritis model, other mutant mice, and the like have been used as disease models. However, these arthritis occurring in small animals such as rats and mice partially resemble the pathological condition of rheumatoid arthritis, but they do not always accurately reproduce human rheumatoid arthritis. In particular, the therapeutic goal of rheumatoid arthritis is to suppress joint destruction due to proliferative synovitis (pannus), but the present situation is that there is no in vitro human-derived cell model that reproduces this synovitis. Recently, Holoshitz et al. Reported a cell model of forming pannus-like tissue from synovial membrane-derived mononuclear cells of patients with rheumatoid arthritis (Arthris).
tis Rheum, 34: 679-686, 199.
1). However, this system was created by artificially adding a specific antigen, an acid-fast bacterium-derived protein and T cell growth factor IL-2, and cannot be said to be a universal pathological model for rheumatoid arthritis. Therefore, even if the drug efficacy of antirheumatic drugs is evaluated using such a cell model, the drug efficacy cannot be accurately evaluated in comparison with the actual pathological condition.
【０００３】[0003]
【発明が解決しようとする問題点】このように、慢性関
節リウマチの疾患モデルは、その病因あるいは病態を解
明するうえで重要であり、かつ新しい抗リウマチ薬を開
発するうえで必要不可欠である。しかしながら、従来用
いられてきた関節炎モデルは、マウスやラットなどの動
物を用いたものであるため、ヒトとは薬剤感受性が異な
る点や、慢性関節リウマチの病態を必ずしも正確に再現
していないことなどの問題があった。したがって、慢性
関節リウマチをより正確に再現するようなヒトの細胞を
用いた疾患モデルの開発が望まれていた。本発明者ら
は、慢性関節リウマチの滑膜炎をより正確に再現し、効
率よく抗リウマチ薬の薬効を評価できる疾患モデルを開
発すべく鋭意検討を重ねた。その結果、慢性関節リウマ
チ患者の滑膜組織に浸潤している単核細胞を採取し、初
代混合培養を行ない、パンヌス様組織を効率よく再構成
させる細胞モデルを確立し、当該細胞モデルを用いて行
なう抗リウマチ薬の薬効評価が、実際の病態に対する薬
効と等しいものであることを確認し、本発明を完成させ
たのである。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention As described above, the disease model of rheumatoid arthritis is important for elucidating the etiology or pathological condition thereof and is essential for developing a new antirheumatic drug. However, since the arthritis model that has been used so far uses animals such as mice and rats, it has different drug sensitivity from humans and that it does not always accurately reproduce the pathological condition of rheumatoid arthritis. There was a problem. Therefore, development of a disease model using human cells that more accurately reproduces rheumatoid arthritis has been desired. The present inventors have earnestly studied to develop a disease model that can more accurately reproduce synovitis of rheumatoid arthritis and efficiently evaluate the efficacy of antirheumatic drugs. As a result, mononuclear cells infiltrating the synovial tissue of patients with rheumatoid arthritis were collected, primary mixed culture was performed, and a cell model for efficiently reconstituting pannus-like tissue was established. It was confirmed that the drug efficacy evaluation of the antirheumatic drug to be performed is equivalent to the drug efficacy against the actual pathological condition, and the present invention has been completed.
【０００４】すなわち、本発明者らは慢性関節リウマチ
患者滑膜由来の単核細胞を、刺激物質を添加することな
く基礎培地のみで初代混合培養したところ、その細胞の
組織培養状態が、客観的な経時的変化として、培養皿中
に細胞の集族が始まり、その周囲に細胞がいなくなった
空白域ができた後、さらに集族すると、それを包むよう
にマトリックス様の構造が形成され、その後徐々に増大
して厚みのある組織となり、その組織の状態は肉眼的に
も観察可能となる段階で進行することを見い出した。そ
してこの組織の培養上での進行状態は、患者の病態の進
行と等しいものであることを確認し、したがって、抗リ
ウマチ薬をこの培養段階に添加してやり、組織の培養状
態のステージをモニタリングとして評価すれば、抗リウ
マチ薬の薬効評価方法となり得ることを確立し、本発明
を完成したのである。That is, the present inventors have performed primary mixed culture of synovial membrane-derived mononuclear cells of patients with rheumatoid arthritis in a basal medium alone without adding a stimulant, and the tissue culture state of the cells was objectively examined. As a time-dependent change, the cells gathered in the culture dish, and a blank area was created around the cells, and when they gathered further, a matrix-like structure was formed to wrap them, and then gradually. It was found that the tissue became thicker and thicker, and the state of the tissue progressed at a stage where it could be visually observed. Then, it was confirmed that the progressing state of this tissue in culture was equivalent to the progression of the pathological condition of the patient.Therefore, an antirheumatic drug was added to this culture stage, and the stage of the tissue culture state was evaluated as monitoring. Then, it was established that it could be a method for evaluating the efficacy of antirheumatic drugs, and the present invention was completed.
【０００５】[0005]
【課題を解決するための手段】しかして本発明は、慢性
関節リウマチ患者の滑膜組織由来の単核細胞を、初代混
合培養し、その経時的培養形態の変化を観察することに
よる、抗リウマチ薬の効果評価方法において、初代混合
培養時に、抗リウマチ薬の効果被検定濃度を添加し、そ
の経時的培養形態をモニタリングし、細胞培養の経時ス
テージを評価すること、による、抗リウマチ薬の効果評
価方法を提供する。[Means for Solving the Problems] The present invention, therefore, provides anti-rheumatoid arthritis by culturing mononuclear cells derived from synovial tissue of patients with rheumatoid arthritis in a primary mixed culture and observing changes in the culture morphology over time. In the method for evaluating the effect of a drug, the effect of an anti-rheumatic drug is obtained by adding an effect concentration to be tested for the anti-rheumatic drug at the time of primary mixed culture, monitoring the time-dependent culture morphology, and evaluating the time-course stage of cell culture. Provide an evaluation method.
【０００６】本発明方法は、後記するごとくその詳細に
おいて、特に単核細胞の培養形態のモニタリングを、 細胞の集族と空白域の出現 マトリクス様構造の形成 肉眼的観察可能な組織形成 の経時ステージとして評価することを特徴とする。The method of the present invention will be described in detail below, in particular, for monitoring the culture morphology of mononuclear cells, for the aggregation of cells and the appearance of blank areas, the formation of matrix-like structures, and the time-course stage of macroscopically observable tissue formation. It is characterized by evaluating as.
【０００７】さらに本発明方法は、慢性関節リウマチ患
者の滑膜組織由来の単核細胞を、初代混合培養するに際
して、人為的な刺激物質を添加することなく、基礎培地
のみで初代混合培養し、部分的ではあるが、患者滑膜炎
の病態そのものを細胞モデルレベルで再現した点に特徴
を有する。Further, the method of the present invention is that, when mononuclear cells derived from synovial tissue of a patient with rheumatoid arthritis are subjected to primary mixed culture, primary mixed culture is performed only with a basal medium without adding an artificial stimulant, Although it is partial, it is characterized in that the pathological condition of patient's synovitis itself is reproduced at the cell model level.
【０００８】[0008]
【作用】以上のように本発明方法は、外来抗原、例えば
従来行なわれていた抗酸菌由来蛋白、Ｔ細胞増殖因子Ｉ
Ｌ−２の添加などの人為的加工を一切行なわず、患者滑
膜由来細胞の自発的増殖とパンヌス様組織形成をｉｎ
ｖｉｔｒｏで再現させたものである。したがって、人為
的に病態を類似させた従来の疾患モデルとは異なり、本
方法は、部分的ではあるが、患者滑膜炎の病態そのもの
を再現した点で画期的なものである。また、一人の滑膜
組織から１０7 ないし１０8 個もの単核細胞を採取で
き、患者間での差異がなく、客観的に再現性がよいこと
などから、抗リウマチ薬の薬効を評価するうえで有用な
ものといえる。As described above, according to the method of the present invention, a foreign antigen, for example, an acid-fast bacterium-derived protein, T cell growth factor I, which has been conventionally used, is used.
No artificial processing such as addition of L-2 was performed, and spontaneous proliferation of pancreatic-like tissue formation and synovium-derived cells in patients
It was reproduced in vitro. Therefore, unlike the conventional disease model in which the pathological condition is artificially made similar, this method is a breakthrough in that the pathological condition of the patient's synovitis itself is reproduced although it is partial. In addition, 10 7 to 10 8 mononuclear cells can be collected from one synovial tissue, and there is no difference between patients, and objectively reproducible. Can be said to be useful.
【０００９】この場合、本発明方法で使用するリウマチ
患者滑膜由来単核細胞モデルは、例えば、以下のように
して構築される。すなわち、慢性関節リウマチ患者の滑
膜切除術、または人工関節置換術施行時に得られた滑膜
組織を細切し、１０％ＦＣＳまたは５％ヒトＡＢ血清を
含むＲＰＭＩ−１６４０培地で培養した。培養開始３な
いし５日目に浮遊細胞を回収し、１〜２×１０6 ／ｍｌ
の濃度で初代混合培養することにより、構築される。In this case, the synovial membrane-derived mononuclear cell model of the rheumatic patient used in the method of the present invention is constructed, for example, as follows. That is, the synovial tissue obtained at the time of performing synovectomy or artificial joint replacement of a rheumatoid arthritis patient was cut into small pieces and cultured in RPMI-1640 medium containing 10% FCS or 5% human AB serum. Floating cells were collected on the 3rd to 5th day after the start of culturing, and 1 to 2 × 10 6 / ml was collected.
It is constructed by primary mixed culture at a concentration of.
【００１０】このようにして構築（ｉｎ ｖｉｔｒｏで
の再構築）された細胞モデルは、経時的に形態観察、培
養上清中のサイトカイン産生量の測定、細胞表面マーカ
ーの測定により、本細胞モデルが、機能的にも患者滑膜
組織を部分的に再現していることが示された。この再構
築された細胞モデルの組織の再構成の確認は、凍結乾燥
保存後免疫組織染色を含む組織学的検索により行ない、
サイトカインの測定は、ＥＬＩＳＡキット、表面マーカ
ーはフローサイトメーターを用いて行なった。それらの
点を以下に記載する。The cell model constructed in this manner (reconstruction in vitro) is obtained by morphological observation with time, measurement of the amount of cytokine produced in the culture supernatant, and measurement of cell surface markers. , It was shown that the patient's synovial tissue was functionally reproduced. Confirmation of the reconstitution of the tissue of this reconstructed cell model was performed by histological search including immunohistological staining after lyophilization preservation,
The cytokine was measured using an ELISA kit, and the surface marker was measured using a flow cytometer. These points are described below.
【００１１】細胞モデルの組織の再構成（肉眼的所見） 患者滑膜由来単核細胞を、刺激物質を添加することなく
基礎培地のみで初代混合培養したところ、１週後より、
培養皿の片隅に細胞が集族しはじめ、その周囲に細胞が
いなくなった空白域ができた。その培養状態写真を図１
として示す。２週後よりさらに集族すると、それを包む
ようにマトリックス様の構造が形成され（その培養状態
写真を図２に示す。）、その後徐々に増大して厚みのあ
る組織となり、３〜４週後にはその組織の状態は肉眼的
にも観察可能となり（その培養状態写真を図３に示
す。）、６週後まで成長を続けた。このような組織の構
築は、１０名の患者全例において認められた。 Reconstruction of Tissue of Cell Model (Macroscopic Findings) Patient synovial membrane-derived mononuclear cells were subjected to primary mixed culture in a basal medium alone without addition of a stimulant.
Cells began to collect in one corner of the culture dish, and there was a blank area around which cells had disappeared. Figure 1 of the culture state photograph
Show as. After 2 weeks, a matrix-like structure was formed so as to wrap it (a photograph of the culture state is shown in FIG. 2), and then gradually increased to become a thick tissue, and after 3 to 4 weeks. The tissue state became visually observable (the photograph of the culture state is shown in FIG. 3), and the growth was continued until 6 weeks later. Such tissue construction was observed in all 10 patients.
【００１２】組織学的対比 培養開始６週後に構築された組織の凍結切片を染色し
て、患者滑膜と組織学的に比較検討した。リウマチ患者
滑膜組織は従来から知られているように、滑膜細胞の絨
毛形成、リンパ濾胞形成、結合織の増生、血管新生、リ
ンパ球や形質細胞の浸潤、活性化されたマクロファージ
や繊維芽細胞からなる肉芽様組織などが特徴である。そ
の染色組織写真を図４に示す。一方、本発明で使用する
ｉｎ ｖｉｔｒｏで形成された滑膜組織細胞モデルにあ
っては、絨毛形成、結合織の増生、リンパ球浸潤がＨＥ
染色で認められ（その染色組織写真を図５に示す。）、
免疫組織染色にて浸潤リンパ球は、ＣＤ４５ＲＯ+ のＴ
細胞、活性化マクロファージならびに繊維芽細胞が多数
認められた。この免疫組織染色状態を図６〜８に示す。
このように、本発明のｉｎ ｖｉｔｒｏで形成された滑
膜組織細胞モデルは、明らかなリンパ濾胞形成や血管新
生は認められなかったものの、リウマチ患者滑膜炎組織
が部分的に再構築されていることが組織学的に明らかで
あることが理解される。 Histological contrast Six weeks after the start of culture, frozen sections of the tissue constructed were stained and examined histologically in comparison with the patient's synovium. Synovial tissue of patients with rheumatoid arthritis is conventionally known, including synovial cell villi formation, lymphoid follicle formation, connective tissue hyperplasia, angiogenesis, infiltration of lymphocytes and plasma cells, activated macrophages and fibroblasts. It is characterized by granulation tissue composed of cells. A photograph of the stained structure is shown in FIG. On the other hand, in the synovial tissue cell model formed in vitro used in the present invention, villi formation, connective tissue growth, and lymphocyte infiltration are
It was observed by staining (a photograph of the stained tissue is shown in FIG. 5),
Immunohistochemical staining revealed that infiltrating lymphocytes were T of CD45RO + .
Many cells, activated macrophages and fibroblasts were found. This immunohistochemical staining state is shown in FIGS.
As described above, in the in vitro synovial tissue cell model of the present invention, although clear lymphoid follicle formation and angiogenesis were not observed, synovitis tissue of a rheumatic patient was partially reconstructed. It is understood that is histologically clear.
【００１３】また４週後に形成された本発明の組織を細
切し、浸潤したリンパ球を回収してその表面マーカーを
測定したところ、ＣＤ４+ 、ＣＤ４５ＲＯ+ のＴ細胞が
大部分であった。その結果を表１にまとめた。When the tissue of the present invention formed after 4 weeks was sliced and infiltrated lymphocytes were collected and the surface marker thereof was measured, the majority of T cells were CD4 + and CD45RO + . The results are summarized in Table 1.
【００１４】[0014]
【表１】 [Table 1]
【００１５】この比率は、患者滑膜組織を細切して採集
した非付着細胞の分画と一致していた。このことは、初
代混合培養４週後においても滑膜由来細胞の細胞分画が
維持されていたことを示している。This ratio was consistent with the fraction of non-adherent cells collected by finely slicing the patient's synovial tissue. This indicates that the cell fraction of synovial membrane-derived cells was maintained even after 4 weeks of the primary mixed culture.
【００１６】産生されるサイトカインの測定 生体内（ｉｎ ｖｉｖｏ）において慢性関節リウマチ患
者滑膜細胞が大量に産生するサイトカインとしては、イ
ンターロイキン−１（ＩＬ−１）、インターロイキン−
６（ＩＬ−６）、ＰＧＥ２などが知られている。本発明
の再構築された細胞モデルの組織が、機能的にも患者滑
膜炎組織を再現しているか否かを検討するため、培養上
清中に産生されるサイトカインを測定した。その結果、
図９および１０に示すように、初代混合培養開始後１週
および２週後の培養上清中にはＩＬ−１βが６０ないし
８０ｐｇ／ｍｌ認められ、２週後には、ＩＬ−６が１６
０ｎｇ／ｍｌ、３週後にはＰＧＥ２が７００〜９５０ｐ
ｇ／ｍｌ検出された。この成績は、本発明の細胞モデル
が機能的にも患者滑膜組織を部分的に再現していること
を示すものである。また、この培養上清中でのサイトカ
インの産生測定は、今後の薬剤の薬効評価のひとつのメ
ルクマールとなるものである。 Measurement of Produced Cytokines As cytokines produced in large quantities by synovial cells of patients with rheumatoid arthritis in vivo, interleukin-1 (IL-1) and interleukin-
6 (IL-6), PGE2 and the like are known. In order to examine whether or not the tissue of the reconstructed cell model of the present invention functionally reproduces the synovitis tissue of the patient, the cytokine produced in the culture supernatant was measured. as a result,
As shown in FIGS. 9 and 10, 60 to 80 pg / ml of IL-1β was observed in the culture supernatant one week and two weeks after the start of the primary mixed culture, and IL-6 was 16% after 2 weeks.
0 ng / ml, PGE2 700-950p after 3 weeks
g / ml was detected. This result shows that the cell model of the present invention functionally partially reproduces the synovial tissue of the patient. In addition, the measurement of cytokine production in this culture supernatant will be one of the mercumars for evaluation of drug efficacy in the future.
【００１７】抗リウマチ薬の薬効評価検討（その１） 以上の様に、本発明の細胞モデルは、組織学的にも、機
能的にも患者滑膜組織を部分的に再現していることが判
明したので、この本発明の細胞モデルを使用した抗リウ
マチ薬の薬効評価の検討を行なった。現在、臨床的に最
も有効な抗リウマチ薬のひとつとして、メトトレキサー
ト（ＭＴＸ）が盛んに用いられている。そこで本発明の
細胞モデルに対するＭＴＸの抑制効果を検討した。 Examination of Evaluation of Anti-rheumatic Drug Efficacy (Part 1) As described above, the cell model of the present invention partially reproduces the synovial tissue of the patient both histologically and functionally. Since it was revealed, the evaluation of the drug efficacy of the antirheumatic drug using this cell model of the present invention was examined. Currently, methotrexate (MTX) is actively used as one of the most clinically effective antirheumatic drugs. Therefore, the inhibitory effect of MTX on the cell model of the present invention was examined.
【００１８】上記した方法により得られた本発明の細胞
モデルを用い、初代混合培養をし、経時的にその培養状
態の変化を観察することにより、ＭＴＸの抗リウマチ薬
としての薬効評価を行なった。初代混合培養にあたり、
ＭＴＸの濃度を０．０１μｇ／ｍｌ、０．１μｇ／ｍ
ｌ、１μｇ／ｍｌ、２μｇ／ｍｌおよび５μｇ／ｍｌ、
ならびにＭＴＸ２μｇ／ｍｌとｌ−ロイコボリン（ＬＶ
Ｎ）５μｇ／ｍｌの混合物を培養液に添加し、その細胞
モデルの組織の形態変化を経時的に観察した。なお、コ
ントロールとして基礎培地のみの培養状態をおいた。培
養後の本発明の細胞モデルの形態変化を半定量的に表わ
すため、以下のように点数化をした。 細胞の集族と空白域の出現： １点 マトリクス様構造の形成 ： ２点 肉眼的観察可能な組織形成： ３点 実験は、２４穴の培養皿３穴の点数の平均値を、組織化
スコアとした。その結果を図１１に示す。Using the cell model of the present invention obtained by the above-mentioned method, primary mixed culture was performed, and changes in the culture state were observed over time to evaluate the efficacy of MTX as an antirheumatic drug. . In the primary mixed culture,
MTX concentration is 0.01μg / ml, 0.1μg / m
1, 1 μg / ml, 2 μg / ml and 5 μg / ml,
And MTX 2 μg / ml and 1-leucovorin (LV
N) A mixture of 5 μg / ml was added to the culture medium, and the morphological change of the tissue of the cell model was observed over time. As a control, the culture state of only the basal medium was set. In order to semi-quantitatively represent the morphological change of the cell model of the present invention after culturing, it was scored as follows. Appearance of cell clusters and blank areas: 1 point Formation of matrix-like structure: 2 points Macroscopically observable tissue formation: 3 points In the experiment, the average score of 3 wells in a 24-well culture dish was used as the organization score. And The result is shown in FIG.
【００１９】図中の結果から明らかなように、基礎培地
のみで培養した対照群（コントロール群）では、１週後
に形態変化が進み、２週後には組織形成が１００％に達
した。これに対してＭＴＸは、用量依存的に組織形成を
抑制し、２週後における５０％抑制濃度は０．１μｇ／
ｍｌ以下であった。この濃度は、臨床的に患者に０．１
〜０．２μｇ／ｋｇを投与した時の血中濃度あるいはそ
れ以下に相当するものである。従来の動物を用いた疾患
モデルでは、０．３μｇ／ｋｇのＭＴＸでは全く関節炎
を抑制できなかった。これはＭＴＸの標的酵素がマウス
ではヒトの１０分の１しか活性がないためである。この
点から見ても、本発明の細胞モデルは、人の関節炎を組
織的に再現したものであり、その感度も従来の動物モデ
ルのものより良好であり、本モデルの卓越性が示唆され
る。As is clear from the results in the figure, in the control group (control group) cultured in the basal medium alone, the morphological change proceeded after 1 week and the tissue formation reached 100% after 2 weeks. On the other hand, MTX suppressed the tissue formation in a dose-dependent manner, and the 50% inhibitory concentration after 2 weeks was 0.1 μg /
It was less than or equal to ml. This concentration is clinically 0.1
It corresponds to the blood concentration at the time of administration of 0.2 μg / kg or less. In a conventional disease model using animals, 0.3 μg / kg of MTX could not suppress arthritis at all. This is because the target enzyme of MTX is only 10 times less active in human than in human. Also from this point of view, the cell model of the present invention systematically reproduces human arthritis, and its sensitivity is also better than that of the conventional animal model, suggesting the excellence of this model. .
【００２０】抗リウマチ薬の薬効評価検討（その２） 一方、デキサメタゾンなどの副腎皮質ホルモンは、大量
に用いると慢性関節リウマチの関節炎を抑制することが
知られている。これに対して、インドメタシンなどの非
ステロイド系抗炎症剤は鎮痛効果は認められるものの、
関節炎を抑制できない。そこで、この２薬剤に対して本
発明の細胞モデルを用いた薬効効果判定方法を適用し、
本発明方法の客観性を確認した。薬効効果判定の方法
は、前記したＭＴＸの場合と同様に行なった。その結
果、デキサメタゾン（ＤＥＸ）は、用量依存的に組織形
成を抑制したが（図１２）、インドメタシン（Ｉｎｄ
ｏ）は、全く抑制できなかった（図１３）。この成績か
らみても、本発明の抗リウマチ薬の薬効評価方法の優れ
ている点が確認された。 Evaluation of drug efficacy of antirheumatic drugs (Part 2) On the other hand, corticosteroids such as dexamethasone are known to suppress arthritis of rheumatoid arthritis when used in large amounts. On the other hand, although non-steroidal anti-inflammatory drugs such as indomethacin have an analgesic effect,
Inability to control arthritis. Therefore, the drug efficacy determination method using the cell model of the present invention is applied to these two drugs,
The objectivity of the method of the present invention was confirmed. The drug efficacy determination method was the same as in the case of MTX described above. As a result, dexamethasone (DEX) suppressed tissue formation in a dose-dependent manner (Fig. 12), but indomethacin (Ind).
o) could not be suppressed at all (Fig. 13). From these results, it was confirmed that the method of evaluating the efficacy of the antirheumatic drug of the present invention is excellent.
【００２１】[0021]
【発明の効果】以上の様に、本発明は、慢性関節リウマ
チ患者滑膜細胞由来の単核細胞を使用することにより、
組織学的にも、機能的にも患者滑膜炎組織を部分的に再
現している細胞モデルを使用した点で、その感度も従来
の動物モデルのものより良好であり、抗リウマチ薬の薬
効評価方法として優れたものである。INDUSTRIAL APPLICABILITY As described above, according to the present invention, by using mononuclear cells derived from synovial cells of patients with rheumatoid arthritis,
The sensitivity of the antirheumatic drug was better than that of the conventional animal model in that it used a cell model that partially reproduced the synovitis tissue of the patient both histologically and functionally. It is an excellent evaluation method.
【図１】本発明の細胞モデルの培養開始１週後の培養状
態の写真である。FIG. 1 is a photograph showing the culture state of the cell model of the present invention one week after the start of culture.
【図２】本発明の細胞モデルの培養開始２週後の培養状
態の写真である。FIG. 2 is a photograph showing the culture state of the cell model of the present invention two weeks after the start of culture.
【図３】本発明の細胞モデルの培養開始３週後の培養状
態の写真である。FIG. 3 is a photograph showing the culture state of the cell model of the present invention 3 weeks after the start of culture.
【図４】患者滑膜細胞のＨＥ染色組織写真である。FIG. 4 is an HE-stained tissue photograph of patient synovial cells.
【図５】本発明の細胞モデルのＨＥ染色写真である。FIG. 5 is an HE-stained photograph of the cell model of the present invention.
【図６】本発明の細胞モデルの抗ＣＤ４５ＲＯ抗体によ
る免疫組織染色写真である。FIG. 6 is a photograph of immunohistological staining of the cell model of the present invention with anti-CD45RO antibody.
【図７】本発明の細胞モデルの抗ＣＤ６８抗体による免
疫組織染色写真である。FIG. 7 is a photograph of immunohistological staining of the cell model of the present invention with anti-CD68 antibody.
【図８】本発明の細胞モデルの抗リメンチン抗体による
免疫組織染色写真である。FIG. 8 is a photograph of immunohistochemical staining with the anti-rementin antibody of the cell model of the present invention.
【図９】本発明の細胞モデルの培養上清中の、ＩＬ−１
β、ＰＧＥ２の測定の結果を示す図である。FIG. 9: IL-1 in the culture supernatant of the cell model of the present invention
It is a figure which shows the result of measurement of (beta) and PGE2.
【図１０】本発明の細胞モデルの培養上清中の、ＩＬ−
６の測定の結果を示す図である。FIG. 10: IL-in the culture supernatant of the cell model of the present invention
It is a figure which shows the result of the measurement of 6.
【図１１】本発明の細胞モデルを用いたＭＴＸの抑制効
果を示す図である。FIG. 11 is a graph showing the inhibitory effect of MTX using the cell model of the present invention.
【図１２】本発明の細胞モデルを用いた薬物の抑制効果
を示す図であり、デキサメタゾンの結果を示す。FIG. 12 is a graph showing the inhibitory effect of drugs using the cell model of the present invention, showing the results of dexamethasone.
【図１３】本発明の細胞モデルを用いた薬物の抑制効果
を示す図であり、インドメタシンの結果を示す。FIG. 13 shows the inhibitory effect of drugs using the cell model of the present invention, showing the results of indomethacin.
Claims (6)
単核細胞を、初代混合培養し、その経時的培養形態の変
化を観察することによる、抗リウマチ薬の効果評価方法
において、 初代混合培養時に、抗リウマチ薬の効果被検定濃度を添
加し、 その経時的培養形態をモニタリングし、 細胞培養の経時ステージを評価すること、による、抗リ
ウマチ薬の効果評価方法。1. A method for evaluating the effect of an anti-rheumatic drug by culturing mononuclear cells derived from synovial tissue of a patient with rheumatoid arthritis in a primary mixed culture and observing changes in the culture morphology over time. A method for evaluating the effect of an antirheumatic drug, comprising adding a concentration to be tested for the effect of an antirheumatic drug, monitoring the time-dependent culturing morphology, and evaluating the time stage of cell culture.
マチ薬の効果評価方法。2. The anti-rheumatic disease according to claim 1, wherein the monitoring of the culture morphology of mononuclear cells is evaluated as a time-course stage of cell aggregation and appearance of blank areas, formation of matrix-like structure, and macroscopically observable tissue formation. How to evaluate the effects of drugs.
となく、基礎培地のみで初代混合培養する、請求項１記
載の抗リウマチ薬の効果評価方法。3. The method for evaluating the effect of an antirheumatic drug according to claim 1, wherein the primary mixed culture is carried out only with the basal medium without adding a stimulating substance.
の濃度で培養する、請求項１記載の抗リウマチ薬の効果
評価方法。4. The primary mixed culture is 1 to 2 × 10 6 / ml.
The method for evaluating the effect of an antirheumatic drug according to claim 1, which comprises culturing at the concentration of.
を細切し、１０％ＦＣＳまたは５％ヒトＡＢ血清を含む
ＲＰＭＩ−１６４０培地で培養し、単核細胞を回収す
る、請求項１記載の抗リウマチ薬の効果評価方法。5. When collecting mononuclear cells, the synovial tissue is minced and cultured in RPMI-1640 medium containing 10% FCS or 5% human AB serum to recover the mononuclear cells. The method for evaluating the effect of the described antirheumatic drug.
細切し、１０％ＦＣＳまたは５％ヒトＡＢ血清を含むＲ
ＰＭＩ−１６４０培地で培養し、培養開始３ないし５日
目に浮遊細胞を回収することにより得た、請求項１記載
の抗リウマチ薬の効果評価用細胞モデル。6. R-containing synovial tissue from a patient with rheumatoid arthritis and containing 10% FCS or 5% human AB serum
The cell model for evaluating the effect of an antirheumatic drug according to claim 1, which was obtained by culturing in a PMI-1640 medium and collecting floating cells 3 to 5 days after the start of culturing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11016794A JPH07289288A (en) | 1994-04-27 | 1994-04-27 | Method for evaluating effect of antirheumatic medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11016794A JPH07289288A (en) | 1994-04-27 | 1994-04-27 | Method for evaluating effect of antirheumatic medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07289288A true JPH07289288A (en) | 1995-11-07 |
Family
ID=14528755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11016794A Pending JPH07289288A (en) | 1994-04-27 | 1994-04-27 | Method for evaluating effect of antirheumatic medicine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07289288A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19632236A1 (en) * | 1996-08-09 | 1998-02-12 | Univ Ludwigs Albert | Production of rheumatic pannus tissue |
| WO1998006825A1 (en) * | 1996-08-08 | 1998-02-19 | KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG | Artificial rheumatic pannus tissues and diagnostic method for detecting rheumatoid arthritis |
| JP2010209119A (en) * | 1996-02-09 | 2010-09-24 | Abbott Biotechnology Ltd | HUMAN ANTIBODY BINDING TO HUMAN TNFalpha |
| US9683033B2 (en) | 2012-04-20 | 2017-06-20 | Abbvie, Inc. | Cell culture methods to reduce acidic species |
| US9688752B2 (en) | 2013-10-18 | 2017-06-27 | Abbvie Inc. | Low acidic species compositions and methods for producing and using the same using displacement chromatography |
| US9708399B2 (en) | 2013-03-14 | 2017-07-18 | Abbvie, Inc. | Protein purification using displacement chromatography |
| US9708400B2 (en) | 2012-04-20 | 2017-07-18 | Abbvie, Inc. | Methods to modulate lysine variant distribution |
-
1994
- 1994-04-27 JP JP11016794A patent/JPH07289288A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010209119A (en) * | 1996-02-09 | 2010-09-24 | Abbott Biotechnology Ltd | HUMAN ANTIBODY BINDING TO HUMAN TNFalpha |
| WO1998006825A1 (en) * | 1996-08-08 | 1998-02-19 | KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG | Artificial rheumatic pannus tissues and diagnostic method for detecting rheumatoid arthritis |
| DE19632236A1 (en) * | 1996-08-09 | 1998-02-12 | Univ Ludwigs Albert | Production of rheumatic pannus tissue |
| US9683033B2 (en) | 2012-04-20 | 2017-06-20 | Abbvie, Inc. | Cell culture methods to reduce acidic species |
| US9708400B2 (en) | 2012-04-20 | 2017-07-18 | Abbvie, Inc. | Methods to modulate lysine variant distribution |
| US9957318B2 (en) | 2012-04-20 | 2018-05-01 | Abbvie Inc. | Protein purification methods to reduce acidic species |
| US9708399B2 (en) | 2013-03-14 | 2017-07-18 | Abbvie, Inc. | Protein purification using displacement chromatography |
| US9688752B2 (en) | 2013-10-18 | 2017-06-27 | Abbvie Inc. | Low acidic species compositions and methods for producing and using the same using displacement chromatography |
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