JP2016148644A - Fibrosis determination method - Google Patents
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Abstract
Description
本発明は、被験者から得られた試料中からCD45+C1q+CCR8+細胞を検出することを特徴とする線維化判定方法に関するものである。 The present invention relates to a fibrosis determination method characterized by detecting CD45 + C1q + CCR8 + cells from a sample obtained from a subject.
(線維化)
線維化は、病因を問わず最終的に臓器不全に至るまでの経過中から共通に観られる現象である。したがって、臓器線維化の早期診断方法とその治療法の確立が、臓器不全への進展阻止を考える上で重要である。
具体例として、近年、疫学研究により慢性腎疾患(Chronic kidney disease, CKD)患者の心疾患発症率はCKDがない患者に比べて約3倍頻度が多いことが明らかになった。このような心疾患で、初期から出現する心臓の線維化は不可逆的な現象であり、組織再生が起こりにくい心臓にとっては特に重要な問題となりえる。
(Fibrosis)
Fibrosis is a phenomenon commonly observed during the course of organ failure regardless of etiology. Therefore, early diagnosis of organ fibrosis and establishment of a treatment method are important in considering the prevention of progression to organ failure.
As a specific example, recent epidemiological studies have revealed that the incidence of heart disease in patients with chronic kidney disease (CKD) is about three times as frequent as that of patients without CKD. In such a heart disease, the fibrosis of the heart that appears from the beginning is an irreversible phenomenon, which can be a particularly important problem for the heart where tissue regeneration is difficult to occur.
(線維化の検査方法)
心臓の線維化に代表されるように、臓器の線維化という状態は機能的な面においても臓器に生じている病態の最終終末像である。つまり、最終終末像に至る前に、事前に線維化が進行するのかどうかを予測し見極める手段・手法の開発が必要である。これまでには、組織生検を行って臓器・組織に現在、炎症や細胞浸潤がどこまで進行しているのか、線維化の状態がどのようになっているのかを判断するしかなかった。特に心臓などでそのような評価を行いたい場合、心筋生検は、得られる情報に比べて侵襲が大きすぎるというリスク面が大きかった。さらに、線維化がこれから進行する状態であるのかどうかや、今生じている線維化の活動性を予測可能にする有効な技術は全く存在していないという現状がある。
肝臓線維化においては、血中のIV 型コラーゲン(以下 IV.C)、プロコラーゲン III ペプチド(以下PIIIP)、ヒアルロン酸(以下HA)をはじめとする血中肝線維化マーカーを測定して線維化の病態を判断しようとすることもされているが病態進行の予測や、活動性の指標には十分になり得ていない。
(Examination method of fibrosis)
As represented by the fibrosis of the heart, the state of organ fibrosis is the final image of the pathological condition occurring in the organ in terms of function. In other words, before reaching the final terminal image, it is necessary to develop means and methods for predicting and determining whether fibrosis progresses in advance. Until now, there has been no choice but to determine how far inflammation or cell infiltration is progressing in the organ / tissue and how the state of fibrosis has been performed by performing a tissue biopsy. In particular, when it is desired to perform such an evaluation on the heart or the like, the risk of myocardial biopsy being too invasive compared to the information obtained is great. Furthermore, there is a current situation that there is no effective technique for predicting whether the fibrosis is in a state of progressing from now on and the activity of the fibrosis occurring now.
In liver fibrosis, fibrosis is measured by measuring blood liver fibrosis markers such as type IV collagen (hereinafter IV.C), procollagen III peptide (hereinafter PIIIP), and hyaluronic acid (HA) in the blood. It is also attempted to determine the pathological condition of the disease, but it cannot be sufficient for predicting the progression of the pathological condition or indicating the activity.
一方で、臓器線維化に対して早期に介入し、積極的な予防処理あるいは治療を開始することで、以降の臓器不全進展に対して抑制可能であることが、腎臓病、心血管病をはじめとして明らかになってきた。しかしながら、各種臓器線維化を正確、迅速、および簡便に評価する技術はこれまで存在しなかった(非特許文献1、2)。 On the other hand, early intervention against organ fibrosis and the start of aggressive preventive treatment or treatment can suppress the subsequent progression of organ failure, including kidney disease and cardiovascular disease. It has become clear as. However, there has been no technology for accurately, rapidly and simply evaluating various organ fibrosis (Non-Patent Documents 1 and 2).
特許文献1では、「末梢血中のfibrocyteの濃度を測定することによる腎線維化検査方法」を開示している。しかし、本発明の線維化判定方法は、特許文献1に記載の腎線維化検査方法とは、使用するマーカーが明らかに異なる。さらに、特許文献1に記載の腎線維化検査方法では、血中fibrocyteの定量化により腎線維化との関連性を証明できるものではなかった。よって本発明では、さらに細胞表面マーカーを追加することで細胞種を絞り込んだ技術を含む。 Patent Document 1 discloses “a renal fibrosis test method by measuring the concentration of fibrocytes in peripheral blood”. However, the fibrosis determination method of the present invention is clearly different from the renal fibrosis test method described in Patent Document 1 in terms of markers used. Further, the renal fibrosis test method described in Patent Document 1 cannot prove the relevance to renal fibrosis by quantifying blood fibrocytes. Therefore, the present invention includes a technique in which cell types are further narrowed down by adding cell surface markers.
現段階において、線維化、特に心臓の線維化を判定する正確、迅速、および簡便な方法はない。そこで、本発明者らは、新規な線維化判定方法を開発することを目的とした。 At present, there is no accurate, rapid and convenient way to determine fibrosis, especially heart fibrosis. Accordingly, the present inventors have aimed to develop a novel fibrosis determination method.
本発明者らは、被験者から得られた試料中からCD45+C1q+CCR8+細胞が線維化のマーカーになることを確認して、本発明の線維化判定方法を完成した。 The present inventors have confirmed that CD45 + C1q + CCR8 + cells serve as fibrosis markers from samples obtained from subjects, and completed the fibrosis determination method of the present invention.
本発明は以下からなる。
1.被験者から得られた試料中から以下のいずれか1以上の細胞を検出することを特徴とする線維化判定方法。
(1)CD45+C1q+CCR8+細胞
(2)CD45+C1q+細胞
(3)C1q+CCR8+細胞
(4)CD45+CCR8+細胞
2.以下の細胞を検出することを特徴とする前項1の線維化判定方法。
(1)CD45+C1q+CCR8+Sca1+細胞
(2)CD45+C1q+Sca1+細胞
(3)C1q+CCR8+Sca1+細胞
(4)CD45+CCR8+Sca1+細胞
(5)CD45+Sca1+細胞
3.前記被験者から得られた試料中の細胞数が、健常者から得られた試料中の細胞数と比較して、高い場合には、線維化していると判定することを特徴とする前項1又は2に記載の線維化判定方法。
4.前記線維化は、以下の臓器の線維化である前項1〜3のいずれか1に記載の線維化判定方法。
(1)心臓
(2)腎臓
(3)肺
(4)肝臓
(5)膵臓
(6)皮膚
(7)動脈
(8)腹膜
(9)がん組織
5.以下のいずれから1以上の抗体を含む線維化判定キット。
(1)抗CD45抗体
(2)抗C1q抗体
(3)抗CCR8抗体
(4)抗Sca1抗体
The present invention comprises the following.
1. A fibrosis determination method comprising detecting any one or more of the following cells from a sample obtained from a subject.
(1) CD45 + C1q + CCR8 + cells (2) CD45 + C1q + cells (3) C1q + CCR8 + cells (4) CD45 + CCR8 + cells 2. The fibrosis determination method according to item 1, wherein the following cells are detected.
(1) CD45 + C1q + CCR8 + Sca1 + cells (2) CD45 + C1q + Sca1 + cells (3) C1q + CCR8 + Sca1 + cells (4) CD45 + CCR8 + Sca1 + cells (5) CD45 + Sca1 + cells 3. The preceding item 1 or 2, wherein when the number of cells in the sample obtained from the subject is higher than the number of cells in the sample obtained from a healthy subject, it is determined as fibrosis The fibrosis determination method according to 1.
4). 4. The fibrosis determination method according to any one of items 1 to 3, wherein the fibrosis is fibrosis of the following organ.
(1) Heart (2) Kidney (3) Lung (4) Liver (5) Pancreas (6) Skin (7) Artery (8) Peritoneum (9) Cancer tissue A fibrosis determination kit comprising one or more antibodies from any of the following.
(1) Anti-CD45 antibody (2) Anti-C1q antibody (3) Anti-CCR8 antibody (4) Anti-Sca1 antibody
本発明の線維化判定方法は、線維化を判定する正確、迅速、および簡便な方法である。 The fibrosis determination method of the present invention is an accurate, rapid and simple method for determining fibrosis.
(本発明の線維化判定方法)
本発明の線維化判定方法は、被験者から得られた試料中から以下のいずれか1以上の細胞(各陽性細胞)を検出することを特徴とする。なお、「+」は、陽性細胞であることを意味する。
(1)CD45+C1q+CCR8+細胞(細胞表面にCD45抗原、C1q抗原及びCCR8抗原が発現している細胞)
(2)CD45+C1q+細胞(細胞表面にCD45抗原及びC1q抗原が発現している細胞)
(3)C1q+CCR8+細胞(細胞表面にC1q抗原及びCCR8抗原が発現している細胞)
(4)CD45+CCR8+細胞(細胞表面にCD45抗原及びCCR8抗原が発現している細胞)
(5)CD45+C1q+CCR8+Sca1+細胞(細胞表面にCD45抗原、C1q抗原、CCR8抗原及びSca1抗原が発現している細胞)
(6)CD45+C1q+Sca1+細胞(細胞表面にCD45抗原、C1q抗原及びSca1抗原が発現している細胞)
(7)C1q+CCR8+Sca1+細胞(細胞表面にC1q抗原、CCR8抗原及びSca1抗原が発現している細胞)
(8)CD45+CCR8+Sca1+細胞(細胞表面にCD45抗原、CCR8抗原及びSca1抗原が発現している細胞)
(9)CD45+Sca1+細胞(細胞表面にCD45抗原及びSca1抗原が発現している細胞)
なお、CD45は、分子量約115kDの糖タンパク質である。Sca1は、グルコシルホスファチジールイノシトールに結合するLy(lymphocyteactivation protein)-6に属する細胞表面タンパク質である。C1qは、補体の一種である。CCR8は、ケモカイン(C-C)レセプター8タンパク質を意味する。
上記した各種細胞の表面に発現している抗原は、該抗原に対する抗体(ポリクローナル抗体でもモノクローナル抗体の何れでもよい)により、容易に検出することができる。なお、すべての抗原に対する抗体は、市販品の抗体が存在するので、当業者なら容易に入手可能である。
(Fibrosis determination method of the present invention)
The fibrosis determination method of the present invention is characterized by detecting any one or more of the following cells (each positive cell) from a sample obtained from a subject. “ + ” Means a positive cell.
(1) CD45 + C1q + CCR8 + cells (cells expressing CD45 antigen, C1q antigen and CCR8 antigen on the cell surface)
(2) CD45 + C1q + cells (cells expressing CD45 antigen and C1q antigen on the cell surface)
(3) C1q + CCR8 + cells (cells that express C1q and CCR8 antigens on the cell surface)
(4) CD45 + CCR8 + cells (cells expressing CD45 antigen and CCR8 antigen on the cell surface)
(5) CD45 + C1q + CCR8 + Sca1 + cells (cells expressing CD45 antigen, C1q antigen, CCR8 antigen and Sca1 antigen on the cell surface)
(6) CD45 + C1q + Sca1 + cells (cells expressing CD45 antigen, C1q antigen and Sca1 antigen on the cell surface)
(7) C1q + CCR8 + Sca1 + cells (cells expressing C1q antigen, CCR8 antigen and Sca1 antigen on the cell surface)
(8) CD45 + CCR8 + Sca1 + cells (cells expressing CD45 antigen, CCR8 antigen and Sca1 antigen on the cell surface)
(9) CD45 + Sca1 + cells (cells expressing CD45 antigen and Sca1 antigen on the cell surface)
CD45 is a glycoprotein having a molecular weight of about 115 kD. Sca1 is a cell surface protein belonging to Ly (lymphocyte activation protein) -6 that binds to glucosylphosphatidylinositol. C1q is a type of complement. CCR8 means chemokine (CC) receptor 8 protein.
Antigens expressed on the surfaces of the above-mentioned various cells can be easily detected with an antibody against the antigen (which may be either a polyclonal antibody or a monoclonal antibody). Since antibodies against all antigens are commercially available, those skilled in the art can easily obtain them.
(被験者)
本発明の被験者は、臓器で線維化が生じる哺乳類であれば特に限定されないが、例えば、ヒト、マウス、ネコ、イヌ、ウマ、ウシ、ラット、ウサギ、フェレット、スンクス、ヒツジ、ロバ、ブタ等を例示することができる。
(subject)
The subject of the present invention is not particularly limited as long as it is a mammal in which fibrosis occurs in an organ. For example, humans, mice, cats, dogs, horses, cows, rats, rabbits, ferrets, sunks, sheep, donkeys, pigs, etc. It can be illustrated.
(試料)
本発明の試料は、被験者から採取され、かつ、CD45+細胞陽性細胞、C1q+細胞陽性細胞、CCR8+細胞及びSca1+細胞が存在していれば特に限定されないが、血液、リンパ液、髄液、骨髄液、唾液、尿、関節液、胸水、腹水、涙液、眼房水、硝子体液、鼻腔液、母乳、精液、前立腺液、膣液、膵液、胆汁、汗、膿、気管支洗浄液、生検サンプル(胃粘膜、大腸粘膜、気管支粘膜、皮膚、筋肉、腫瘍、リンパ節、子宮粘膜)、解剖検体(病理組織、法医学的組織)等を例示することができ、好ましくは、血液、リンパ液、髄液、唾液、気管支洗浄液、骨髄液、及び生検サンプルである。
(sample)
The sample of the present invention is not particularly limited as long as it is collected from a subject and CD45 + cell positive cells, C1q + cell positive cells, CCR8 + cells and Sca1 + cells are present, but blood, lymph, cerebrospinal fluid, Bone marrow fluid, saliva, urine, joint fluid, pleural effusion, ascites, tear fluid, aqueous humor, nasal fluid, breast milk, semen, prostate fluid, vaginal fluid, pancreatic fluid, bile, sweat, pus, bronchial lavage fluid, biopsy Samples (gastric mucosa, large intestine mucosa, bronchial mucosa, skin, muscle, tumor, lymph node, uterine mucosa), anatomical specimen (pathological tissue, forensic tissue), etc. can be exemplified, preferably blood, lymph fluid, marrow Fluid, saliva, bronchial lavage fluid, bone marrow fluid, and biopsy sample.
(各陽性細胞の検出方法)
本発明の線維化判定方法は、試料中の各陽性細胞を検出することができれば、特に限定されないが、好ましくはフローサイトメトリーで行うことができる。フローサイトメトリーを用いて各陽性細胞の蛍光発光を検出・測定する場合、各抗原を特異的に認識可能な各種抗体は、蛍光標識物質で標識した標識抗体として使用することが好ましい。本発明で用いる蛍光標識物質の種類は、フローサイトメトリーで検出できるものであれば特に限定されず、例えば、フィコエリスリン、FITC等を例示することができる。
(Method for detecting each positive cell)
The fibrosis determination method of the present invention is not particularly limited as long as each positive cell in a sample can be detected, but can be preferably performed by flow cytometry. When detecting and measuring the fluorescence emission of each positive cell using flow cytometry, various antibodies capable of specifically recognizing each antigen are preferably used as labeled antibodies labeled with a fluorescent labeling substance. The type of fluorescent labeling substance used in the present invention is not particularly limited as long as it can be detected by flow cytometry, and examples thereof include phycoerythrin and FITC.
(指標)
本発明の「指標(Cut off(カットオフ)値)」とは、線維化を有する患者と健常者を区別するための試料中の各陽性細胞数を意味する。例えば、被験者の試料中の各陽性細胞が、予め設定した試料中の各陽性細胞数以上の場合には、臓器で線維化が発症している、進行している、重篤である、及び/又は今後の発症の可能性が高いと判定することができる。Cut off(カットオフ)値の設定方法としては、線維化を有しない被験者の試料中の各陽性細胞数の平均値から算出する。通常、予め決定した線維化を有しない被験者の試料中の各陽性細胞数の平均値の標準偏差の90%以下、好ましくは80%以下、より好ましくは70%以下、さらに好ましくは60%以下、最も好ましくは50%以下の範囲の細胞数を指標とする。
また、別の指標の設定方法として、被験者の試料中の各陽性細胞数が、線維化を有しない被験者の試料中の各陽性細胞数の平均値に対して、105%以上、110%以上、120%以上、130%以上、140%以上、150%以上、180%以上、200%以上、250%以上、300%以上、400%以上、500%以上、600%以上、700%以上、800%以上、900%以上又は1000%以上の場合には、臓器で線維化が発症している、進行している、重篤である、及び/又は今後の発症の可能性が高いと判定することができる。
さらに、別のcut off値の設定方法としては、予め線維化を有していることを確認している患者及び線維化を有しない被験者において、試料中の各陽性細胞数を測定して得られた値に基づき、市販の統計解析ソフトを使用してROC(Receiver Operating Characteristic)曲線を作成し、最適な感度及び特異度を求める。例えば、一次スクリーニング等の目的では感度が高い方を優先し、精査目的では特異度が高くなるようなカットオフ値を設定することが可能である。
(index)
The “index (Cut off (cutoff) value)” of the present invention means the number of positive cells in a sample for distinguishing a patient having fibrosis from a healthy person. For example, if each positive cell in the subject's sample is greater than or equal to a preset number of positive cells in the sample, fibrosis has developed in the organ, is progressing, severe, and / or Or it can determine with the possibility of future onset being high. As a method for setting the cut off value, the cut off value is calculated from the average value of the number of positive cells in the sample of the subject without fibrosis. Usually, 90% or less, preferably 80% or less, more preferably 70% or less, more preferably 60% or less, of the standard deviation of the average value of the number of each positive cell in a sample of a subject having no predetermined fibrosis, Most preferably, the number of cells in the range of 50% or less is used as an index.
In addition, as another index setting method, the number of positive cells in the subject sample is 105% or more, 110% or more with respect to the average value of the number of positive cells in the subject sample without fibrosis, 120% or more, 130% or more, 140% or more, 150% or more, 180% or more, 200% or more, 250% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% As mentioned above, when it is 900% or more or 1000% or more, it may be determined that fibrosis has developed in the organ, has progressed, is severe, and / or has a high possibility of future onset. it can.
Furthermore, another cut-off value setting method is obtained by measuring the number of positive cells in a sample in a patient who has been confirmed to have fibrosis in advance and a subject who has no fibrosis. Based on the measured values, ROC (Receiver Operating Characteristic) curves are created using commercially available statistical analysis software, and optimum sensitivity and specificity are obtained. For example, it is possible to set a cut-off value that gives priority to a higher sensitivity for the purpose of primary screening or the like and has a higher specificity for the purpose of examination.
本発明の線維化判定方法では、試料中の各陽性細胞を検出することにより、心臓、腎臓、肺、肝臓、膵臓、皮膚、動脈、がん組織(スキルス胃がん、乳がん、膵臓がん、肝がん、肺がんなど)及び/又は、腹膜の線維化を判定することができるが、特に線維化の検出感度が高いのは、下記の実施例の結果より、心臓及び腎臓である。 In the fibrosis determination method of the present invention, heart, kidney, lung, liver, pancreas, skin, artery, cancer tissue (skills gastric cancer, breast cancer, pancreatic cancer, liver are detected by detecting each positive cell in the sample. , Lung cancer, etc.) and / or peritoneal fibrosis can be determined, but the detection sensitivity of fibrosis is particularly high in the heart and kidney from the results of the following examples.
(試料中の各陽性細胞の検出方法)
本発明の線維化判定方法における試料中の各陽性細胞の検出方法の工程を下記に例示するが特に限定されない。
(1)被験者から血液(全血)を取得する。
(2)取得した血液を緩衝液で希釈する。
(3)希釈した血液を、市販の蛍光したCD45抗体(APC-Cy7)、C1q抗体(APC)、CCR8抗体(PE)及びSca1+抗体(V450)を用いてフローサイトメトリーにより各陽性細胞の蛍光発光を検出・測定する。APC-Cy7、APC、PE、V450などの蛍光色素は、解析方法としてその他のものと置き換えることができる。
(4)測定した各陽性細胞数を、カットオフ値と比較して、線維化を判定する。
(Method for detecting each positive cell in the sample)
Although the process of the detection method of each positive cell in the sample in the fibrosis determination method of this invention is illustrated below, it is not specifically limited.
(1) Obtain blood (whole blood) from the subject.
(2) The obtained blood is diluted with a buffer solution.
(3) Fluorescence of each positive cell by flow cytometry using diluted CD45 antibody (APC-Cy7), C1q antibody (APC), CCR8 antibody (PE) and Sca1 + antibody (V450) Detect and measure luminescence. Fluorescent dyes such as APC-Cy7, APC, PE, and V450 can be replaced with other analysis methods.
(4) Fibrosis is determined by comparing each measured number of positive cells with a cutoff value.
(線維化判定キット)
本発明の線維化判定キットは、CD45+C1q+CCR8+細胞、CD45+C1q+細胞、C1q+CCR8+細胞、CD45+CCR8+細胞、CD45+C1q+CCR8+Sca1+細胞、CD45+C1q+Sca1+細胞、C1q+CCR8+Sca1+細胞、CD45+CCR8+Sca1+細胞、及び/又はCD45+Sca1+細胞を被験者から得られた試料中から検出するために必要な構成を含む。
例えば、本発明の線維化判定キットは、下記のようないずれか1以上の抗体を含む。
(1)抗CD45抗体
(2)抗C1q抗体
(3)抗CCR8抗体
(4)抗Sca1抗体
なお、上記すべての抗体は、自体公知の市販品でもよりが、各抗原を標的として作製したモノクローナル抗体又はポリクローナル抗体でも良い。
(Fibrosis determination kit)
The fibrosis determination kit of the present invention comprises CD45 + C1q + CCR8 + cells, CD45 + C1q + cells, C1q + CCR8 + cells, CD45 + CCR8 + cells, CD45 + C1q + CCR8 + Sca1 + cells, CD45 + C1q + Sca1 A configuration necessary for detecting + cells, C1q + CCR8 + Sca1 + cells, CD45 + CCR8 + Sca1 + cells, and / or CD45 + Sca1 + cells from a sample obtained from a subject.
For example, the fibrosis determination kit of the present invention includes any one or more of the following antibodies.
(1) Anti-CD45 antibody (2) Anti-C1q antibody (3) Anti-CCR8 antibody (4) Anti-Sca1 antibody All of the above-mentioned antibodies are monoclonal antibodies produced by targeting each antigen, even if they are commercially available products. Or a polyclonal antibody may be sufficient.
以下、実施例を挙げて本発明を詳細に説明するが、本発明の範囲はこれらの実施例により限定されるものではない。なお、本実施例は、金沢大学動物実験規程に従って行った。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited by these Examples. This example was performed according to the Kanazawa University animal experiment regulations.
(心線維化モデルの作成と心線維化の評価)
心臓の線維化が予測可能なバイオマーカーを新たに同定するために、心線維化が進行する臓器線維化モデルを作成し、さらにparabiosisモデル、骨髄移植モデルを使用し実験を行った。バイオマーカーの中でも心線維化の進行によりその数が変化する細胞種(群)を、抗体を使用した細胞表面マーカーを使って調べた。詳細は、以下の通りである。
(Creation of cardiac fibrosis model and evaluation of cardiac fibrosis)
In order to newly identify biomarkers that can predict cardiac fibrosis, an organ fibrosis model in which cardiac fibrosis progresses was created, and further experiments were performed using a parabiosis model and a bone marrow transplantation model. Among the biomarkers, cell types (groups) whose numbers change with the progression of cardiac fibrosis were examined using cell surface markers using antibodies. Details are as follows.
(臓器線維化モデルにおける心線維化の評価)
生後8週齢のC57BL/6Jマウス(B6 マウス)に、一側尿管結紮(Unilateral ureteralobstruction: UUO)および浸透圧ポンプを用いてアンジオテンシンII(AII)を2.8 mg/kg/dayにて持続静注したモデルを作成した(参照:図1)。対照群としてAII単独群、UUO単独群も作成した。第14日にと殺を行い、解析した。
tail-cuff法を用いて、各群の収縮期血圧を測定した。第14日において、AII+UUO群が最も血圧が高値であったが、AII群との間に差を認めなかった(参照:図2)。心臓のアザン染色像を示す(参照:図3)。線維化面積率および心体重比ともにAII+UUO群が最も大きく、線維化が進行していた(参照:図4)。
心組織よりRNAを抽出し、collagen type 1αおよびTGF-β1のmRNA発現を検討した(参照:図5)。
また、ハイドロキシプロリン法を行い、心線維化を半定量的に評価した。いずれにおいてもAII+UUO群にて最も上昇していた(参照:図6)。
経皮的心エコーを行い、心機能を評価した(参照:図7)。左にM mode像を示す。左室駆出率(Ejection Fraction: EF)はAII+UUO群にて有意に低下していた。
以上の結果により、心線維化モデルにおいて、AII+UUO群は、AII群と比較しても血圧非依存性に心線維化が進行した。これにより、心線維化が進行する臓器線維化モデルが作成できたことを確認した。以降はこのマウスを心線維化モデルとして使用した。
(Evaluation of cardiac fibrosis in organ fibrosis model)
Continuous intravenous infusion of angiotensin II (AII) at 2.8 mg / kg / day using unilateral ureteralobstruction (UUO) and osmotic pump in C57BL / 6J mice (B6 mice) 8 weeks old The model was created (see: FIG. 1). As a control group, AII single group and UUO single group were also prepared. On day 14, it was killed and analyzed.
The systolic blood pressure of each group was measured using the tail-cuff method. On day 14, the AII + UUO group had the highest blood pressure, but there was no difference from the AII group (see: FIG. 2). An Azan staining image of the heart is shown (see: FIG. 3). The AII + UUO group had the largest fibrosis area ratio and heart-weight ratio, and fibrosis was progressing (see: FIG. 4).
From cardiac tissue extracts RNA, it was examined mRNA expression of collagen type l [alpha] and TGF-beta 1 (see FIG. 5).
Moreover, the hydroxyproline method was performed and the cardiac fibrosis was evaluated semi-quantitatively. In any case, the increase was highest in the AII + UUO group (see FIG. 6).
Percutaneous echocardiography was performed to evaluate cardiac function (see: FIG. 7). The M mode image is shown on the left. Left ventricular ejection fraction (EF) was significantly reduced in the AII + UUO group.
Based on the above results, in the cardiac fibrosis model, cardiac fibrosis progressed in the AII + UUO group in a blood pressure-independent manner as compared with the AII group. This confirmed that an organ fibrosis model in which cardiac fibrosis progressed could be created. Thereafter, this mouse was used as a cardiac fibrosis model.
(parabiosisモデルにおける心線維化の評価)
B6マウスとC57BL/6-Tg (CAG-EGFP:全身のすべての細胞でenhanced GFPが発現する)マウスを用いてparabiosisモデルを作成した。麻酔導入後に、皮膚および腹膜を長軸方向に切開し、各々縫合した。キメラ完成後にB6マウス側にAII+UUOを作成した(参照:図8)。AII+UUO作成後、第7日及び第14日にと殺を行い、解析した。
心臓のアザン染色像を示す(参照:図9)。線維化面積率は、第7日においてAII+UUO群が最も大きく、線維化が進行しており、第14日にさらに進行した(参照:図10)。
以上により、parabiosisモデルにおいても、AII+UUOによって心線維化の進行を認めた。加えて、蛍光抗体法による免疫染色を行ったところ、心筋内にGFP陽性細胞が浸潤していた(参照:図11)。次に、マルチカラーフローサイトメトリー法にて、GFP陽性細胞のキャラクタリゼーションを行った(参照:図12)。GFP陽性細胞は、白血球のマーカーであるCD45とstem cellの代表的マーカーであるSca1の2つを用いて大きく3分類できた(参照:図13)。さらに、線維化の進行に伴ってCD45+Sca1-細胞とともにCD45+Sca1+細胞が増加した(参照:図14)。
(Evaluation of cardiac fibrosis in parabiosis model)
A parabiosis model was created using B6 mice and C57BL / 6-Tg (CAG-EGFP: enhanced GFP is expressed in all cells throughout the body). After induction of anesthesia, the skin and peritoneum were incised in the longitudinal direction and each sutured. After completion of the chimera, AII + UUO was prepared on the B6 mouse side (see: FIG. 8). After AII + UUO creation, the slaughter was performed on the 7th and 14th days and analyzed.
An Azan staining image of the heart is shown (see: FIG. 9). The fibrosis area rate was the largest in the AII + UUO group on the 7th day, and the fibrosis had progressed, and further progressed on the 14th day (see: FIG. 10).
As described above, in the parabiosis model, the progression of cardiac fibrosis was recognized by AII + UUO. In addition, immunostaining by the fluorescent antibody method revealed that GFP-positive cells infiltrated into the myocardium (see FIG. 11). Next, GFP positive cells were characterized by multicolor flow cytometry (see: FIG. 12). GFP-positive cells could be roughly classified into three types using two markers, CD45, which is a leukocyte marker, and Sca1, which is a typical stem cell marker (see FIG. 13). Further, with the progress of fibrosis CD45 + Sca1 - CD45 + Sca1 + cells increased with the cells (see Figure 14).
(骨髄移植モデルにおける心線維化の評価)
C57BL/6-Tg(CAG-EGFP)マウスの骨髄を、B6マウスに移植し、骨髄移植モデルを作成し、加えてAII+UUOを作成した(参照:図15)。AII+UUO作成後、第7日にと殺を行い、解析した。
心浸潤GFP細胞のキャラクターをparabiosisモデル同様にフローサイトメトリー法にて解析した(参照:図16)。骨髄移植モデルにおいてもGFP陽性細胞はCD45とSca1の2つを用いて3つに分類することできた(参照:図17)。また線維化の進行に伴ってCD45+Sca1-細胞とともにCD45+Sca1+細胞が増加した(参照:図18)。
以上により、本モデルでは、骨髄由来の細胞が流血を介して線維化部分に浸潤していくことを確認した。
(Evaluation of cardiac fibrosis in bone marrow transplantation model)
The bone marrow of C57BL / 6-Tg (CAG-EGFP) mice was transplanted into B6 mice, a bone marrow transplantation model was created, and AII + UUO was also created (see: FIG. 15). After making AII + UUO, killed on day 7 and analyzed.
Cardiac infiltrating GFP cell characters were analyzed by flow cytometry as in the parabiosis model (see FIG. 16). In the bone marrow transplantation model, GFP-positive cells could be classified into three using CD45 and Sca1 (see: FIG. 17). Also with the progress of fibrosis CD45 + Sca1 - CD45 + Sca1 + cells increased with the cells (see Figure 18).
As described above, in this model, it was confirmed that cells derived from bone marrow infiltrated into the fibrotic part via blood flow.
(CD45+Sca1+細胞の線維化誘導能の確認)
機能的にCD45+Sca1+細胞が線維化を誘導する細胞であるのかを解析するために、胚性線維芽細胞との共培養を行い、胚性線維芽細胞のコラーゲン1産生を定量評価することによる線維化誘導能を調べた。さらに、その後、CD45+Sca1+細胞はまだヘテロな集団からなる細胞群であり、線維化を誘導する細胞種をさらに絞り込むためにCD45+Sca1+細胞の遺伝子発現解析を行い線維化誘導に関与する候補遺伝子を調べた。その結果をもとに、フローサイトメトリー及び共培養にてCD45+Sca1+細胞に加え、さらに絞り込んだ細胞表面マーカーの同定と確認を行った。詳細は、以下の通りである。
(Confirmation of CD45 + Sca1 + cell fibrosis-inducing ability)
In order to analyze whether CD45 + Sca1 + cells are functionally fibrotic cells, co-culture with embryonic fibroblasts and quantitatively evaluate collagen 1 production in embryonic fibroblasts The fibrosis-inducing ability was investigated. In addition, CD45 + Sca1 + cells are still a heterogeneous group of cells that are involved in the induction of fibrosis through gene expression analysis of CD45 + Sca1 + cells to further narrow down the cell types that induce fibrosis Candidate genes were examined. Based on the results, in addition to CD45 + Sca1 + cells, further narrowed cell surface markers were identified and confirmed by flow cytometry and co-culture. Details are as follows.
(CD45+Sca1+細胞と胚性線維芽細胞の共培養を用いた線維化誘導能の評価)
COL/LUC-Tg (コラーゲン1α2のプロモーターの下流にルシフェラーゼ遺伝子を融合した遺伝子を過剰発現したマウス)マウスより、胚性線維芽細胞(mouse embryonic fibroblast: MEF)を採取し、フローサイトメトリーを用いたソーティングにて得られたCD45+Sca1+細胞と共培養を行った(参照:図19、図20)。48時間後にルシフェラーゼアッセイにて線維化を引き起こすcollagen1α2の発現を評価した。
CD45+Sca1+細胞は、CD45-Sca1+細胞と比較して、線維化誘導能を有することを確認した(参照:図21)。TGF-βは陽性コントロールとして使用した。
(Evaluation of fibrosis-inducing ability using co-culture of CD45 + Sca1 + cells and embryonic fibroblasts)
Mouse embryonic fibroblast (MEF) was collected from COL / LUC-Tg (a mouse overexpressing a gene with a luciferase gene fused downstream of the collagen 1α2 promoter), and flow cytometry was used. Co-culture with CD45 + Sca1 + cells obtained by sorting was performed (see: FIGS. 19 and 20). 48 hours later, the expression of collagen1α2 causing fibrosis was evaluated by luciferase assay.
CD45 + Sca1 + cells were confirmed to have fibrosis-inducing ability compared to CD45 − Sca1 + cells (see: FIG. 21). TGF-β was used as a positive control.
(CD45+Sca1+細胞と胚性線維芽細胞の共培養を用いた表面マーカーの検討)
CD45+Sca1+細胞よりRNAを抽出し、genetipによる発現解析をCD45-Sca1+細胞、CD45+Sca1-細胞と比較し、同細胞のキャラクターを評価した。約4万個の遺伝子の中から、31個の候補遺伝子を抽出した(参照:図22)。
抽出した候補遺伝子の中から細胞表面に発現するもので、表面マーカーとして使用可能なものを選定し、CD45+Sca1+というカテゴリーに加え、さらなる表面マーカーの追加を行った。CD45+Sca1+X+ 細胞(Xは、図25中の13種類)およびCD45+Sca1+X-細胞をセルソーターを使用してソーティングした。上記と同様にMEFと共培養を行い、48時間後のルシフェラーゼアッセイにて線維化誘導能を評価した(参照:図23、図24)。その結果、CD45+Sca1+X+細胞は、CD45+Sca1+X-細胞と比較して、強い線維化誘導能を有している細胞群は存在しなかった(参照:図25)。しかしながら、C1q抗体陽性でソーティングした細胞は常に線維化誘導能がコントロールレベルまで完全に抑制されている事実が判明した。
(Examination of surface markers using co-culture of CD45 + Sca1 + cells and embryonic fibroblasts)
RNA was extracted from CD45 + Sca1 + cells, and expression analysis using genetip was compared with CD45 − Sca1 + cells and CD45 + Sca1 − cells, and the characters of the cells were evaluated. Thirty-one candidate genes were extracted from about 40,000 genes (see FIG. 22).
Among the extracted candidate genes, those that are expressed on the cell surface and that can be used as surface markers were selected, and in addition to the category of CD45 + Sca1 + , additional surface markers were added. CD45 + Sca1 + X + cells (X is 13 types in FIG. 25) and CD45 + Sca1 + X − cells were sorted using a cell sorter. Co-culture with MEF was performed in the same manner as described above, and the fibrosis-inducing ability was evaluated by luciferase assay 48 hours later (see: FIGS. 23 and 24). As a result, there was no cell group in which CD45 + Sca1 + X + cells had a strong fibrosis-inducing ability compared to CD45 + Sca1 + X − cells (see FIG. 25). However, it was found that C1q antibody-positive cells sorted always had fibrosis induction ability completely suppressed to the control level.
(CD45+Sca1+細胞と胚性線維芽細胞の共培養を用いた抗C1q抗体投与の影響の検討)
上記の結果をさらに検証するために以下の実験を行った。
ソーティングして回収したCD45+Sca1+細胞とCOL/LUC-Tg 由来MEFを共培養し、そこに抗C1q抗体の濃度を振って添加し、48時間後のルシフェラーゼアッセイにて線維化誘導能を評価した(参照:図26、図27)。
抗C1q抗体の投与により、CD45+Sca1+細胞のcollagen1α2の発現誘導が濃度依存的に抑制された(参照:図28)。TGF-βは陽性コントロールとして使用した。
(Examination of the effects of anti-C1q antibody administration using coculture of CD45 + Sca1 + cells and embryonic fibroblasts)
The following experiment was conducted to further verify the above results.
CD45 + Sca1 + cells collected by sorting and MEF derived from COL / LUC-Tg were co-cultured, added with anti-C1q antibody at different concentrations, and assessed for fibrillation induction ability by luciferase assay 48 hours later (Reference: FIGS. 26 and 27).
Administration of anti-C1q antibody suppressed the expression of collagen1α2 expression in CD45 + Sca1 + cells in a concentration-dependent manner (see FIG. 28). TGF-β was used as a positive control.
(CD45+Sca1+C1q+細胞の表面マーカーの検討)
以上の結果から、骨髄由来のCD45+Sca1+C1q+細胞が線維化を誘導する細胞であると判断し、さらに、その性質について検討を行った。
野生型のC57BL/6JマウスにCAG-EGFPマウスの骨髄を移植した骨髄移植モデルを作成し、マルチカラーフローサイトメトリー法にて検討を行った(参照:図29)。
CD45+Sca1+C1q+細胞には、さらに、CCR8が発現していることを確認した(参照:図30、31、32)。
(Examination of CD45 + Sca1 + C1q + cell surface markers)
Based on the above results, it was determined that CD45 + Sca1 + C1q + cells derived from bone marrow were fibrosis-inducing cells, and their properties were examined.
A bone marrow transplantation model in which bone marrow of CAG-EGFP mice was transplanted into wild-type C57BL / 6J mice was prepared and examined by a multicolor flow cytometry method (see FIG. 29).
It was further confirmed that CCR8 was expressed in CD45 + Sca1 + C1q + cells (see: FIGS. 30, 31, and 32).
(CD45+Sca1+C1q+CCR8+細胞の表面マーカーの検討)
CD45+Sca1+C1q+CCR8+細胞に着目し、その発現を腎臓、脾臓、骨髄および血液で検討した。前記骨髄移植モデルを用いて、マルチカラーフローサイトメトリー法にて検討を行った(参照:図33)。
同細胞は、腎臓および血液においても検出できた(参照:図34)。
(Examination of CD45 + Sca1 + C1q + CCR8 + cell surface markers)
Focusing on CD45 + Sca1 + C1q + CCR8 + cells, its expression was examined in kidney, spleen, bone marrow and blood. Using the bone marrow transplantation model, examination was performed by a multicolor flow cytometry method (see FIG. 33).
The cells could also be detected in kidney and blood (see: FIG. 34).
(CD45+Sca1+C1q+CCR8+細胞の臓器線維化のマーカーとしての評価)
前記臓器線維化モデル(参照:図35)を使用して、血液中のCD45+Sca1+C1q+CCR8+細胞が、臓器線維化のバイオマーカーとなりうるかについて検討した。心臓より採血を行い、フローサイトメトリー法を用いて解析した。
CD45+Sca1+C1q+CCR8+細胞は、線維化の進行に伴い有意に増加しており、臓器線維化のマーカーとなることを確認した(参照:図36)。
(Evaluation as a marker of organ fibrosis of CD45 + Sca1 + C1q + CCR8 + cells)
Using the organ fibrosis model (see FIG. 35), it was examined whether CD45 + Sca1 + C1q + CCR8 + cells in blood could be biomarkers of organ fibrosis. Blood was collected from the heart and analyzed using flow cytometry.
CD45 + Sca1 + C1q + CCR8 + cells were significantly increased as fibrosis progressed, confirming that they became markers of organ fibrosis (see: FIG. 36).
(ヒトにおける線維化マーカーの確認)
ヒトにおいてもCD45+Sca1+C1q+CCR8+細胞が検出可能かを評価した。なお、Sca1は、ヒトには存在しないため、CD45+C1q+ CCR8 + 細胞で検討した。
(Confirmation of fibrosis markers in humans)
Whether human could detect CD45 + Sca1 + C1q + CCR8 + cells was also evaluated. Since Sca1 does not exist in humans, it was examined using CD45 + C1q + CCR8 + cells.
(ヒトにおける線維化評価)
維持透析症例及び対照健常者より得た血液試料を用いて、フローサイトメトリー法にて検討を行った。
健常者においてもCD45+C1q+ CCR8 + 細胞は、検出可能であり、腎不全状態にて増加した(参照:図37)。透析症例は、腎線維化および心線維化が進行した症例群であり、ヒトにおいてもCD45+C1q+CCR8+細胞が、臓器線維化のマーカーとなることを確認した(参照:図37)。
(Evaluation of fibrosis in humans)
Using blood samples obtained from maintenance dialysis cases and healthy controls, flow cytometry was used.
CD45 + C1q + CCR8 + cells were detectable even in healthy subjects and increased in the renal failure state (see: FIG. 37). Dialysis cases are a group of cases in which renal fibrosis and cardiac fibrosis have progressed, and it was confirmed that CD45 + C1q + CCR8 + cells also serve as markers of organ fibrosis in humans (see FIG. 37).
(総論)
以上の実施例1〜3の結果により、以下の各陽性細胞を被験者の試料中から検出することにより、臓器の線維化を判定することができる。
(1)CD45+C1q+CCR8+細胞
(2)CD45+C1q+細胞
(3)C1q+CCR8+細胞
(4)CD45+CCR8+細胞
(5)CD45+C1q+CCR8+Sca1+細胞
(6)CD45+C1q+Sca1+細胞
(7)C1q+CCR8+Sca1+細胞
(8)CD45+CCR8+Sca1+細胞
(9)CD45+Sca1+細胞
(General)
From the results of Examples 1 to 3 described above, organ fibrosis can be determined by detecting each of the following positive cells from the sample of the subject.
(1) CD45 + C1q + CCR8 + cells (2) CD45 + C1q + cells (3) C1q + CCR8 + cells (4) CD45 + CCR8 + cells (5) CD45 + C1q + CCR8 + Sca1 + cells (6) CD45 + C1q + Sca1 + cells (7) C1q + CCR8 + Sca1 + cells (8) CD45 + CCR8 + Sca1 + cells (9) CD45 + Sca1 + cells
本発明では、新規な線維化判定方法を提供できる。 In the present invention, a novel fibrosis determination method can be provided.
Claims (5)
(1)CD45+C1q+CCR8+細胞
(2)CD45+C1q+細胞
(3)C1q+CCR8+細胞
(4)CD45+CCR8+細胞
A fibrosis determination method comprising detecting any one or more of the following cells from a sample obtained from a subject.
(1) CD45 + C1q + CCR8 + cells (2) CD45 + C1q + cells (3) C1q + CCR8 + cells (4) CD45 + CCR8 + cells
(1)CD45+C1q+CCR8+Sca1+細胞
(2)CD45+C1q+Sca1+細胞
(3)C1q+CCR8+Sca1+細胞
(4)CD45+CCR8+Sca1+細胞
(5)CD45+Sca1+細胞
The fibrosis determination method according to claim 1, wherein the following cells are detected.
(1) CD45 + C1q + CCR8 + Sca1 + cells (2) CD45 + C1q + Sca1 + cells (3) C1q + CCR8 + Sca1 + cells (4) CD45 + CCR8 + Sca1 + cells (5) CD45 + Sca1 + cells
When the number of cells in the sample obtained from the subject is higher than the number of cells in the sample obtained from a healthy subject, it is determined that the cells are fibrotic. 2. The fibrosis determination method according to 2.
(1)心臓
(2)腎臓
(3)肺
(4)肝臓
(5)膵臓
(6)皮膚
(7)動脈
(8)腹膜
(9)がん組織
The fibrosis determination method according to claim 1, wherein the fibrosis is fibrosis of the following organ.
(1) Heart (2) Kidney (3) Lung (4) Liver (5) Pancreas (6) Skin (7) Artery (8) Peritoneum (9) Cancer tissue
(1)抗CD45抗体
(2)抗C1q抗体
(3)抗CCR8抗体
(4)抗Sca1抗体 A fibrosis determination kit comprising one or more antibodies from any of the following.
(1) Anti-CD45 antibody (2) Anti-C1q antibody (3) Anti-CCR8 antibody (4) Anti-Sca1 antibody
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