JP6539511B2 - Marker for judgment of motor function - Google Patents
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- JP6539511B2 JP6539511B2 JP2015122455A JP2015122455A JP6539511B2 JP 6539511 B2 JP6539511 B2 JP 6539511B2 JP 2015122455 A JP2015122455 A JP 2015122455A JP 2015122455 A JP2015122455 A JP 2015122455A JP 6539511 B2 JP6539511 B2 JP 6539511B2
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Description
本発明は、運動機能の判定用マーカー、筋量判定用マーカー、骨格筋細胞の分化の判定用マーカー、又は筋管肥大の判定用マーカーに関する。 The present invention relates to a marker for determination of motor function, a marker for determination of muscle mass, a marker for determination of differentiation of skeletal muscle cells, or a marker for determination of myotube hypertrophy.
ロコモティブシンドロームは、加齢などが原因とされる運動器の疾患をいう。ロコモティブシンドロームの有病率は加齢に伴い上昇し、ロコモティブシンドロームが進行すると、要介護になるリスクが大きく高まる。
これまでに、ロコモティブシンドロームなどの運動器の加齢性疾患に関連する遺伝子やタンパク質の研究が進んでいる。しかし、これまでに運動器の疾患に関連する因子の特定には至っていない。
Locomotive syndrome refers to a musculoskeletal disorder caused by aging and the like. The prevalence of locomotive syndrome increases with age, and as locomotive syndrome progresses, the risk of needing nursing care is greatly increased.
So far, studies on genes and proteins related to age-related diseases of exercise organs such as locomotive syndrome have been advanced. However, to date no factor has been identified that is related to motor disease.
一方ミオシン結合タンパク質H(以下、単に「MYBPH」ともいう)については、ヒト肺腺癌細胞株においてMYBPHをノックダウンすると細胞遊走が促進されること、及びイヌ腎臓尿細管上皮由来細胞株においてMYBPHを過剰発現させると細胞遊走及び浸潤が抑制されることが知られている(非特許文献1参照)。また、ラット心臓由来細胞株においてMYBPHとミオシン結合タンパク質Cをダブルノックダウンすると、アドレナリン刺激による細胞収縮が抑制されることが知られている(非特許文献2参照)。さらに、医薬品などの投与による副作用(筋肉毒性)により生じる骨格筋障害を検出するためのタンパク質バイオマーカーとしてMYBPHを使用することも開示されている(特許文献1参照)。
しかし、日常生活に必要な運動機能や運動器の加齢性疾患を判定、評価するためのバイオマーカーとしてMYBPHを使用できることについては知られていない。
On the other hand, with regard to myosin binding protein H (hereinafter also referred to simply as "MYBPH"), knocking down MYBPH in human lung adenocarcinoma cell lines promotes cell migration, and MYBPH in canine renal tubular epithelium-derived cell lines It is known that overexpression suppresses cell migration and invasion (see Non-patent Document 1). In addition, it is known that double knockdown of MYBPH and myosin binding protein C in a rat heart-derived cell line suppresses cell contraction due to adrenergic stimulation (see non-patent document 2). Furthermore, the use of MYBPH as a protein biomarker for detecting skeletal muscle damage caused by side effects (myotoxicity) by administration of pharmaceuticals and the like is also disclosed (see Patent Document 1).
However, it is not known that MYBPH can be used as a biomarker for determining and evaluating age-related diseases of motor function and exercise equipment necessary for daily life.
本発明は、個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価するためのバイオマーカーの提供を課題とする。
また本発明は、前記バイオマーカーを用いた、運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度の評価方法の提供を課題とする。
An object of the present invention is to provide a biomarker for evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy in an individual.
Another object of the present invention is to provide a method for evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy using the above-mentioned biomarkers.
本発明者等は上記課題に鑑み鋭意検討を行った。
運動器官の1つであり身体を動かす骨格に結合する骨格筋には、多くのタンパク質が発現することが知られている。これらのタンパク質のうち、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子(以下、「MYBPH遺伝子」ともいう)の発現レベルが、個体の運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大との間で相関性を有することを見出した。そして、このMYBPH又はMYBPH遺伝子が、個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価するためのバイオマーカーとして有用であることを見出した。さらに、個体から採取されたMYBPH又はMYBPH遺伝子の発現レベルを測定し、測定した発現レベルに基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大の程度を評価できることを見出した。
本発明はこれらの知見に基づいて完成されたものである。
The present inventors diligently studied in view of the above problems.
It is known that many proteins are expressed in skeletal muscle which is one of the movement organs and is connected to a skeleton that moves the body. Among these proteins, the expression level of skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same (hereinafter, also referred to as "MYBPH gene") indicates the motor function of the individual, muscle mass, differentiation of skeletal muscle cells It was found that there is a correlation between the degree of hypertrophy and myotube hypertrophy. And, it was found that this MYBPH or MYBPH gene is useful as a biomarker for evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy in an individual. Furthermore, the expression level of the MYBPH or MYBPH gene collected from the individual can be measured, and the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy can be evaluated based on the measured expression level. Found out.
The present invention has been completed based on these findings.
本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子を含む、運動機能の評価用マーカーに関する。
また本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子を含む、筋量の評価用マーカーに関する。
また本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子を含む、骨格筋細胞の分化評価用マーカーに関する。
また本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子を含む、筋管肥大の評価用マーカーに関する。
The present invention relates to a marker for evaluating motor function, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
The present invention also relates to a marker for evaluating muscle mass, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
The present invention also relates to a marker for evaluating differentiation of skeletal muscle cells, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
The present invention also relates to a marker for evaluating myotube hypertrophy, which comprises skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same.
また本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子の発現レベルを測定し、測定した発現レベルに基づいて個体の運動機能を評価する、運動機能の評価方法に関する。
また本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子の発現レベルを測定し、測定した発現レベルに基づいて筋量を評価する、筋量の評価方法に関する。
また本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子の発現レベルを測定し、測定した発現レベルに基づいて骨格筋細胞の分化の程度を評価する、骨格筋細胞の分化の評価方法に関する。
さらに本発明は、個体から採取された骨格筋由来のMYBPH又はこれをコードする遺伝子の発現レベルを測定し、測定した発現レベルに基づいて筋管肥大の程度を評価する、筋管肥大の評価方法に関する。
The present invention also relates to a method of evaluating motor function, which measures the expression level of skeletal muscle-derived MYBPH or a gene encoding the same collected from an individual, and evaluates the motor function of the individual based on the measured expression level.
The present invention also relates to a method for evaluating muscle mass, which measures the expression level of skeletal muscle-derived MYBPH or a gene encoding the same collected from an individual, and evaluates the muscle mass based on the measured expression level.
Furthermore, the present invention measures skeletal muscle-derived MYBPH collected from an individual or the expression level of a gene encoding the same, and evaluates the degree of differentiation of skeletal muscle cells based on the measured expression level. It relates to a method of evaluating differentiation.
Furthermore, the present invention measures the expression level of skeletal muscle-derived MYBPH collected from an individual or a gene encoding the same, and evaluates the degree of myotube hypertrophy on the basis of the measured expression level. About.
本発明のバイオマーカーは、個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度の評価に使用することができる。
また本発明の評価方法は、運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を的確かつ簡便に評価することができる。さらに本発明の評価方法に基づいて、運動機能を向上させる物質、筋量を増加させる物質、骨格筋細胞の分化を促進させる物質、又は筋管肥大を促進させる物質のスクリーニングなどに好適に適用することができる。
The biomarkers of the invention can be used to assess the individual's motor function, muscle mass, degree of differentiation of skeletal muscle cells, or degree of myotube hypertrophy.
The evaluation method of the present invention can accurately and simply evaluate the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy. Further, based on the evaluation method of the present invention, the present invention is suitably applied to screening of substances that improve motor function, substances that increase muscle mass, substances that promote differentiation of skeletal muscle cells, or substances that promote myotube hypertrophy be able to.
本明細書において、データ又は測定値等に関する「評価」又は「判定」とは、あるサンプル(群)から取得したデータ又は測定値を所定の目的に応じて分析し、分析した情報に基づいて当該サンプル(群)が特定の性状、動向、傾向を有するか否かを結論づけることを意味する。
また本発明において「運動機能」とは、身体を構成し、支え、身体運動を可能にする運動器官の運動能力を指す。
In the present specification, "evaluation" or "determination" regarding data or measured values etc. refers to analysis of data or measured values obtained from a certain sample (group) according to a predetermined purpose, and based on the analyzed information. It is meant to conclude whether the sample (s) have specific characteristics, trends, trends.
Further, in the present invention, the term "motor function" refers to the exercise ability of a motion organ that constitutes, supports and enables physical exercise.
本発明の運動機能の評価用マーカー、筋量の評価用マーカー、骨格筋細胞の分化評価用マーカー、及び筋管肥大の評価用マーカー(以下、これらをまとめて単に「本発明のマーカー」ともいう)は、個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子を含む。 The marker for evaluating motor function, the marker for evaluating muscle mass, the marker for evaluating differentiation of skeletal muscle cells, and the marker for evaluating myotube hypertrophy according to the present invention (hereinafter collectively referred to simply as "the marker of the present invention" 2.) contains skeletal muscle-derived MYBPH or MYBPH gene collected from an individual.
後述の実施例で示すように、本発明のマーカーに含まれる、個体から採取された骨格筋由来のMYBPH及びMYBPH遺伝子の発現レベルは、個体の運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大との間で相関性を有する。そして、運動機能の向上、筋量の増加、骨格筋細胞の分化の進行、及び筋管肥大に伴い、MYBPH及びMYBPH遺伝子の発現レベルが有意に上昇することを本発明者らは見出した。
したがって、本発明のマーカーは、運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大の程度の指標とすることができる。さらに本発明のマーカーは、運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大の程度を簡便かつ的確に評価する方法に好適に用いることができる。
As shown in Examples described later, the expression levels of skeletal muscle-derived MYBPH and MYBPH genes contained in the marker of the present invention are the motor function of the individual, muscle mass, and the degree of differentiation of skeletal muscle cells. And myotube hypertrophy have correlation. The present inventors found that the expression levels of MYBPH and MYBPH genes are significantly increased with the improvement of motor function, the increase of muscle mass, the progress of differentiation of skeletal muscle cells, and myotube hypertrophy.
Therefore, the marker of the present invention can be used as an indicator of motor function, muscle mass, degree of differentiation of skeletal muscle cells, and degree of myotube hypertrophy. Furthermore, the marker of the present invention can be suitably used in a method for simply and accurately evaluating the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.
なお、特許文献1には、MYBPHから構成される、医薬品などの投与による副作用(筋肉毒性)により生じる骨格筋の障害を検出するためのバイオマーカーが記載されている。これに対して本発明のマーカーは、日常生活に必要な運動機能や運動器の加齢性疾患を判定、評価するためのバイオマーカーである。このように、本発明において判定又は評価の対象が、特許文献1に記載の発明とは全く異なるものである。 Patent Document 1 describes a biomarker composed of MYBPH for detecting a skeletal muscle disorder caused by a side effect (muscle toxicity) by administration of a drug or the like. On the other hand, the marker of the present invention is a biomarker for determining and evaluating age-related diseases of motor function and exercise equipment necessary for daily life. Thus, the object of determination or evaluation in the present invention is completely different from the invention described in Patent Document 1.
前記MYBPHのアミノ酸配列の情報は、各種データベースから入手可能である。例えば、マウス由来のMYBPHのアミノ酸配列はNCBI RefSeqにID:NP_058029として、ゼブラフィッシュ由来のMYBPH(MYBPHa及びMYBPHb)のアミノ酸配列はNCBI RefSeqにID:NP_956852.1及びNM_001100137.1として(http://www.ncbi.nlm.nih.gov/gene/393530、http://www.ncbi.nlm.nih.gov/gene/321053参照)、ヒト由来のMYBPHのアミノ酸配列はNCBI RefSeqにID:NP_004988として、それぞれ登録されている。
なお本発明で用いるMYBPHはこれらに限定するものではなく、これらのアミノ酸配列において、1又は数個、通常1〜100個、好ましくは1〜75個、より好ましくは1〜50個、さらに好ましくは1〜25個、特に好ましくは1〜10個、最も好ましくは1〜5個、のアミノ酸が欠失、置換、挿入及び/又は付加されたアミノ酸配列からなり、かつ機能的に同等なタンパク質も含まれる。また、これらのアミノ酸配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有するアミノ酸配列からなり、かつ機能的に同等なタンパク質も含まれる。ここで本明細書においてアミノ酸配列の相同性は、Lipman-Pearson法(Science,227,1435,(1985))によって計算される。具体的には、遺伝情報処理ソフトウェアGenetyx-Win(ソフトウェア開発)のホモロジー解析(Search homology)プログラムを用いて、Unit size to compare(ktup)を2として解析を行うことにより算出される。
Information on the amino acid sequence of MYBPH is available from various databases. For example, the amino acid sequence of mouse-derived MYBPH is NCBI RefSeq as ID: NP_058029, the amino acid sequence of zebrafish-derived MYBPH (MYBPHa and MYBPHb) as NCBI RefSeq as ID: NP_956852.1 and NM_001100137.1 (http: // www.ncbi.nlm.nih.gov/gene/393530 (see http://www.ncbi.nlm.nih.gov/gene/321053), the amino acid sequence of human-derived MYBPH is NCBI RefSeq as ID: NP_004988, Each is registered.
The MYBPH used in the present invention is not limited to these, and in these amino acid sequences, one or several, usually 1 to 100, preferably 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids consist of amino acid sequences with deletion, substitution, insertion and / or addition, and functionally equivalent proteins are also included Be In addition, it has homology of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, most preferably 99% or more to these amino acid sequences. Also included are proteins that consist of amino acid sequences and that are functionally equivalent. Here, the homology of amino acid sequences herein is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with Unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).
本発明のマーカーは、前述のMYBPHのアミノ酸配列の一部を有するポリペプチドからなり、MYBPHの発現レベルの情報を取得するための抗体又はペプチドアプタマーにより特異的に認識される長さのポリペプチドであることが好ましい。ここで「MYBPHのアミノ酸配列の一部を有するポリペプチド」とは、前述のMYBPHのアミノ酸配列の一部、好ましくは5個以上、より好ましくは7個以上、さらに好ましくは10個以上、特に好ましくは12個以上、好ましくは40個以下、より好ましくは30個以下、さらに好ましくは20個以下、特に好ましくは15個以下、を連続して有するポリペプチドを意味する。 The marker of the present invention consists of a polypeptide having a part of the amino acid sequence of MYBPH as described above, and is a polypeptide of a length specifically recognized by an antibody or peptide aptamer for obtaining information on the expression level of MYBPH. Is preferred. Here, “a polypeptide having a portion of the amino acid sequence of MYBPH” is a portion of the amino acid sequence of MYBPH described above, preferably 5 or more, more preferably 7 or more, still more preferably 10 or more, particularly preferably Means a polypeptide having 12 or more, preferably 40 or less, more preferably 30 or less, still more preferably 20 or less, particularly preferably 15 or less.
前記MYBPH遺伝子の塩基配列の情報は、各種データベースから入手可能である。例えば、マウス由来のMYBPH遺伝子の塩基配列はNCBI RefSeqにID:NM_016749として、ゼブラフィッシュ由来のMYBPH遺伝子(MYBPHa遺伝子及びMYBPHb遺伝子)の塩基配列はNCBI RefSeqにID:NM_200558.1及びNP_001093607.1として(http://www.ncbi.nlm.nih.gov/gene/393530、http://www.ncbi.nlm.nih.gov/gene/321053参照)、ヒト由来のMYBPHの塩基配列はNCBI RefSeqにID:NM_004997として、それぞれ登録されている。
なお本発明で用いるMYBPH遺伝子はこれらに限定するものではなく、これらの塩基配列において、1又は数個、通常1〜389個、好ましくは1〜292個、より好ましくは1〜195個、さらに好ましくは1〜98個、特に好ましくは1〜39個、最も好ましくは1〜20個、の塩基が欠失、置換、挿入又は付加された塩基配列からなり、かつ前述のMYBPHをコードする核酸も含まれる。また、これらの塩基配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有する塩基配列からなり、かつ前述のMYBPHをコードする核酸も含まれる。ここで本明細書において塩基配列の相同性は、Lipman-Pearson法(Science,227,1435,(1985))によって計算される。具体的には、遺伝情報処理ソフトウェアGenetyx-Win(ソフトウェア開発)のホモロジー解析(Search homology)プログラムを用いて、Unit size to compare(ktup)を2として解析を行うことにより算出される。
また本発明において、MYBPH遺伝子はDNA及びRNAのいずれであってもよく、MYBPH遺伝子そのもの(DNA)、mRNA、cDNA、及びcRNAのいずれであってもよい。
Information on the nucleotide sequence of the MYBPH gene is available from various databases. For example, the nucleotide sequence of mouse-derived MYBPH gene is NCBI RefSeq as ID: NM_016749, the nucleotide sequence of zebrafish-derived MYBPH gene (MYBPHa gene and MYBPHb gene) is NCBI RefSeq as ID: NM_200558.1 and NP_001093607.1 ( Refer to http://www.ncbi.nlm.nih.gov/gene/339530, http://www.ncbi.nlm.nih.gov/gene/321053), and the nucleotide sequence of human-derived MYBPH as the ID of NCBI RefSeq. : Registered as NM_004997, respectively.
The MYBPH gene used in the present invention is not limited to these, and in these base sequences, one or several, usually 1 to 389, preferably 1 to 292, more preferably 1 to 195, more preferably Is composed of a nucleotide sequence having 1 to 98, particularly preferably 1 to 39, and most preferably 1 to 20, deletions, substitutions, insertions or additions of bases, and also includes the above-mentioned nucleic acid encoding MYBPH Be In addition, they have homology of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, most preferably 99% or more to these base sequences. Also included are nucleic acids consisting of base sequences and encoding the aforementioned MYBPH. Here, the homology of the nucleotide sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with Unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).
In the present invention, the MYBPH gene may be either DNA or RNA, and may be any of MYBPH gene itself (DNA), mRNA, cDNA, and cRNA.
本発明のマーカーは、前述のMYBPH遺伝子の塩基配列の一部を有するポリオリゴヌクレオチドからなり、MYBPH遺伝子の発現レベルの情報を取得するための核酸プローブや核酸アプタマー、核酸プライマーとストリンジェントな条件でハイブリダイズ可能な長さのポリヌクレオチドであることが好ましい。ここで「ストリンジェントな条件」としては、例えばMolecular Cloning−A LABORATORY MANUAL THIRD EDITION[Joseph Sambrook,David W.Russell.,Cold Spring Harbor Laboratory Press]記載の方法が挙げられ、例えば、6×SSC(1×SSCの組成:0.15M塩化ナトリウム、0.015Mクエン酸ナトリウム、pH7.0)、0.5%SDS、5×デンハート及び100mg/mLニシン精子DNAを含む溶液にプローブとともに65℃で8〜16時間恒温し、ハイブリダイズさせる条件が挙げられる。 The marker of the present invention is composed of a polyoligonucleotide having a part of the base sequence of the MYBPH gene described above, and is under stringent conditions with nucleic acid probes and nucleic acid aptamers for obtaining information on the expression level of the MYBPH gene It is preferred that the polynucleotide is a hybridizable length. Here, as "stringent conditions", for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. et al. Russell, Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% Conditions in which a solution containing SDS, 5 × Denhardt and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized are mentioned.
本発明の運動機能の評価方法、筋量の評価方法、骨格筋細胞の分化の評価方法及び筋管肥大の評価方法(以下、これらをまとめて「本発明の評価方法」ともいう)は、本発明のマーカーを用いて個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子の発現レベルを測定する。そして、測定したMYBPH又はMYBPH遺伝子の発現レベルに基づいて、個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価する。 The method of evaluating motor function, the method of evaluating muscle mass, the method of evaluating differentiation of skeletal muscle cells, and the method of evaluating myotube hypertrophy according to the present invention (hereinafter collectively referred to as "the evaluation method of the present invention") The expression level of skeletal muscle-derived MYBPH or MYBPH gene collected from an individual is measured using the marker of the invention. Then, based on the measured expression level of the MYBPH or MYBPH gene, the motor function of the individual, the muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy is evaluated.
本発明の評価方法においてまず、動物、好ましくはヒトや非ヒト動物、より好ましくは、ヒトや実験動物(マウス、ラット、モルモット、ハムスター、ウサギ、ゼブラフィッシュなど)、から採取した生体試料や、人工的に培養した細胞株から、MYBPH又はMYBPH遺伝子を検出する。MYBPH又はMYBPH遺伝子を検出するための生体試料に特に制限はないが、骨格筋、好ましくはヒラメ筋、ヒフク筋又は足底筋、からMYBPH又はMYBPH遺伝子を採取する。 In the evaluation method of the present invention, first, a biological sample collected from an animal, preferably a human or non-human animal, more preferably a human or an experimental animal (mouse, rat, guinea pig, hamster, rabbit, zebrafish, etc.) The MYBPH or MYBPH gene is detected from cell lines cultured in culture. There is no particular limitation on the biological sample for detecting the MYBPH or MYBPH gene, but the MYBPH or MYBPH gene is collected from skeletal muscle, preferably soleus muscle, hip muscle or plantar muscle.
MYBPH又はMYBPH遺伝子を個体から採取するタイミングは、適宜選択することができる。例えば、運動など筋量を増加させる外部刺激を個体に負荷してから1〜6時間後にMYBPH又はMYBPH遺伝子を採取することが好ましい。 The timing at which the MYBPH or MYBPH gene is collected from the individual can be selected appropriately. For example, it is preferable to collect the MYBPH or MYBPH gene 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise.
このように採取したMYBPH又はMYBPH遺伝子について、その発現レベルを測定する。発現レベルを測定する方法としては、MYBPH又はMYBPH遺伝子の発現を定性的又は定量的、すなわちMYBPH又はMYBPH遺伝子の発現の有無や発現量、を測定する常法より適宜選択することができる。 The expression level of the MYBPH or MYBPH gene thus collected is measured. As the method of measuring the expression level, the expression of MYBPH or MYBPH gene can be selected appropriately from the usual method of measuring qualitatively or quantitatively, that is, the presence or absence and the expression amount of expression of MYBPH or MYBPH gene.
個体から採取された骨格筋由来のMYBPHの発現レベルを測定する方法としては、常法に従い作製した、MYBPHと特異的に結合する抗体やペプチドアプタマーを用いて、免疫沈降法、質量分析法、RIA(ラジオイムノアッセイ)、ウェスタンブロット法、ELISA(酵素結合免疫吸着)法、ECLIA(電気化学発光免疫測定)法、マイクロアレイなどの常法に従い測定する方法が挙げられる。 As a method of measuring the expression level of skeletal muscle-derived MYBPH collected from an individual, immunoprecipitation, mass spectrometry, RIA using an antibody or peptide aptamer specifically binding to MYBPH prepared according to a conventional method (Radioimmunoassay), Western blotting, ELISA (enzyme-linked immunosorbent assay), ECLIA (electrochemiluminescence immunoassay), a method of measuring according to a conventional method such as microarray, etc. may be mentioned.
個体から採取された骨格筋由来のMYBPH遺伝子の発現レベルを測定する方法としては、常法に従い作製した、MYBPH遺伝子とストリンジェントな条件下で結合する核酸プローブ、核酸アプタマー、又は核酸プライマーを用いて、ポリメラーゼ連鎖反応(PCR)法、RT-PCR法、リアルタイムPCR法、LAMP(Loop-mediated isothermal amplification)法などの核酸増幅法、サザンハイブリダイゼーション、ノザンハイブリダイゼーションなどのハイブリダイゼーション法、マイクロアレイ法などの常法に従い測定する方法が挙げられる。また前記核酸プローブ、核酸アプタマー及び核酸プライマーは、放射性同位体、蛍光物質、酵素などの標識物質で標識されていてもよい。
このうち本発明では、核酸プライマーを用いてPCR法によりMYBPH遺伝子の発現レベルを測定することが好ましい。また前記核酸プライマーとしては、下記表1に示す核酸プライマーセット1〜3のいずれか1つを好ましく用いることができる。
As a method of measuring the expression level of the MYBPH gene derived from skeletal muscle collected from an individual, a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer which binds to the MYBPH gene under stringent conditions prepared according to a conventional method is used , Polymerase chain reaction (PCR) method, RT-PCR method, real-time PCR method, nucleic acid amplification method such as LAMP (Loop-mediated isothermal amplification) method, Southern hybridization, hybridization method such as Northern hybridization, microarray method, etc. The method of measuring according to a conventional method is mentioned. The nucleic acid probe, the nucleic acid aptamer and the nucleic acid primer may be labeled with a labeling substance such as a radioactive isotope, a fluorescent substance, or an enzyme.
Among them, in the present invention, it is preferable to measure the expression level of MYBPH gene by PCR using a nucleic acid primer. Moreover, as said nucleic acid primer, any one of the nucleic acid primer sets 1-3 shown to following Table 1 can be used preferably.
運動機能の向上、筋量の増加、骨格筋細胞の分化の進行、及び筋管肥大に伴い、MYBPH及びMYBPH遺伝子の発現レベルが上昇する。これに対して、運動機能の低下、筋量の減少、骨格筋細胞の分化の不進行、及び筋管縮小に伴い、MYBPH及びMYBPH遺伝子の発現レベルは低下する。
よって、個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子の発現レベルを測定し、運動機能、筋量、骨格筋細胞の分化の程度、又は筋管に関して標準的な個体から採取されたMYBPH又はMYBPH遺伝子の発現レベルと比較を行う。そして、測定した発現レベルが上昇した場合には、運動機能が向上した、筋量が増加した、骨格筋細胞の分化が進行した、又は筋管が肥大した、と判断する。一方、MYBPH又はMYBPH遺伝子の発現レベルが低下した場合には、運動機能が低下した、筋量が減少した、骨格筋細胞の分化が進行していない、又は筋管が縮小した、と判断する。また、MYBPH又はMYBPH遺伝子の発現レベルに変化が確認できない場合は、運動機能、筋量、骨格筋細胞の分化の程度、及び筋管は標準的な状態であると判断する。あるいは、運動など筋量を増加させるような外部刺激を個体に行ってもMYBPH又はMYBPH遺伝子の発現レベルが増加しない場合には、その個体がロコモティブシンドロームに罹患している、又はロコモティブシンドロームに罹患するリスクが高い、と判断する。
The expression levels of the MYBPH and MYBPH genes rise with the improvement of motor function, the increase of muscle mass, the progress of differentiation of skeletal muscle cells, and myotube hypertrophy. On the other hand, the expression levels of the MYBPH and MYBPH genes decrease with the decrease in motor function, the decrease in muscle mass, the non-progression of differentiation of skeletal muscle cells, and the contraction of myotubes.
Therefore, the expression level of the MYBPH or MYBPH gene derived from skeletal muscle collected from an individual is measured, and the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, or MYBPH collected from a standard individual with respect to myotubes Compare with the expression level of MYBPH gene. Then, when the measured expression level is increased, it is determined that the motor function has been improved, the muscle mass has been increased, the differentiation of skeletal muscle cells has progressed, or the myotube has been enlarged. On the other hand, when the expression level of the MYBPH or MYBPH gene is reduced, it is judged that the motor function is reduced, the muscle mass is reduced, the differentiation of skeletal muscle cells is not progressing, or the myotubes are contracted. Also, if no change in the expression level of MYBPH or MYBPH gene can be confirmed, the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and myotubes are judged to be in a standard state. Alternatively, if the individual does not increase the expression level of the MYBPH or MYBPH gene even if an external stimulus such as exercise to increase muscle mass is given to the individual, the individual suffers from locomotive syndrome or suffers from locomotive syndrome Decide that the risk is high.
本発明でMYBPHの発現レベルを測定し、測定した発現レベルに基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価する際、骨格筋のタンパク質分子マーカーとして通常用いられる物質の発現レベルとMYBPHの発現レベルとを比較し、測定したMYBPHの発現レベルを補正することが好ましい。このように発現レベルの補正を行うことで、個体の運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大の程度それぞれを正確に評価することができる。
通常用いられる、前記骨格筋のタンパク質分子マーカーとしては、β-アクチンなどが挙げられる。
In the present invention, the expression level of MYBPH is measured, and a protein molecule of skeletal muscle is used to evaluate the motor function, muscle mass, the degree of differentiation of skeletal muscle cells or the degree of myotube hypertrophy based on the measured expression level. It is preferable to compare the expression level of a substance usually used as a marker with the expression level of MYBPH and correct the measured expression level of MYBPH. By correcting the expression level in this manner, it is possible to accurately evaluate the individual's motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.
Examples of the protein molecule marker for skeletal muscle that is usually used include β-actin and the like.
本発明でMYBPH遺伝子の発現レベルを測定し、測定した発現レベルに基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価する際、ハウスキーピング遺伝子(内部標準遺伝子)の発現レベルと、MYBPH遺伝子の発現レベルとを比較し、測定したMYBPH遺伝子の発現レベルを補正することすることが好ましい。具体的には、MYBPH遺伝子の発現量を測定し、別途測定したハウスキーピング遺伝子の発現量で補正を行い、補正した値に基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価することが好ましい。このように発現レベルの比較を行うことで、個体の運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大の程度それぞれを正確に評価することができる。
通常用いられるハウスキーピング遺伝子としては、36B4遺伝子、グリセルアルデヒド-3-リン酸デヒドロゲナーゼ(Glyceraldehyde-3-phosphate dehydrogenase、GAPDH)遺伝子、β-アクチン遺伝子などが挙げられる。
In the present invention, the expression level of the MYBPH gene is measured, and the housekeeping gene (the muscle function, muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy is assessed based on the measured expression level. It is preferable to compare the expression level of the internal control gene) with the expression level of the MYBPH gene and correct the measured expression level of the MYBPH gene. Specifically, the expression level of the MYBPH gene is measured, corrected with the expression level of the housekeeping gene measured separately, and based on the corrected value, the exercise function of the individual, the muscle mass, the degree of differentiation of skeletal muscle cells, Alternatively, it is preferable to evaluate the degree of myotube hypertrophy. By comparing the expression levels in this manner, it is possible to accurately evaluate the individual's motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.
Examples of commonly used housekeeping genes include 36B4 gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, β-actin gene and the like.
本発明は、MYBPH又はMYBPH遺伝子の発現レベルを測定するための抗体、ペプチドアプタマー、核酸プローブ、核酸アプタマー又は核酸プライマーを含んでなる、個体の運動機能、筋量、骨格筋細胞の分化の程度又は筋管肥大の程度の評価用キットも提供する。
このキットは、前述した、タンパク質分子マーカーやハウスキーピング遺伝子の発現レベルを測定するための抗体、ペプチドアプタマー、核酸プローブ、核酸アプタマー又は核酸プライマーを含んでもよい。
The present invention relates to an antibody for measuring the expression level of MYBPH or MYBPH gene, a peptide aptamer, a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, the exercise function of an individual, the muscle mass, the degree of differentiation of skeletal muscle cells or Also provided is a kit for evaluation of the degree of myotube hypertrophy.
This kit may contain the aforementioned antibody for measuring the expression level of a protein molecule marker or a housekeeping gene, a peptide aptamer, a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer.
本発明は、前記評価方法に基づいて、ロコモティブシンドロームの予防又は改善剤、運動機能向上の促進剤、運動機能低下の予防又は改善剤、筋量増加の促進剤、筋量低下の予防又は改善剤、骨格筋細胞の分化促進剤、筋管肥大の促進剤、筋管縮小の予防又は改善剤をスクリーニングするスクリーニング方法も提供する。このスクリーニング方法では、ヒト若しくは非ヒト動物、又は骨格筋由来の細胞株に前記剤の候補となる物質を投与又は摂取させ、物質の投与又は摂取の前後で本発明の評価方法に基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価し、運動機能を向上させる物質、筋量を増加させる物質、骨格筋細胞の分化を促進させる物質、又は筋管を肥大させる物質をこれらの剤として選択することができる。
なお本明細書において「予防」とは、個体における疾患若しくは症状の発症の防止若しくは遅延、又は個体の疾患若しくは症状の発症の危険性を低下させることをいう。また、本明細書において「改善」とは、疾患、症状若しくは状態の好転、疾患、症状若しくは状態の悪化の防止若しくは遅延、又は疾患、症状若しくは状態の進行の逆転、防止若しくは遅延をいう。
The present invention provides a preventive or ameliorating agent for locomotive syndrome, a promoter for improving the motor function, a promoter for preventing or improving the motor function decline, a promoter for increasing the muscle mass, a promoter for preventing or improving the muscle mass loss based on the above evaluation method. Also provided are screening methods for screening for skeletal muscle cell differentiation promoting agents, myotube hypertrophy promoting agents, and agents for preventing or improving myotube contraction. In this screening method, a human or non-human animal, or a skeletal muscle-derived cell line is administered or ingested a candidate substance of the agent, and before or after administration or intake of the substance, the individual evaluates the individual according to the evaluation method of the present invention. Substances that improve motor function, substances that increase muscle mass, substances that promote differentiation of skeletal muscle cells, or muscle that evaluate motor function, muscle mass, degree of differentiation of skeletal muscle cells, or degree of myotube hypertrophy Substances that enlarge the tube can be selected as these agents.
As used herein, "prevention" refers to preventing or delaying the onset of a disease or condition in an individual, or reducing the risk of developing an individual's disease or condition. Moreover, in the present specification, “improvement” refers to amelioration of disease, symptom or condition, prevention or delay of deterioration of disease, symptom or condition, or reversal, prevention or delay of progression of disease, symptom or condition.
本発明のバイオマーカーは、分化が進行した骨格筋細胞の検出方法及び筋管が肥大した骨格筋細胞の検出方法にも好適に用いることができる。この検出方法では、骨格筋細胞に発現するMYBPH又はMYBPH遺伝子の発現レベルを測定し、発現レベルの高い細胞を分化が進行した骨格筋細胞又は筋管が肥大した骨格筋細胞として検出することができる。
また本発明のバイオマーカーは、ロコモティブシンドロームの罹患の有無又は罹患するリスクの有無の評価方法にも好適に用いることができる。この評価方法では、運動など筋量を増加させるような外部刺激を行った個体の骨格筋細胞由来のMYBPH又はMYBPH遺伝子の発現レベルを測定し、MYBPH又はMYBPH遺伝子の発現レベルが増加しない場合に、ロコモティブシンドロームに罹患している、又は罹患するリスクが高い、と評価することができる。
さらに本発明のバイオマーカーは、ロコモティブシンドロームの治療又は予防効果の評価方法にも好適に用いることができる。ロコモティブシンドロームに罹患した被験者にロコモティブシンドロームの治療又は予防措置が効果的であった場合、それに応じてMYBPH又はMYBPH遺伝子の発現レベルも上昇する。したがって、ロコモティブシンドロームの治療又は予防措置の前後でMYBPH又はMYBPH遺伝子の発現レベルを測定し、発現レベルが上昇した場合には治療又は予防措置が効果的であると評価することができる。
The biomarker of the present invention can also be suitably used for a method of detecting skeletal muscle cells in which differentiation has progressed and a method of detecting skeletal muscle cells in which myotubes are enlarged. In this detection method, the expression level of MYBPH or MYBPH gene expressed in skeletal muscle cells can be measured, and cells with high expression levels can be detected as skeletal muscle cells in which differentiation has progressed or skeletal muscle cells in which myotubes are enlarged .
The biomarkers of the present invention can also be suitably used as a method for evaluating the presence or absence of the risk of having the locomotive syndrome. In this evaluation method, the expression level of MYBPH or MYBPH gene derived from skeletal muscle cells of an individual subjected to external stimulation such as exercise to increase muscle mass is measured, and the expression level of MYBPH or MYBPH gene does not increase. It can be evaluated that the patient is suffering from or at high risk of suffering from locomotive syndrome.
Furthermore, the biomarkers of the present invention can also be suitably used in methods for evaluating the therapeutic or prophylactic effect of locomotive syndrome. When a subject suffering from locomotive syndrome is effective in the treatment or prevention of locomotive syndrome, the expression level of the MYBPH or MYBPH gene is also elevated accordingly. Therefore, the expression level of MYBPH or MYBPH gene can be measured before and after treatment or preventive measures for locomotive syndrome, and treatment or preventive measures can be evaluated to be effective if the expression levels are elevated.
上述した実施形態に関し、本発明はさらに以下のマーカー、使用、方法、評価方法、スクリーニング方法、及びキットを開示する。 With regard to the embodiments described above, the present invention further discloses the following markers, uses, methods, evaluation methods, screening methods, and kits.
<1>個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子を含む、運動機能の判定用マーカー、筋量判定用マーカー、骨格筋細胞の分化の判定用マーカー、又は筋管肥大の判定用マーカー。 A marker for determination of motor function, a marker for determination of muscle mass, a marker for determination of differentiation of skeletal muscle cells, or a marker for determination of myotube hypertrophy including the skeletal muscle-derived MYBPH or MYBPH gene collected from an individual <1> .
<2>前記MYBPHのアミノ酸配列の一部を有するポリペプチド、又は前記MYBPH遺伝子の塩基配列の一部を有するポリオリゴヌクレオチドからなる、前記<1>項に記載のマーカー。
<3>前記MYBPH又はMYBPH遺伝子がヒラメ筋、ヒフク筋又は足底筋由来のMYBPH又はMYBPH遺伝子である、前記<1>又は<2>項に記載のマーカー。
<4>前記MYBPHが、NCBI RefSeqにNP_058029、NP_956852.1、NM_001100137.1、若しくはNP_004988で登録されているタンパク質、これらのタンパク質のアミノ酸配列において、1又は数個、通常1〜100個、好ましくは1〜75個、より好ましくは1〜50個、さらに好ましくは1〜25個、特に好ましくは1〜10個、最も好ましくは1〜5個、のアミノ酸が欠失、置換、挿入及び/若しくは付加されたアミノ酸配列からなり、かつ機能的に同等なタンパク質、又はこれらのタンパク質のアミノ酸配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有するアミノ酸配列からなり、かつ機能的に同等なタンパク質、である、前記<1>〜<3>のいずれか1項に記載のマーカー。
<5>前記MYBPH遺伝子が、NCBI RefSeqにNM_016749、NM_200558.1、NP_001093607.1、若しくはNM_004997で登録されている遺伝子、これらの遺伝子の塩基配列において、1又は数個、通常1〜389個、好ましくは1〜292個、より好ましくは1〜195個、さらに好ましくは1〜98個、特に好ましくは1〜39個、最も好ましくは1〜20個、の塩基が欠失、置換、挿入又は付加された塩基配列からなり、かつMYBPHをコードする核酸、又はこれらの遺伝子の塩基配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有する塩基配列からなり、かつMYBPHをコードする核酸、である、前記<1>〜<4>のいずれか1項に記載のマーカー。
<6>前記MYBPHが、MYBPHと特異的に認識される抗体又はペプチドアプタマー、好ましくは抗体、により検出されたものである、前記<1>〜<5>のいずれか1項に記載のマーカー。
<7>前記MYBPH遺伝子が、核酸プローブ、核酸アプタマー又は核酸プライマー、好ましくは核酸プライマー、により検出されたものである、前記<1>〜<6>のいずれか1項に記載のマーカー。
<8>前記MYBPH遺伝子が、前記表1に示す核酸プライマーセット1〜3のいずれか1つにより検出されたものである、前記<7>項に記載のマーカー。
<9>前記MYBPH又はMYBPH遺伝子が、運動など筋量を増加させる外部刺激を個体に負荷してから1〜6時間後に採取されたMYBPH又はMYBPH遺伝子である、前記<1>〜<7>のいずれか1項に記載のマーカー。
The marker as described in said <1> which consists of a polypeptide which has a part of amino acid sequence of <2> said MYBPH, or the polynucleotide which has a part of base sequence of said MYBPH gene.
<3> The marker according to <1> or <2>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene derived from flatfish muscle, hippocampus muscle or plantar muscle.
<4> Proteins whose MYBPH is registered in NCBI RefSeq as NP_058029, NP_956852.1, NM_001100137.1, or NP_004988, and one or several, usually 1 to 100, preferably the amino acid sequences of these proteins 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids are deleted, substituted, inserted and / or added Or a functionally equivalent protein, or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably an amino acid sequence of these proteins A functionally equivalent protein consisting of an amino acid sequence having a homology of 98% or more, most preferably 99% or more, which is the above <1> to Marker according to any one of 3>.
<5> Genes whose MYBPH genes are registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, and the nucleotide sequences of these genes: one or several, usually 1 to 389, preferably Is 1 to 292, more preferably 1 to 195, still more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. And a nucleic acid encoding MYBPH, or a nucleotide sequence of these genes, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% The marker according to any one of the above <1> to <4>, which is a nucleic acid consisting of a nucleotide sequence having homology of 99% or more, most preferably 99% or more, and encoding MYBPH.
<6> The marker according to any one of <1> to <5>, wherein the MYBPH is detected by an antibody or peptide aptamer that is specifically recognized as MYBPH, preferably an antibody.
<7> The marker according to any one of <1> to <6>, wherein the MYBPH gene is detected by a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, preferably a nucleic acid primer.
<8> The marker according to the above <7>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1 above.
<9> The above <1> to <7>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise. The marker according to any one of the preceding claims.
<10>個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子の、運動機能の判定用マーカー、筋量判定用マーカー、骨格筋細胞の分化の判定用マーカー、又は筋管肥大の判定用マーカーとしての使用。
<11>個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子を、運動機能の判定用マーカー、筋量判定用マーカー、骨格筋細胞の分化の判定用マーカー、又は筋管肥大の判定用マーカーとして使用する方法。
<12>個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子を使用する、運動機能の判定方法、筋量判定方法、骨格筋細胞の分化の判定方法、又は筋管肥大の判定方法。
<10> A marker for determination of motor function, a marker for determination of muscle mass, a marker for determination of differentiation of skeletal muscle cells, or a marker for determination of myotube hypertrophy of skeletal muscle-derived MYBPH or MYBPH gene collected from an individual Use of.
The skeletal muscle-derived MYBPH or MYBPH gene collected from an individual <11> is used as a marker for determination of motor function, a marker for determination of muscle mass, a marker for determination of differentiation of skeletal muscle cells, or a marker for determination of myotube hypertrophy How to use.
The determination method of the motor function, the muscle amount determination method, the determination method of a skeletal muscle cell differentiation, or the determination method of myotube hypertrophy using the skeletal muscle-derived MYBPH or MYBPH gene collected from <12> individual.
<13>前記MYBPHのアミノ酸配列の一部を有するポリペプチド、又は前記遺伝子の塩基配列の一部を有するポリオリゴヌクレオチドを使用する、前記<10>〜<12>のいずれか1項に記載の使用又は方法。
<14>前記MYBPH又はMYBPH遺伝子がヒラメ筋、ヒフク筋又は足底筋由来のMYBPH又はMYBPH遺伝子である、前記<10>〜<13>のいずれか1項に記載の使用又は方法。
<15>前記MYBPHが、NCBI RefSeqにNP_058029、NP_956852.1、NM_001100137.1、若しくはNP_004988で登録されているタンパク質、これらのタンパク質のアミノ酸配列において、1又は数個、通常1〜100個、好ましくは1〜75個、より好ましくは1〜50個、さらに好ましくは1〜25個、特に好ましくは1〜10個、最も好ましくは1〜5個、のアミノ酸が欠失、置換、挿入及び/若しくは付加されたアミノ酸配列からなり、かつ機能的に同等なタンパク質、又はこれらのタンパク質のアミノ酸配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有するアミノ酸配列からなり、かつ機能的に同等なタンパク質、である、前記<10>〜<14>のいずれか1項に記載の使用又は方法。
<16>前記MYBPH遺伝子が、NCBI RefSeqにNM_016749、NM_200558.1、NP_001093607.1、若しくはNM_004997で登録されている遺伝子、これらの遺伝子の塩基配列において、1又は数個、通常1〜389個、好ましくは1〜292個、より好ましくは1〜195個、さらに好ましくは1〜98個、特に好ましくは1〜39個、最も好ましくは1〜20個、の塩基が欠失、置換、挿入又は付加された塩基配列からなり、かつMYBPHをコードする核酸、又はこれらの遺伝子の塩基配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有する塩基配列からなり、かつMYBPHをコードする核酸、である、前記<10>〜<15>のいずれか1項に記載の使用又は方法。
<17>前記MYBPHが、MYBPHと特異的に認識される抗体又はペプチドアプタマー、好ましくは抗体、により検出されたものである、前記<10>〜<16>のいずれか1項に記載の使用又は方法。
<18>前記MYBPH遺伝子が、核酸プローブ、核酸アプタマー又は核酸プライマー、好ましくは核酸プライマー、により検出されたものである、前記<10>〜<17>のいずれか1項に記載の使用又は方法。
<19>前記MYBPH遺伝子が、前記表1に示す核酸プライマーセット1〜3のいずれか1つにより検出されたものである、前記<18>項に記載の使用又は方法。
<20>前記MYBPH又はMYBPH遺伝子が、運動など筋量を増加させる外部刺激を個体に負荷してから1〜6時間後に採取されたMYBPH又はMYBPH遺伝子である、前記<10>〜<19>のいずれか1項に記載の使用又は方法。
<13> The polypeptide according to any one of <10> to <12>, wherein a polypeptide having a portion of the amino acid sequence of MYBPH or a polyoligonucleotide having a portion of the base sequence of the gene is used Use or method.
<14> The use or method according to any one of the above <10> to <13>, wherein the MYBPH or MYBPH gene is an MYBPH or MYBPH gene derived from flatfish muscle, hippocampus muscle or plantar muscle.
<15> Proteins whose MYBPH is registered in NCBI RefSeq as NP_058029, NP_956852.1, NM_001100137.1, or NP_004988, and one or several, usually 1 to 100, preferably the amino acid sequences of these proteins 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids are deleted, substituted, inserted and / or added Or a functionally equivalent protein, or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably an amino acid sequence of these proteins A functionally equivalent protein consisting of an amino acid sequence having a homology of 98% or more, most preferably 99% or more, Use or method according to any one of - <14>.
<16> Genes whose MYBPH genes are registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, and the nucleotide sequences of these genes: one or several, usually 1 to 389, preferably Is 1 to 292, more preferably 1 to 195, still more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. And a nucleic acid encoding MYBPH, or a nucleotide sequence of these genes, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% The use according to any one of the above <10> to <15>, which is a nucleic acid consisting of a nucleotide sequence having the homology as described above, most preferably 99% or more and encoding MYBPH Method.
<17> The use according to any one of the above <10> to <16>, wherein the MYBPH is detected by an antibody or peptide aptamer specifically recognized as MYBPH, preferably an antibody. Method.
<18> The use or the method according to any one of <10> to <17>, wherein the MYBPH gene is detected by a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, preferably a nucleic acid primer.
<19> The use or method according to <18>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1 above.
<20> The above <10> to <19>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise. Use or method according to any one of the preceding paragraphs.
<21>個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子の発現レベルを測定し、
測定した発現レベルに基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価する、運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度の評価方法。
<21> measuring the expression level of MYBPH or MYBPH gene derived from skeletal muscle collected from an individual;
Evaluate the motor function, muscle mass, degree of differentiation of skeletal muscle cells, or degree of myotube hypertrophy based on the expression level measured, motor function, muscle mass, degree of differentiation of skeletal muscle cells, or myotube How to assess the degree of hypertrophy.
<22>前記MYBPHのアミノ酸配列の一部を有するポリペプチド、又は前記MYBPH遺伝子の塩基配列の一部を有するポリオリゴヌクレオチドの発現レベルを測定し、測定した発現レベルに基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価する、前記<21>項に記載の方法。
<23>前記発現レベルが、前記MYBPH又はMYBPH遺伝子の発現の有無、又は前記MYBPH又はこれをコードする遺伝子の発現量である、前記<21>又は<22>項に記載の方法。
<24>前記MYBPH又はMYBPH遺伝子がヒラメ筋、ヒフク筋又は足底筋由来のMYBPH又はMYBPH遺伝子である、前記<21>〜<23>のいずれか1項に記載の方法。
<25>前記MYBPHが、NCBI RefSeqにNP_058029、NP_956852.1、NM_001100137.1、若しくはNP_004988で登録されているタンパク質、これらのタンパク質のアミノ酸配列において、1又は数個、通常1〜100個、好ましくは1〜75個、より好ましくは1〜50個、さらに好ましくは1〜25個、特に好ましくは1〜10個、最も好ましくは1〜5個、のアミノ酸が欠失、置換、挿入及び/若しくは付加されたアミノ酸配列からなり、かつ機能的に同等なタンパク質、又はこれらのタンパク質のアミノ酸配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有するアミノ酸配列からなり、かつ機能的に同等なタンパク質、である、前記<21>〜<24>のいずれか1項に記載の方法。
<26>前記MYBPH遺伝子が、NCBI RefSeqにNM_016749、NM_200558.1、NP_001093607.1、若しくはNM_004997で登録されている遺伝子、これらの遺伝子の塩基配列において、1又は数個、通常1〜389個、好ましくは1〜292個、より好ましくは1〜195個、さらに好ましくは1〜98個、特に好ましくは1〜39個、最も好ましくは1〜20個、の塩基が欠失、置換、挿入又は付加された塩基配列からなり、かつMYBPHをコードする核酸、又はこれらの遺伝子の塩基配列と80%以上、好ましくは85%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上、最も好ましくは99%以上、の相同性を有する塩基配列からなり、かつMYBPHをコードする核酸、である、前記<21>〜<25>のいずれか1項に記載の方法。
<27>MYBPHと特異的に認識される抗体又はペプチドアプタマー、好ましくは抗体、によりMYBPHを検出し、MYBPHの発現レベルを測定する、前記<21>〜<26>のいずれか1項に記載の方法。
<28>核酸プローブ、核酸アプタマー又は核酸プライマー、好ましくは核酸プライマー、によりMYBPH遺伝子を検出し、MYBPH遺伝子の発現レベルを測定する、前記<21>〜<27>のいずれか1項に記載の方法。
<29>前記表1に示す核酸プライマーセット1〜3のいずれか1つによりMYBPH遺伝子を検出し、MYBPH遺伝子の発現レベルを測定する、前記<28>項に記載の方法。
<30>測定したMYBPH又はMYBPH遺伝子の発現レベルが上昇した場合に、運動機能が向上した、筋量が増加した、骨格筋細胞の分化が進行した、又は筋管が肥大した、と判断する、前記<21>〜<29>のいずれか1項に記載の方法。
<31>測定したMYBPH又はMYBPH遺伝子の発現レベルが低下した場合に、運動機能が低下した、筋量が減少した、骨格筋細胞の分化が進行していない、又は筋管が縮小した、と判断する、前記<21>〜<30>のいずれか1項に記載の方法。
<32>測定したMYBPH又はMYBPH遺伝子の発現レベルに変化が確認できない場合に、運動機能、筋量、骨格筋細胞の分化の程度、又は筋管は標準的な状態であると判断する、前記<21>〜<31>のいずれか1項に記載の方法。
<33>筋量を増加させるような外部刺激を行った個体から採取された骨格筋由来のMYBPH又はMYBPH遺伝子の発現レベルを測定し、測定した発現レベルが増加しない場合に、その個体がロコモティブシンドロームに罹患している又はロコモティブシンドロームに罹患するリスクが高いと判断する、前記<21>〜<32>のいずれか1項に記載の方法。
<34>骨格筋のタンパク質分子マーカー、好ましくはβ-アクチン、の発現レベルとMYBPHの発現レベルとを比較し、測定したMYBPHの発現レベルを補正する、前記<21>〜<33>のいずれか1項に記載の方法。
<35>ハウスキーピング遺伝子、好ましくは36B4遺伝子、GAPDH遺伝子、又はβ-アクチン遺伝子、の発現レベルとMYBPH遺伝子の発現レベルとを比較し、測定したMYBPH遺伝子の発現レベルを補正する、前記<21>〜<34>のいずれか1項に記載の方法。
<36>前記MYBPH又はMYBPH遺伝子が、運動など筋量を増加させる外部刺激を個体に負荷してから1〜6時間後に採取されたMYBPH又はMYBPH遺伝子である、前記<21>〜<35>のいずれか1項に記載の方法。
≪ 22 > Motor function of the individual based on the expression level measured by measuring the expression level of the polypeptide having a part of the amino acid sequence of MYBPH or the polynucleotide having a part of the base sequence of the MYBPH gene The method according to <21>, wherein the muscle mass, the degree of differentiation of skeletal muscle cells, or the degree of myotube hypertrophy is evaluated.
<23> The method according to <21> or <22>, wherein the expression level is the presence or absence of expression of the MYBPH or MYBPH gene, or the expression amount of the MYBPH or a gene encoding the same.
<24> The method according to any one of <21> to <23>, wherein the MYBPH or MYBPH gene is an MYBPH or MYBPH gene derived from a flatfish muscle, a calf muscle or a plantar muscle.
<25> Proteins whose MYBPH is registered in NCBI RefSeq as NP_058029, NP_956852.1, NM_001100137.1, or NP_004988, and one or several, usually 1 to 100, preferably the amino acid sequences of these proteins 1 to 75, more preferably 1 to 50, more preferably 1 to 25, particularly preferably 1 to 10, most preferably 1 to 5 amino acids are deleted, substituted, inserted and / or added Or a functionally equivalent protein, or an amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably an amino acid sequence of these proteins A functionally equivalent protein consisting of an amino acid sequence having a homology of 98% or more, most preferably 99% or more, The method according to any one of - <24>.
<26> Genes whose MYBPH genes are registered in NCBI RefSeq as NM_016749, NM_200558.1, NP_001093607.1, or NM_004997, and one or several, usually 1 to 389, in the base sequences of these genes, preferably Is 1 to 292, more preferably 1 to 195, still more preferably 1 to 98, particularly preferably 1 to 39, most preferably 1 to 20 bases are deleted, substituted, inserted or added. And a nucleic acid encoding MYBPH, or a nucleotide sequence of these genes, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% The method according to any one of <21> to <25> above, which is a nucleic acid encoding a MYBPH-encoding nucleic acid consisting of a nucleotide sequence having homology of 99% or more, most preferably 99% or more.
The antibody or peptide aptamer specifically recognized as <27> MYBPH, preferably an antibody, detects MYBPH and measures the expression level of MYBPH according to any one of the above <21> to <26>. Method.
<28> The method according to any one of <21> to <27>, wherein the MYBPH gene is detected by a nucleic acid probe, a nucleic acid aptamer or a nucleic acid primer, preferably a nucleic acid primer, and the expression level of the MYBPH gene is measured. .
<29> The method according to <28>, wherein the MYBPH gene is detected by any one of the nucleic acid primer sets 1 to 3 shown in Table 1 above, and the expression level of the MYBPH gene is measured.
<30> When the expression level of the measured MYBPH or MYBPH gene is increased, it is judged that the motor function is improved, the muscle mass is increased, the differentiation of skeletal muscle cells is advanced, or the myotube is expanded. The method according to any one of <21> to <29>.
<31> It is judged that the motor function was reduced, the muscle mass was decreased, the differentiation of skeletal muscle cells was not progressed, or the myotube was contracted when the expression level of the measured MYBPH or MYBPH gene was decreased The method according to any one of <21> to <30>.
<32> If no change is observed in the measured expression level of MYBPH or MYBPH gene, motor function, muscle mass, degree of differentiation of skeletal muscle cells, or myotubes are judged to be in a standard state, 21. The method according to any one of <31>.
<33> The expression level of skeletal muscle-derived MYBPH or MYBPH gene collected from an individual subjected to external stimulation to increase muscle mass is measured, and if the measured expression level does not increase, the individual has locomotive syndrome The method according to any one of <21> to <32>, which is judged to be afflicted with or at high risk of suffering from locomotive syndrome.
Any one of the above <21> to <33> which compares the expression level of a protein molecule marker of <34> skeletal muscle, preferably β-actin, with the expression level of MYBPH and corrects the measured expression level of MYBPH Method according to paragraph 1.
<35> The expression level of the MYBPH gene is compared with the expression level of the MYBPH gene by comparing the expression level of the housekeeping gene, preferably the 36B4 gene, preferably the GAPDH gene, or the β-actin gene, with the above <21>. The method according to any one of <34>.
<36> The above <21> to <35>, wherein the MYBPH or MYBPH gene is a MYBPH or MYBPH gene collected 1 to 6 hours after the individual is loaded with an external stimulus that increases muscle mass such as exercise. The method according to any one of the preceding claims.
<37>MYBPH又はMYBPH遺伝子の発現レベルを測定するための抗体、ペプチドアプタマー、核酸プローブ、核酸アプタマー又は核酸プライマーを含んでなる、個体の運動機能、筋量、骨格筋細胞の分化の程度又は筋管肥大の程度の評価用キット。
<38>骨格筋のタンパク質分子マーカー、好ましくはβ-アクチン、又はハウスキーピング遺伝子、好ましくは36B4遺伝子、GAPDH遺伝子、又はβ-アクチン遺伝子、の発現レベルを測定するための抗体、ペプチドアプタマー、核酸プローブ、核酸アプタマー又は核酸プライマーをさらに含む、前記<37>に記載のキット。
<37> Antibody for measuring the expression level of MYBPH or MYBPH gene, peptide aptamer, nucleic acid probe, nucleic acid aptamer or nucleic acid primer, motor function of the individual, muscle mass, degree of differentiation of skeletal muscle cells or muscle Evaluation kit for the degree of ductal hypertrophy.
<38> Antibody, peptide aptamer, nucleic acid probe for measuring the expression level of protein molecule marker of skeletal muscle, preferably β-actin, or housekeeping gene, preferably 36B4 gene, GAPDH gene, or β-actin gene The kit according to <37>, further comprising a nucleic acid aptamer or a nucleic acid primer.
<39>ロコモティブシンドロームの予防又は改善剤、運動機能向上の促進剤、運動機能低下の予防又は改善剤、筋量増加の促進剤、筋量低下の予防又は改善剤、骨格筋細胞の分化促進剤、筋管肥大の促進剤、筋管縮小の予防又は改善剤をスクリーニングするスクリーニング方法であって、
ヒト若しくは非ヒト動物、又は骨格筋由来の細胞株に前記剤の候補となる物質を投与又は摂取させ、
物質の投与又は摂取の前後で前記<21>〜<35>のいずれか1項に記載の評価方法に基づいて個体の運動機能、筋量、骨格筋細胞の分化の程度、又は筋管肥大の程度を評価し、
運動機能を向上させる物質、筋量を増加させる物質、骨格筋細胞の分化を促進させる物質、又は筋管を肥大させる物質を前記剤として選択する、スクリーニング方法。
<40>前記<1>〜<9>のいずれか1項に記載のマーカーを用いて、骨格筋細胞に発現するMYBPH又はMYBPH遺伝子の発現レベルを測定し、
MYBPH又はMYBPH遺伝子の発現レベルの高い細胞を分化が進行した骨格筋細胞又は筋管が肥大した骨格筋細胞として検出する、
分化が進行した骨格筋細胞又は筋管が肥大した骨格筋細胞の検出方法。
<41>前記<1>〜<9>のいずれか1項に記載のマーカーを用いて、骨格筋細胞由来のMYBPH又はMYBPH遺伝子の発現レベルを測定し、
骨格筋細胞由来のMYBPH又はMYBPH遺伝子の発現レベルを測定し、
筋量を増加させるような外部刺激を個体に行ってもMYBPH又はMYBPH遺伝子の発現レベルが増加しない場合に、その個体がロコモティブシンドロームに罹患している、又は罹患するリスクが高い、と評価する、
ロコモティブシンドロームの罹患の有無又は罹患するリスクの評価方法。
<42>前記<1>〜<9>のいずれか1項に記載のマーカーを用いて、骨格筋細胞に発現するMYBPH又はMYBPH遺伝子の発現レベルを測定し、
MYBPH又はMYBPH遺伝子の発現レベルが上昇した場合には前記治療又は予防措置が効果的であると評価する、
ロコモティブシンドロームの治療又は予防効果の評価方法。
<39> An agent for preventing or improving locomotive syndrome, an agent for improving motor function, an agent for preventing or improving motor function decrease, an agent for increasing muscle mass, an agent for preventing or improving muscle mass loss, a differentiation promoter for skeletal muscle cells A screening method for screening an agent for promoting myotube hypertrophy and an agent for preventing or improving myotube contraction,
Administration or ingestion of a candidate for the agent to a human or non-human animal or a cell line derived from skeletal muscle,
Before and after administration or intake of substance Based on the evaluation method according to any one of the above <21> to <35>, the individual's motor function, muscle mass, degree of differentiation of skeletal muscle cells, or myotube hypertrophy Evaluate the degree,
A screening method, wherein a substance that improves motor function, a substance that increases muscle mass, a substance that promotes differentiation of skeletal muscle cells, or a substance that enlarges a myotube is selected as the agent.
<40> The expression level of MYBPH or MYBPH gene expressed in skeletal muscle cells is measured using the marker described in any one of <1> to <9>.
Detecting cells with high expression level of MYBPH or MYBPH gene as skeletal muscle cells or myotubes with enlarged differentiation;
A method of detecting a skeletal muscle cell in which differentiation has progressed or a skeletal muscle cell in which a myotube is enlarged.
<41> The expression level of MYBPH or MYBPH gene derived from a skeletal muscle cell is measured using the marker according to any one of <1> to <9>.
Measuring the expression level of MYBPH or MYBPH gene derived from skeletal muscle cells,
If external stimulation to increase muscle mass does not increase the expression level of MYBPH or MYBPH gene, the individual is evaluated as suffering from or at high risk of suffering from locomotive syndrome.
Method of evaluating presence or absence of or risk of suffering from locomotive syndrome.
<42> The expression level of the MYBPH or MYBPH gene expressed in skeletal muscle cells is measured using the marker described in any one of <1> to <9>.
If the expression level of MYBPH or MYBPH gene is increased, the treatment or preventive measure is evaluated as effective.
A method for evaluating the therapeutic or preventive effect of locomotive syndrome.
以下、本発明を実施例に基づきさらに詳細に説明するが、本発明はこれに限定されるものではない。
ここで、下記試験例及び比較例で用いた、各種遺伝子の増幅に用いたプライマーの塩基配列を表2に示す。なお下記プライマーは、標的遺伝子の断片を約100〜200bpのサイズで増幅するように設計した。
EXAMPLES The present invention will be described in more detail based on examples given below, but the invention is not meant to be limited by these.
Here, the base sequences of the primers used for amplification of various genes used in the following test examples and comparative examples are shown in Table 2. The following primers were designed to amplify a target gene fragment at a size of about 100 to 200 bp.
試験例1 マウスの運動試験後のヒラメ筋の筋量及びヒラメ筋におけるMYBPH遺伝子の発現量の測定
(1)ヒラメ筋の筋量及び筋力の測定
室温を23±2℃、湿度を55±10%とし、照明時間を7〜19時と設定した飼育環境下で、7週齢の雄性C57BL/6Jマウス(オリエンタルバイオサービス社より購入)に対して一週間の環境馴化を行った後、個別飼育に馴化させた。その後、体重を基準に32匹のマウスを選抜した。これら32匹のマウスを非運動条件飼育群(対象群:個別飼育)16匹と、自発運動条件飼育群(運動群:回転カゴ付ケージで個別飼育)16匹とに分けた。試験食(商品名:CE-2、日本クレア社製)自由摂食下でこれら2群のマウスを飼育し、運動開始より1ヶ月目及び2ヶ月目に、各群8匹ずつの解剖を実施した。解剖は自由摂食条件下でセボフレン(商品名、丸石製薬社製)を麻酔した後、下腹部大静脈より全採血し、ヒラメ筋を採取した。採取したヒラメ筋の筋量及び筋力を測定し、すみやかに液体窒素で凍結し-80℃で保存した。運動開始より2カ月後のヒラメ筋の筋量の結果を図1に、運動開始より2カ月後のヒラメ筋の筋力の結果を図2に、それぞれ示す。
なおヒラメ筋の筋力測定は、左肢のヒラメ筋を縫合糸(#5-0 silk)でトランスデューサー(商品名:FORT100、World Precision Instruments社製)に固定し、37℃のKrebs溶液(95%O2、5%CO2通気、pH7.2)に浸し、2本のプラチナ電極より電気刺激を与えて単収縮刺激を施した後、トランスデューサーより得られるシグナルを筋力として評価した。
Test Example 1 Measurement of muscle mass of soleus muscle and expression level of MYBPH gene in solei muscle after exercise test of mouse (1) Measurement of muscle mass and muscle strength of solei muscle room temperature 23 ± 2 ° C., humidity 55 ± 10% Under the breeding environment where the lighting time is set to 7 to 19 o'clock, one week of environmental acclimation is performed on 7-week-old male C57BL / 6J mice (purchased from Oriental Bioservices Co., Ltd.), and then individualized breeding I got used to it. Thereafter, 32 mice were selected based on body weight. These 32 mice were divided into 16 non-motor condition rearing groups (target group: individual rearing) and 16 locomotion condition rearing groups (exercise group: individual rearing in a cage with a rotating cage). These two groups of mice are bred under a free diet on a test meal (trade name: CE-2, manufactured by CLEA Japan, Inc.), and eight animals in each group are dissected one month and two months after the start of exercise. did. The autopsy was performed under anesthesia with sebofurene (trade name, manufactured by Maruishi Pharmaceutical Co., Ltd.) under free feeding conditions, and then whole blood was collected from the lower abdominal vena cava to collect soleus muscle. The muscle mass and muscle strength of the collected soleus muscle were measured, immediately frozen with liquid nitrogen and stored at -80 ° C. The results of muscle mass of the flatfish muscle two months after the start of exercise are shown in FIG. 1, and the results of muscle strength of the flatfish muscle two months after the start of exercise are shown in FIG.
To measure the muscle strength of the soleus muscle, fix the soleus muscle of the left leg with a suture (# 5-0 silk) to a transducer (trade name: FORT 100, manufactured by World Precision Instruments), and use a 37 ° C Krebs solution (95%) After soaking in O 2 , 5% CO 2 aeration, pH 7.2) and electrical stimulation from two platinum electrodes to give twitch stimulation, the signal obtained from the transducer was evaluated as muscle force.
(2)MYBPH遺伝子の発現量の測定
保存したヒラメ筋から、RNeasy Fibrous Mini Kit(商品名、Qiagen社製)を使用してTotal RNAを抽出した。抽出したTotal RNAの濃度を揃え、65℃にて10分間の熱処理を行い、急冷後下記に示す逆転写反応に使用した。
125ng相当のRNAを鋳型とし、逆転写反応液{1×PCR buffer 2(商品名、Applied Biosystems社製)、5mM MgCl2、1mM dNTP mix、2.5μM oligo d(T) 18(商品名、New England Biolabs社製)、1U/μL Rnase inhibitor(商品名、タカラバイオ社製)}20μLと混合して、(42℃、1時間)→(52℃、30分)→(99℃、5分)→4℃の条件下で逆転写反応を行った。得られたcDNAサンプルを-20℃で保存した。
また、下記に示すreal-time PCRのスタンダード用として、500ng相当のRNAに対して同様の反応系で逆転写反応を行った。
(2) Measurement of Expression Level of MYBPH Gene From the stored flatfish muscle, total RNA was extracted using RNeasy Fibrous Mini Kit (trade name, manufactured by Qiagen). The concentration of extracted total RNA was made uniform, heat treatment was carried out at 65 ° C. for 10 minutes, and after rapid cooling, it was used for reverse transcription reaction shown below.
Reverse transcription reaction solution {1 × PCR buffer 2 (trade name, manufactured by Applied Biosystems), 5 mM MgCl 2 , 1 mM dNTP mix, 2.5 μM oligo d (T) 18 (trade name, New England) using 125 ng of RNA as a template Biolabs), 1 U / μL Rnase inhibitor (trade name, manufactured by Takara Bio)} mixed with 20 μL, (42 ° C., 1 hour) → (52 ° C., 30 minutes) → (99 ° C., 5 minutes) → Reverse transcription was performed at 4 ° C. The resulting cDNA samples were stored at -20.degree.
In addition, reverse transcription was performed in the same reaction system on 500 ng of RNA as a standard for real-time PCR shown below.
得られたcDNAを鋳型とし、PCR反応液{Fast SYBR(商品名、Applied Biosystems社製)、1μM Forward Primer(配列番号1)、1μM Reverse Primer(配列番号2)}20μLと混合して、7500 Fast Real-Time PCR System(商品名、Applied Biosystems社製)を用いて、MYBPHのmRNAに対するreal-time PCRを行った。スタンダード用に逆転写し得られたcDNAを7段階希釈して調製したcDNAをスタンダードcDNAとしてreal-time PCRを行い、作製した検量線に基づき、MYBPHのmRNAの発現量の解析を行った。得られた解析結果は、配列番号3及び4に示す塩基配列からなるプライマーを用いて測定した内部標準遺伝子(36B4遺伝子)の発現量で補正し、相対的なmRNA発現量として表した。得られた結果を図3に示す。 Using the obtained cDNA as a template, it is mixed with 20 μL of PCR reaction solution {Fast SYBR (trade name, manufactured by Applied Biosystems), 1 μM Forward Primer (SEQ ID NO: 1), 1 μM Reverse Primer (SEQ ID NO: 2)} to obtain 7500 Fast. Real-time PCR was performed on MYBPH mRNA using Real-Time PCR System (trade name, manufactured by Applied Biosystems). Real-time PCR was performed using as a standard cDNA a cDNA prepared by 7-step dilution of reverse-transcribed cDNA for a standard, and analysis of the expression level of MYBPH mRNA was performed based on the prepared calibration curve. The analysis results obtained were corrected with the expression level of the internal standard gene (36B4 gene) measured using the primers consisting of the nucleotide sequences shown in SEQ ID NOS: 3 and 4, and expressed as relative mRNA expression levels. The obtained result is shown in FIG.
図1に示すように、運動開始から2か月目に、運動群でヒラメ筋重量が有意に増加した。しかし、図2に示すように、運動開始から2か月であっても、ヒラメ筋の筋力に有意差は確認できなかった。
また、図3に示すように、運動開始から1か月目に、運動群でMYBPHのmRNAの発現量が有意に増加した。
これらの結果は、筋形成時(運動開始から1か月程度)にMYBPH遺伝子の発現量が増加すると、運動開始から2か月目にヒラメ筋の重量が増加することを示している。すなわちこれらの結果は、ヒラメ筋の筋量が増加する過程のいずれかの段階でMYBPHの一過的な発現量の増加が必要であり、筋形成時にMYBPHの発現を増加させることができない個体は筋量及び運動機能を維持又は増強できないことを示唆するものである。
As shown in FIG. 1, the flatfish muscle weight significantly increased in the exercise group two months after the start of exercise. However, as shown in FIG. 2, no significant difference could be confirmed in the muscle power of the flatfish muscle even two months after the start of exercise.
In addition, as shown in FIG. 3, the expression level of MYBPH mRNA was significantly increased in the exercise group one month after the start of exercise.
These results indicate that when the expression level of the MYBPH gene increases during myogenesis (approximately one month from the start of exercise), the weight of soleus muscle increases at two months after the start of exercise. That is, these results indicate that the transient expression level of MYBPH needs to be increased at any stage of the process of increasing the mass of soleus muscle, and individuals who can not increase the expression of MYBPH during myogenesis It suggests that muscle mass and motor function can not be maintained or enhanced.
試験例2 協働筋を切除した場合のMYBPHの発現量の測定
(1)足底筋の筋量の測定
C57BL/6Jマウス(オリエンタルバイオサービス社より購入、16週齢)を非施術群3匹と施術群3匹とに分けた。施術群のマウスのヒフク筋及びヒラメ筋を切除し(SA,Synergist Ablation手術)、足底筋への負荷を増加させた。
非施術群及び施術群のマウスを飼育し、施術群のマウスのヒフク筋及びヒラメ筋の切除から3日後に、各群のマウスから足底筋を採取した。採取した足底筋の筋量を測定し、すみやかに液体窒素で凍結し-80℃で保存した。測定した足底筋の筋量の結果を図4に示す。
Test Example 2 Measurement of the expression level of MYBPH when excising muscle was removed (1) Measurement of muscle mass of plantar muscle
C57BL / 6J mice (purchased from Oriental Bioservices, 16 weeks old) were divided into 3 non-treatment groups and 3 treatment groups. The muscle of the treatment group mice were resected (SA, Synergist Ablation operation), and the load on the plantar muscles was increased.
The mice in the non-operation group and the operation group were bred, and the plantar muscles were collected from the mice in each group three days after the removal of the hippocampus muscle and soleus muscle of the operation group mice. The muscle mass of the plantar muscle collected was measured, immediately frozen with liquid nitrogen and stored at -80.degree. The results of the measured plantar muscle mass are shown in FIG.
(2)MYBPH遺伝子の発現量の測定
保存した足底筋におけるMYBPHのmRNAの発現量を試験例1と同様にして測定した。得られた結果を図5に示す。
(2) Measurement of expression level of MYBPH gene The expression level of MYBPH mRNA in the stored plantar muscle was measured in the same manner as in Test Example 1. The obtained result is shown in FIG.
図4及び5に示すように、施術群で足底筋の重量が有意に増加するとともに、MYBPHのmRNAの発現量が有意に増加した。
足底筋の協働筋であるヒラメ筋及びヒフク筋を切除した場合、足底筋に大きな負荷がかかる。よって図4及び5に示す結果は、足底筋に大きな負荷をかけて短期間で筋量を増加させると、MYBPH遺伝子の発現量が増加することを示している。
As shown in FIGS. 4 and 5, the weight of the plantar muscles significantly increased in the treatment group, and the expression level of MYBPH mRNA significantly increased.
When the sole and muscle muscles which are the co-operating muscles of the plantar muscles are excised, the plantar muscles are heavily loaded. Therefore, the results shown in FIGS. 4 and 5 indicate that when a large load is applied to the plantar muscles to increase muscle mass in a short period, the expression level of the MYBPH gene increases.
試験例3 尾懸垂後再接地した場合のMYBPHの発現量の測定
Balb/cマウス(チャールス・リバー社より購入、10週齢)を1週間の尾懸垂により後肢不活動状態とし、その後尾懸垂を解除した(再接地)。再接地から3日後にヒラメ筋を採取した。
尾懸垂直後のマウス、及び再接地後のヒラメ筋の筋量を測定し、すみやかに液体窒素で凍結し-80℃で保存した。測定したヒラメ筋の筋量の結果を図6に示す。
Test Example 3 Measurement of the expression level of MYBPH when regrounding after tail suspension
Balb / c mice (purchased from Charles River, 10 weeks old) were rendered hindlimb inactive by 1 week of tail suspension, and then the tail suspension was released (reground). The soleus muscle was collected three days after re-contacting.
The muscle mass of the mouse after tail suspension vertical and reground was measured, and immediately frozen with liquid nitrogen and stored at -80 ° C. The results of the measured muscle mass of the flatfish muscle are shown in FIG.
(2)MYBPH遺伝子の発現量の測定
保存したヒラメ筋におけるMYBPHのmRNAの発現量を試験例1と同様にして測定した。得られた結果を図7に示す。
(2) Measurement of the expression level of MYBPH gene The expression level of MYBPH mRNA in the stored flatfish muscle was measured in the same manner as in Test Example 1. The obtained result is shown in FIG.
図6及び7に示すように、再接地後にヒラメ筋の重量が有意に増加するとともに、MYBPHのmRNAの発現量が有意に増加した。
これらの結果は、尾懸垂直後に再接地させることで短期間で筋量を増加させた場合、筋量増加とともにMYBPH遺伝子の発現量も増加することを示している。
As shown in FIGS. 6 and 7, the weight of soleus muscle significantly increased and the expression level of MYBPH mRNA significantly increased after re-grounding.
These results indicate that when the muscle mass is increased in a short period by re-grounding after tail-clamp vertical, the expression level of the MYBPH gene increases with the muscle mass increase.
試験例4 骨格筋細胞の増殖・分化過程におけるMYBPHの発現量の測定
(1)mRNAの発現量の測定
マウス骨格筋由来細胞株C2C12を0.5×105cells/well又は1.5×105cells/wellとなるように24wellプレートに播種し(n=3)、10%ウシ胎児血清(FBS、AusGeneX)を含むDMEM培地{増殖培地、10%FBS-DMEM;10%FBS、1%Pen Strep(Gibco社製)}を用いて維持した。細胞の播種から24時間後に、増殖区の細胞サンプルを回収した(細胞密度:80%コンフルエント)。さらに、分化誘導区の細胞サンプルに対して、播種から48時間後(細胞密度:100%コンフルエント)に2%ウマ血清(HS、Kohjin Bio社製)を含むDMEM培地{分化誘導培地、2%HS-DMEM;2%HS、1%Pen Strep(Gibco社製)}を用いて分化誘導を施した。分化誘導から0時間後、8時間後、24時間後、48時間後、72時間後、及び120時間後に細胞サンプルを回収した。
Test Example 4 Measurement of expression level of MYBPH during proliferation and differentiation of skeletal muscle cells (1) Measurement of expression level of mRNA 0.5 × 10 5 cells / well or 1.5 × 10 5 cells / well of mouse skeletal muscle cell line C2C12 Seed in a 24-well plate (n = 3) to obtain 10% fetal bovine serum (FBS, AusGeneX) in DMEM medium {growth medium, 10% FBS-DMEM; 10% FBS, 1% Pen Strep (Gibco) Made) to maintain. Twenty four hours after cell seeding, cell samples in the growth zone were collected (cell density: 80% confluence). Furthermore, DMEM medium containing 2% horse serum (HS, manufactured by Kohjin Bio) at 48 hours after seeding (cell density: 100% confluent) with respect to the cell sample of differentiation induction zone {differentiation induction medium, 2% HS Differentiation induction was performed using -DMEM; 2% HS, 1% Pen Strep (manufactured by Gibco)}. Cell samples were collected at 0, 8, 24, 48, 72 and 120 hours after induction of differentiation.
回収した細胞サンプルからRNeasy Mini Kit(商品名、QIAGEN社製)を使用してTotal RNAを抽出し、試験例1と同様に逆転写反応を行い、MYBPHのmRNAの発現量の解析を行った。得られた解析結果は、配列番号5及び6に示す塩基配列からなるプライマーを用いて測定した内部標準遺伝子(GAPDH)の発現量で補正し、相対的なmRNA発現量として表した。このようにして得られた結果を図8に示す。
図8に示すように、MYBPHのmRNAの発現量は、骨格筋細胞の分化と共に増加した。
Total RNA was extracted from the collected cell sample using RNeasy Mini Kit (trade name, manufactured by QIAGEN), reverse transcription was performed in the same manner as in Test Example 1, and expression amount of MYBPH mRNA was analyzed. The analysis results obtained were corrected with the expression level of the internal standard gene (GAPDH) measured using the primers consisting of the base sequences shown in SEQ ID NOS: 5 and 6, and expressed as relative mRNA expression levels. The results obtained in this way are shown in FIG.
As shown in FIG. 8, the expression level of MYBPH mRNA increased with the differentiation of skeletal muscle cells.
(2)タンパク質の発現量の測定
マウス骨格筋由来細胞株C2C12を0.5×105cells/well又は1.5×105cells/wellとなるように6wellプレートに播種し(n=3)、10%ウシ胎児血清(FBS、AusGeneX)を含むDMEM培地{増殖培地、10%FBS-DMEM;10%FBS、1%Pen Strep(Gibco社製)}を用いて維持した。細胞の播種から24時間後に、増殖区の細胞サンプルを回収した(細胞密度:80%コンフルエント)。さらに、分化誘導区の細胞サンプルに対して、播種から48時間後(細胞密度:100%コンフルエント)に2%ウマ血清(HS、Kohjin Bio社製)を含むDMEM培地{分化誘導培地、2%HS-DMEM;2%HS、1%Pen Strep(Gibco社製)}を用いて分化誘導を施した。分化誘導から0時間後、8時間後、24時間後、48時間後、72時間後、及び120時間後に細胞サンプルを回収した。
(2) Measurement of protein expression level Seed the mouse skeletal muscle cell line C2C12 in a 6-well plate at 0.5 × 10 5 cells / well or 1.5 × 10 5 cells / well (n = 3), 10% bovine Maintenance was carried out using DMEM medium (growth medium, 10% FBS-DMEM; 10% FBS, 1% Pen Strep (Gibco)) containing fetal serum (FBS, AusGeneX). Twenty four hours after cell seeding, cell samples in the growth zone were collected (cell density: 80% confluence). Furthermore, DMEM medium containing 2% horse serum (HS, manufactured by Kohjin Bio) at 48 hours after seeding (cell density: 100% confluent) with respect to the cell sample of differentiation induction zone {differentiation induction medium, 2% HS Differentiation induction was performed using -DMEM; 2% HS, 1% Pen Strep (manufactured by Gibco)}. Cell samples were collected at 0, 8, 24, 48, 72 and 120 hours after induction of differentiation.
回収した各細胞サンプルと、1% Protease Inhibitor Cocktail(Sigma社製)を含むCelLytic M(商品名、Sigma社製)100μLとを混合し、-80℃で保存した。得られた混合液を15,000rpm、4℃で15分間遠心し、上清を回収してタンパク質溶液を調製した。タンパク質溶液の濃度を揃え、4×SDS Sample Bufferを添加し{タンパク質溶液:(Sample Buffer)=3:1(体積比))、99℃で10分間の熱処理を行った。 Each collected cell sample was mixed with 100 μL of CelLytic M (trade name, manufactured by Sigma) containing 1% Protease Inhibitor Cocktail (manufactured by Sigma), and stored at -80 ° C. The resulting mixture was centrifuged at 15,000 rpm and 4 ° C. for 15 minutes, and the supernatant was collected to prepare a protein solution. The concentration of the protein solution was made uniform, 4 × SDS Sample Buffer was added, {protein solution: (Sample Buffer) = 3: 1 (volume ratio), and heat treatment was performed at 99 ° C. for 10 minutes.
SDSで処理したタンパク質溶液20μgについてSDS-PAGEを行い、メタノールで1分間処理したImmobilon-P(PVDF膜、Millipore社製)にタンパク質を転写した。転写後のPVDF膜をPVDF Blocking Reagent(商品名、TOYOBO社製)に浸し、室温にて1時間ブロッキング処理を行った。その後、抗MYBPH抗体(1:1000、Aviva Systems Biology社製)、抗ミオシン重鎖(以下、「MyHC」ともいう)抗体(1:600、Developmental studies hybridoma bank社製;MF20)、抗β-actin抗体(1:1000、Cell Signaling社製)を、それぞれに適した濃度になるようCan Get Signal Solution 1(商品名、TOYOBO社製)で希釈し、これらの溶液を用いて4℃、1晩の1次抗体処理を行った。
0.1%Tween20-TBS(T-TBS、Bio-Rad社製)でメンブレンを洗浄した後、抗ウサギIgG抗体(MYBPH及びβ-actin検出用、Cell signaling社製)又は抗マウスIgG抗体(MyHC検出用、Cell signaling社製)をCan Get Signal Solution 2(商品名、TOYOBO社製)で1000倍に希釈して2次抗体溶液を作成し、室温にて1時間の2次抗体処理を行った。T-TBSによる洗浄後、LumiGLO Reagent and Peroxide(商品名、Cell signaling社製)で5分間処理して化学発光を起こし、発光強度をChemiDoc XRS(商品名、Bio Rad社製)で測定し、MYBPH、MyHC及びβ-actinの発現量とした。
The SDS-PAGE was performed on 20 μg of the protein solution treated with SDS, and the protein was transferred to Immobilon-P (PVDF membrane, manufactured by Millipore) treated with methanol for 1 minute. The PVDF membrane after transfer was immersed in PVDF Blocking Reagent (trade name, manufactured by TOYOBO) and subjected to blocking treatment at room temperature for 1 hour. After that, anti-MYBPH antibody (1: 1000, manufactured by Aviva Systems Biology), anti-myosin heavy chain (hereinafter also referred to as "MyHC") antibody (1: 600, Developmental studies hybrida bank; MF20), anti-β-actin The antibody (1: 1000, made by Cell Signaling) is diluted with Can Get Signal Solution 1 (trade name, made by TOYOBO) to a concentration suitable for each, and these solutions are used overnight at 4 ° C. Primary antibody treatment was performed.
After washing the membrane with 0.1% Tween 20-TBS (T-TBS, manufactured by Bio-Rad), an anti-rabbit IgG antibody (for detecting MYBPH and β-actin, manufactured by Cell signaling) or an anti-mouse IgG antibody (for detecting MyHC) A second antibody solution was prepared by diluting Cell Signaling Co., Ltd.) with Can Get Signal Solution 2 (trade name, manufactured by TOYOBO) 1000 times to prepare a secondary antibody solution, and the secondary antibody treatment was performed at room temperature for 1 hour. After washing with T-TBS, treatment with LumiGLO Reagent and Peroxide (trade name, manufactured by Cell signaling) for 5 minutes causes chemiluminescence, and the luminescence intensity is measured by ChemiDoc XRS (trade name, manufactured by Bio Rad), MYBPH , MyHC and β-actin expression levels.
このようにして得られた結果を図9に示す。ここで図9の上段はβ-actinの発現量に対するMYBPHの発現量の相対値を示すグラフであり、下段はSDS-PAGE後にウェスタンブロッティングを実施し化学発光でバンドを検出した結果を示す図である。
図9に示すように、MYBPHの発現量は筋細胞の分化と共に増加した。
The results obtained in this way are shown in FIG. Here, the upper part of FIG. 9 is a graph showing the relative value of the expression level of MYBPH to the expression level of β-actin, and the lower part is a chart showing the result of Western blotting performed after SDS-PAGE and the bands detected by chemiluminescence. is there.
As shown in FIG. 9, the expression level of MYBPH increased with myocyte differentiation.
試験例5 骨格筋細胞におけるMYBPHの発現量及び筋管肥大に対する、RNA干渉の影響
(1)siRNA溶液の調製
MYBPHのmRNAに特異的に設計された、2種類のsmall interfering RNA(siRNA)溶液(MYBPH siRNA1及びMYBPH siRNA2)(Silencer Select Pre-designed and Validated siRNA、Ambion社製)及びネガティブコントロールsiRNA溶液(Silencer Select Negative Control #2 siRNA、Ambion社製)を終濃度25nMとなるよう調製した。
Test Example 5 Effect of RNA interference on expression level of MYBPH in skeletal muscle cells and myotube hypertrophy (1) Preparation of siRNA solution
Two small interfering RNA (siRNA) solutions (MYBPH siRNA 1 and MYBPH siRNA 2) (Silencer Select Pre-designed and Validated siRNA, manufactured by Ambion) and negative control siRNA solution (Silencer Select) specifically designed for mRNA of MYBPH Negative Control # 2 siRNA (manufactured by Ambion) was prepared to a final concentration of 25 nM.
(2)mRNAの発現量の測定
マウス骨格筋由来細胞株C2C12を0.5×105cells/well又は1.5×105cells/wellとなるように24wellプレートに播種した(n=3)。
細胞の播種から24時間後に、前記siRNA溶液及びLipofectamine RNAiMAX Transfection Reagent(商品名、Invitrogen社製)を用い、添付のプロトコルに従って下記に示すように、C2C12へsiRNAのトランスフェクションを実施した。
まず抗生物質無添加の10% FBS-DMEM 500μLを24wellプレートに添加した。そして、20μM siRNA保存溶液をOpti-MEM I Reduced-Serum Medium(商品名、Invitrogen社製)で300μM(終濃度の12倍)となるよう希釈した。一方Lipofectamine RNAiMAX Transfection ReagentをOpti-MEM I Reduced-Serum Medium(商品名、Invitrogen社製)で2%となるよう希釈した。これらの溶液それぞれを5分間静置した。その後両溶液を混合し、10分間反応させた。反応後の混合液100μLを24wellプレートに添加した。
(2) Measurement of mRNA Expression The mouse skeletal muscle cell line C2C12 was seeded on a 24 well plate at 0.5 × 10 5 cells / well or 1.5 × 10 5 cells / well (n = 3).
Twenty-four hours after seeding of the cells, C2C12 was transfected with siRNA using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen) according to the attached protocol as described below.
First, 500 μL of 10% FBS-DMEM without antibiotics was added to a 24 well plate. Then, the 20 μM siRNA stock solution was diluted with Opti-MEM I Reduced-Serum Medium (trade name, manufactured by Invitrogen) to a concentration of 300 μM (12 times the final concentration). On the other hand, Lipofectamine RNAiMAX Transfection Reagent was diluted to 2% with Opti-MEM I Reduced-Serum Medium (trade name, manufactured by Invitrogen). Each of these solutions was allowed to sit for 5 minutes. Both solutions were then mixed and allowed to react for 10 minutes. 100 μL of the mixed solution after reaction was added to a 24 well plate.
siRNAトランスフェクション開始から24時間後(細胞密度:100%コンフルエント)、2%HS-DMEMを用いて細胞に分化誘導を施し、48、72、120時間後に細胞サンプルを回収した。
回収した細胞サンプルから、RNeasy Mini Kit(商品名、QIAGEN社製)を使用してTotal RNAを抽出し、試験例1と同様に逆転写反応を行い、MYBPHのmRNAの発現量の解析を行った。得られた解析結果は、内部標準遺伝子(GAPDH)の発現量で補正し、相対的なmRNA発現量として表した。
このようにして得られた結果を図10に示す。図10に示すように、RNA干渉により、MYBPHのmRNAの発現量は減少した。
Twenty-four hours after initiation of siRNA transfection (cell density: 100% confluence), the cells were induced to differentiate using 2% HS-DMEM, and cell samples were collected 48, 72, 120 hours later.
Total RNA was extracted from the collected cell sample using RNeasy Mini Kit (trade name, manufactured by QIAGEN), reverse transcription was performed in the same manner as in Test Example 1, and expression amount of MYBPH mRNA was analyzed. . The analysis results obtained were corrected with the expression level of the internal standard gene (GAPDH) and expressed as a relative mRNA expression level.
The results obtained in this way are shown in FIG. As shown in FIG. 10, RNA interference reduced the expression level of MYBPH mRNA.
(3)タンパク質の発現量の測定
マウス骨格筋由来細胞株C2C12を0.5×105cells/well又は1.5×105cells/wellとなるように6wellプレートに播種した(n=3)。細胞の播種から24時間後に、前記siRNA溶液及びLipofectamine RNAiMAX Transfection Reagent(商品名、Invitrogen社製)を用いて前述の方法と同様にC2C12へsiRNAのトランスフェクションを実施した。そして、siRNAトランスフェクション開始から24時間後(細胞密度:100%コンフルエント)、2%HS-DMEMを用いて細胞に分化誘導を施し、48、72、120時間後に細胞サンプルを回収した。
回収した各細胞サンプルについて、試験例2と同様にMYBPH、MyHC及びβ-actinの発現量を測定した。このようにして得られた結果を図11(MYBPHの発現量)及び図12(MyHCの発現量)に示す。
図11及び12に示すように、RNA干渉により、MYBPH、及び骨格筋細胞の分化マーカーであるMyHCの発現量は減少した。
(3) Measurement of protein expression amount The mouse skeletal muscle-derived cell line C2C12 was seeded on a 6-well plate at 0.5 × 10 5 cells / well or 1.5 × 10 5 cells / well (n = 3). Twenty-four hours after seeding of the cells, C2C12 was transfected with siRNA in the same manner as described above using the siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, Invitrogen). Then, 24 hours after initiation of siRNA transfection (cell density: 100% confluence), differentiation induction was performed on the cells using 2% HS-DMEM, and cell samples were collected 48, 72, 120 hours later.
For each cell sample collected, the expression levels of MYBPH, MyHC and β-actin were measured in the same manner as in Test Example 2. The results thus obtained are shown in FIG. 11 (expression level of MYBPH) and FIG. 12 (expression level of MyHC).
As shown in FIGS. 11 and 12, RNA interference reduced the expression levels of MYBPH and MyHC, which is a differentiation marker of skeletal muscle cells.
(4)ミオシン重鎖の検出
マウス骨格筋由来細胞株C2C12を0.5×105cells/well又は1.5×105cells/wellとなるように6wellプレートに播種した(n=3)。細胞の播種から24時間後に、前記siRNA溶液及びLipofectamine RNAiMAX Transfection Reagent(商品名、Invitrogen社製)を用い前述の方法と同様にC2C12へsiRNAのトランスフェクションを実施した。そして、siRNAトランスフェクション開始から24時間後(細胞密度:100%コンフルエント)、2%HS-DMEMを用いて細胞に分化誘導を施し、48、72、120時間後に細胞サンプルを回収した。
(4) Detection of Myosin Heavy Chain The mouse skeletal muscle-derived cell line C2C12 was seeded on a 6-well plate at 0.5 × 10 5 cells / well or 1.5 × 10 5 cells / well (n = 3). Twenty-four hours after seeding of the cells, C2C12 was transfected with siRNA using the above-mentioned siRNA solution and Lipofectamine RNAiMAX Transfection Reagent (trade name, manufactured by Invitrogen) in the same manner as described above. Then, 24 hours after initiation of siRNA transfection (cell density: 100% confluence), differentiation induction was performed on the cells using 2% HS-DMEM, and cell samples were collected 48, 72, 120 hours later.
カルチャーカバーグラス(Poly-L-Lysine coat、松浪硝子工業社製)を底面に置き、Fibronectin(100倍希釈で使用、Sigma社製)でコーティングを行った6wellプレートに、回収した細胞を播種した。4%パラホルムアルデヒド・りん酸緩衝液(和光純薬工業社製)で細胞の固定した後、PBS(GIBCO社製)で細胞の洗浄を行った。その後、0.2% TritonX-100(Sigma社製)-PBS溶液を用いて、室温にて10分間細胞の可溶化処理を行った。PBSによる洗浄後、5%BSA(Sigma社製)-PBS溶液を用いて、室温にて30分間細胞のブロッキング処理を行った。その後、6μg/mLとなるよう抗MyHC抗体(1:600、Developmental studies hybridoma bank社製;MF20)を5%BSA-PBSで希釈し、この溶液を用いて室温にて1時間処理した。
PBSで洗浄した後、2次抗体溶液{Alexa Fluor 488 Donkey Anti-mouseIgG(Invitrogen社製)を5%BSA-PBSにより500倍に希釈した溶液}を用い、1時間の2次抗体処理を行った。
PBSによる洗浄後、Prolong Gold antifade reagent with DAPI(Invitrogen社製)を用いて細胞を封入し、細胞の形態観察と写真撮影を行い、筋管直径を測定した。筋管直径の測定は、オールインワン顕微鏡(商品名:BZ-9000、キーエンス社製)と付属の画像解析ソフトウェアを用い、1wellにつき5枚撮影した画像について、画像1枚あたり30本程度の筋管直径をそれぞれ測定し、平均値を算出した。
Culture cover glass (Poly-L-Lysine coat, manufactured by Matsunami Glass Co., Ltd.) was placed on the bottom, and the recovered cells were seeded on a 6-well plate coated with Fibronectin (used at 100-fold dilution, manufactured by Sigma). After fixing the cells with 4% paraformaldehyde phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.), the cells were washed with PBS (manufactured by GIBCO). Thereafter, the cells were subjected to a solubilization treatment for 10 minutes at room temperature using a 0.2% Triton X-100 (manufactured by Sigma) -PBS solution. After washing with PBS, the cells were blocked for 30 minutes at room temperature using a 5% BSA (Sigma) -PBS solution. Thereafter, anti-MyHC antibody (1: 600, Developmental studies hybrid bank; MF20) was diluted with 5% BSA-PBS to 6 μg / mL, and this solution was used at room temperature for 1 hour.
After washing with PBS, secondary antibody treatment was performed for 1 hour using a secondary antibody solution {Alexa Fluor 488 Donkey Anti-mouse IgG (manufactured by Invitrogen) diluted 500 folds with 5% BSA-PBS} .
After washing with PBS, the cells were sealed using Prolong Gold antifade reagent with DAPI (manufactured by Invitrogen), and morphological observation and photography of the cells were performed to measure the myotube diameter. The myotube diameter was measured using an all-in-one microscope (trade name: BZ-9000, manufactured by Keyence Corporation) and the attached image analysis software. About 30 images of myotube diameter per image for 5 images taken per well Were measured, and the average value was calculated.
分化誘導から48時間後、72時間後及び120時間後の細胞の顕微鏡写真を図13に示す。ネガティブコントロールsiRNA溶液で処理した場合、分化誘導に伴い筋管が太くなった(例えば図13(b))。これに対し、MYBPH siRNA溶液で処理した場合、筋管が太くなりきれないまま、筋管が培養限界に達して剥がれてしまった(例えば図13(c)参照)。このように、MYBPHのmRNAの発現をRNA干渉により抑制することで、筋管の肥大が抑制された。
また、分化誘導から72時間後及び120時間後の平均筋管直径を図14に示す。図14に示すように、MYBPHのmRNAの発現をRNA干渉により抑制することで平均筋管直径が減少し、筋管の肥大が抑制された。
Photomicrographs of the cells 48 hours, 72 hours and 120 hours after induction of differentiation are shown in FIG. When treated with a negative control siRNA solution, myotubes became thick with induction of differentiation (for example, FIG. 13 (b)). On the other hand, when treated with the MYBPH siRNA solution, the myotube reached the culture limit and peeled off without being able to thicken the myotube (see, for example, FIG. 13 (c)). Thus, by suppressing the expression of MYBPH mRNA by RNA interference, hypertrophy of myotubes was suppressed.
Also, the mean myotube diameter 72 hours and 120 hours after induction of differentiation is shown in FIG. As shown in FIG. 14, by suppressing the expression of MYBPH mRNA by RNA interference, the average myotube diameter was decreased, and myotube hypertrophy was suppressed.
試験例6 ゼブラフィッシュにおけるMYBPH遺伝子のノックアウト試験
(1)ゼブラフィッシュのMYBPH遺伝子の検索
マウス由来のMYBPH遺伝子と相同性を有する遺伝子をNCBI(National Center for Biotechnology Information、http://www.ncbi.nlm.nih.gov/)のHomoloGene検索を利用し、ゼブラフィッシュのMYBPH遺伝子の検索を行った。その結果ゼブラフィッシュは、それぞれ異なる染色体に存在する、2つの相同遺伝子(MYBPHa遺伝子及びMYBPHb遺伝子)を有することを見出した。さらに、MYBPHa遺伝子及びMYBPHb遺伝子の開始コドン近傍のシークエンスを行った。その結果、これらの遺伝子の塩基配列が、NCBI(http://www.ncbi.nlm.nih.gov/)及びEnsembl(http://asia.ensembl.org/index.html)の情報と差異がないことを確認した。
そこで下記に示すように、これら2つの遺伝子全てをノックアウトした、ダブルノックアウトゼブラフィッシュを作製した。
Test Example 6 Knockout Test of MYBPH Gene in Zebrafish (1) Search for MYBPH Gene in Zebrafish A gene having homology to the mouse-derived MYBPH gene is identified by NCBI (National Center for Biotechnology Information, http: //www.ncbi.nlm The Zebrafish MYBPH gene was searched using the HomoloGene search of .nih.gov /). As a result, it was found that zebrafish has two homologous genes (MYBPHa gene and MYBPHb gene) present in different chromosomes. Furthermore, sequencing was performed in the vicinity of the start codons of the MYBPHa gene and the MYBPHb gene. As a result, the base sequences of these genes are different from the information and differences of NCBI (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://asia.ensembl.org/index.html). I confirmed that there was not.
Therefore, as shown below, a double knockout zebrafish was created in which all these two genes were knocked out.
(2)TALEN mRNAの合成
MYBPHa遺伝子及びMYBPHb遺伝子のノックアウト用TALエフェクターヌクレアーゼ(TALEN)をコードしたプラスミド(和光純薬工業社製)を増幅し、増幅したTALENプラスミドを制限酵素処理反応液{各TALENプラスミド10μL、10×M buffer(TOYOBO社製)5μL、dH2O 34μL、制限酵素HindIII(TOYOBO社製)1μL}を用いて37℃で一晩制限酵素処理した。
得られた消化物をQIAquick PCR Purification Kit(商品名、Qiagen社製)を用いて精製した。そして、精製液を鋳型とし、mMESSAGE mMACHINE Kit(商品名、Applied Biosystems社製)を用いてRNA合成、キャップ構造付加、及びPolyA付加を行ったTALEN mRNAを作製した。ここで、混入したプラスミドを分解するためにDNase処理を行なった。
このようにして得られた産物をRNeasy MiniElute Cleanup Kit(商品名、Qiagen社製)を用いて精製し、TALEN mRNA精製液を得た。なお、TALEN mRNA精製液についてAgilent 2100 バイオアナライザ(商品名、Agilent Technologies社製)にて解析し、RNAが分解されていないことを確認した。
(2) Synthesis of TALEN mRNA
A plasmid (manufactured by Wako Pure Chemical Industries, Ltd.) encoding a TAL effector nuclease (TALEN) for knockout of MYBPHa gene and MYBPHb gene is amplified, and the amplified TALEN plasmid is treated with a restriction enzyme-treated reaction solution {10 μL of each TALEN plasmid, 10 × M buffer The mixture was subjected to restriction enzyme treatment overnight at 37 ° C. using 5 μL (manufactured by TOYOBO), 34 μL dH 2 O, and 1 μL of restriction enzyme Hin dIII (manufactured by TOYOBO).
The obtained digested product was purified using QIAquick PCR Purification Kit (trade name, manufactured by Qiagen). Then, using the purified solution as a template, TALEN mRNA to which RNA synthesis, cap structure addition, and PolyA addition were performed using mMESSAGE mMACHINE Kit (trade name, manufactured by Applied Biosystems) was prepared. Here, DNase treatment was performed to degrade the contaminating plasmid.
The product thus obtained was purified using RNeasy MiniElute Cleanup Kit (trade name, manufactured by Qiagen) to obtain a TALEN mRNA purified solution. The TALEN mRNA purified solution was analyzed with an Agilent 2100 bioanalyzer (trade name, manufactured by Agilent Technologies) to confirm that the RNA was not degraded.
(3)TALEN mRNAのマイクロインジェクション
MYBPHa特異的TALEN mRNA及びMYBPHb特異的TALEN mRNAの両方を含むインジェクション溶液{TALEN(L)MYBPHa mRNA 3μL、TALEN(R)MYBPHa mRNA 3μL、TALEN(L)MYBPHb mRNA 3μL、TALEN(R)MYBPHb mRNA 3μL、フェノールレッド溶液 2μL、dH2O 6μL}を調製し、1細胞期の胚(卵黄を除く)に数nLずつゼブラフィッシュ100匹程度にインジェクションした。ここで、TALEN mRNAを含まないインジェクション溶液を打ち込むコントロール群100匹程度に対しても、同様のインジェクション操作を行った。
インジェクション後の胚(受精卵)には、未受精卵が混入することがある。また、インジェクション操作によりダメージを受け、死卵が発生することがある。そこで、1日につき2回ずつ、死卵の除去を行なった。
(3) Microinjection of TALEN mRNA
Injection solution containing both MYBPHa-specific TALEN mRNA and MYBPHb-specific TALEN mRNA {3 μL of TALEN (L) MYBPHa mRNA, 3 μL TALEN (R) MYBPHa mRNA, 3 μL TALEN (L) MYBPHb mRNA, 3 μL TALEN (R) MYBPHb mRNA 3 μL, 2 μL of phenol red solution, 6 μL of dH 2 O} was prepared, and several nL of each was injected into an embryo of one cell stage (excluding egg yolk) into about 100 zebrafish. Here, the same injection operation was performed on about 100 control groups into which an injection solution containing no TALEN mRNA was injected.
The embryo after injection (fertilized egg) may be contaminated with unfertilized egg. Moreover, damage may be caused by the injection operation and dead eggs may be generated. Therefore, dead eggs were removed twice a day.
受精から5日後に、顕微鏡下で仔魚の形態及び行動を観察し、動画撮影を行った。その結果を図15に示す。ここで図15(a)はコントロール群のゼブラフィッシュの顕微鏡写真を示し、図15(b)はTALEN mRNAをマイクロインジェクションしたゼブラフィッシュの顕微鏡写真を示す。
図15(a)に示すコントロール群のゼブラフィッシュは形態及び行動はいずれも正常であった。これに対して図15(b)に示すように、ゼブラフィッシュにTALEN mRNAをマイクロインジェクションした場合、運動能力の低い個体が出現することが確認された。
Five days after fertilization, the morphology and behavior of the larva were observed under a microscope, and a moving image was taken. The results are shown in FIG. Here, FIG. 15 (a) shows a photomicrograph of the zebrafish in the control group, and FIG. 15 (b) shows a photomicrograph of the zebrafish microinjected with TALEN mRNA.
The morphology and behavior of the zebrafish in the control group shown in FIG. 15 (a) were both normal. On the other hand, as shown in FIG. 15 (b), it was confirmed that individuals with low motor ability appeared when microinjection of TALEN mRNA into zebrafish.
(4)T7エンドヌクレアーゼ1アッセイ
TALEN mRNAのマイクロインジェクション後5日間生育した仔魚のうち、コントロール群の個体から1匹、ダブルノックアウト群の個体から運動機能が低下した個体を3匹回収した。
回収したサンプルから、Dneasy blood & tissue mini kit(商品名、Qiagen社製)を用いて、ゲノムDNAを抽出した。抽出したゲノムDNAを鋳型とし、Tks Gflex DNA Polymerase(商品名、タカラバイオ社製)、並びにMYBPHa遺伝子検出用プライマー(配列番号7及び8)及びMYBPHb遺伝子検出用プライマー(配列番号9及び10)を含むPCR反応液{2×Gflex PCR buffer(商品名、タカラバイオ社製)1μL、100μM Forwardプライマー0.1μL、100μM Reverseプライマー0.1μL、dH2O 23μL、Tks Gflex DNA Polymerase(商品名、タカラバイオ社製)1μL}を用いてPCRを行い、MYBPHa遺伝子及びMYBPHb遺伝子の第1エキソン周辺を増幅した。PCR条件は、(94℃1分)→(98℃10秒→MYBPHa:54℃/MYBPHb:58℃ 15秒→68℃1分20秒)×38サイクル→68℃3分→4℃とした。
(4) T7 endonuclease 1 assay
Among the larvae grown for 5 days after microinjection of TALEN mRNA, 1 individual was recovered from the control group and 3 from the individual of the double knockout group with reduced motor function.
Genomic DNA was extracted from the collected sample using Dneasy blood & tissue mini kit (trade name, manufactured by Qiagen). Using extracted genomic DNA as a template, Tks Gflex DNA Polymerase (trade name, manufactured by Takara Bio Inc.), and primers for detecting MYBPHa gene (SEQ ID Nos. 7 and 8) and primers for detecting MYBPHb gene (SEQ ID Nos. 9 and 10) PCR reaction solution {2 x Gflex PCR buffer (trade name, manufactured by Takara Bio Inc.) 1 μL, 100 μM Forward primer 0.1 μL, 100 μM Reverse primer 0.1 μL, dH 2 O 23 μL, Tks Gflex DNA Polymerase (trade name, manufactured by Takara Bio Inc.) PCR was performed using 1 μl to amplify the vicinity of the first exon of the MYBPHa gene and the MYBPHb gene. The PCR conditions were (94 ° C. for 1 minute) → (98 ° C. for 10 seconds → MYBPHa: 54 ° C./MYBPHb: 58 ° C. for 15 seconds → 68 ° C. for 1 minute 20 seconds) × 38 cycles → 68 ° C. for 3 minutes → 4 ° C.
得られたPCR産物をQIAquick PCR Purification Kit(Qiagen社製)を用いて精製した。精製したPCR産物200ngを含むT7エンドヌクレアーゼ1反応液{10×NEB2 Buffer(NEW ENGLAND BioLabs社製)1.9μL、(dH2O+サンプル溶液):17.1μL}を調製し、95℃10分→(95℃から85℃まで-2℃/秒で冷却)→(85℃から25℃まで-0.3℃/秒で冷却)→25℃10秒→22℃のMelting反応及びre-annealing反応を行った。その後、各サンプル(19μL)にT7エンドヌクレアーゼ1(NEW ENGLAND BioLabs社製)を1μLずつ添加し、37℃15分のT7エンドヌクレアーゼ1処理を行った。
T7エンドヌクレアーゼ1処理を行ったサンプルについて、1%アガロースゲルを用いて電気泳動を行い、BioDoc-It 220 Imaging System(商品名、ビーエム機器社製)を用いてDNAバンドを撮影した。その結果を図16に示す。なお図16(a)はMYBPHa遺伝子について、図16(b)はMYBPHb遺伝子についての結果をそれぞれ示す。
The obtained PCR product was purified using QIAquick PCR Purification Kit (manufactured by Qiagen). Prepare T7 endonuclease 1 reaction solution {10 × NEB2 Buffer (manufactured by NEW ENGLAND BioLabs) 1.9 μL, (dH 2 O + sample solution): 17.1 μL} containing 200 ng of the purified PCR product, 95 ° C. for 10 minutes → (95 Melting reaction and re-annealing reaction were carried out from ° C to 85 ° C at -2 ° C / sec cooling → (cooling from 85 ° C to 25 ° C at -0.3 ° C / sec) → 25 ° C 10 sec to 22 ° C. Thereafter, 1 μL each of T7 endonuclease 1 (manufactured by NEW ENGLAND BioLabs) was added to each sample (19 μL), and T7 endonuclease 1 treatment was performed at 37 ° C. for 15 minutes.
The sample subjected to the T7 endonuclease 1 treatment was subjected to electrophoresis using a 1% agarose gel, and a DNA band was photographed using BioDoc-It 220 Imaging System (trade name, manufactured by BM Instruments Co., Ltd.). The results are shown in FIG. FIG. 16 (a) shows the results for the MYBPHa gene, and FIG. 16 (b) shows the results for the MYBPHb gene.
図16に示すように、コントロール個体から抽出したゲノムDNAには、ミスマッチのある2本鎖DNAが存在しない。すなわち、T7エンドヌクレアーゼ1によるゲノムDNAのミスマッチでの切断は起こらず、MYBPHa遺伝子及びMYBPHb遺伝子由来のバンドが複数検出されることはなかった。
一方、Talen mRNAによりMYBPHa遺伝子及びMYBPHb遺伝子をノックアウトした場合、運動機能が低下した全ての個体で2本鎖DNAにはミスマッチが存在する。そのため、ミスマッチが存在するDNAのミスマッチ部でT7エンドヌクレアーゼにより切断されるため、PCR増幅産物のバンドの他に、低分子量の小さいバンドが2本検出された。
As shown in FIG. 16, there is no mismatched double-stranded DNA in genomic DNA extracted from control individuals. That is, cleavage at a mismatch of genomic DNA by T7 endonuclease 1 did not occur, and multiple bands from MYBPHa gene and MYBPHb gene were not detected.
On the other hand, when the MYBPHa gene and the MYBPHb gene are knocked out by Talen mRNA, there is a mismatch in double-stranded DNA in all individuals with reduced motor function. Therefore, in addition to the band of the PCR amplification product, two small bands of low molecular weight were detected because T7 endonuclease is cleaved at the mismatched portion of the DNA where the mismatch exists.
以上のように、運動機能が低下した個体では骨格筋由来のMYBPH遺伝子の発現が抑制される。したがって、運動機能とMYBPH遺伝子の発現レベルとの間に相関性があることが確認された。 As described above, the expression of the MYBPH gene derived from skeletal muscle is suppressed in individuals with reduced motor function. Therefore, it was confirmed that there is a correlation between motor function and the expression level of the MYBPH gene.
試験例7 マウスの運動試験後のヒフク筋におけるMYBPH遺伝子の発現量の測定
10週齢の雄性C57BL/6Jマウス(日本チャールズリバーより購入)に対して1週間の予備飼育を行って環境に馴化させた後、トレッドミル走行運動に慣れさせるためのトレーニングを下記の通り5日間行った。
1日目:10m/分(15分)→15m/分(15分)
2日目:10m/分(10分)→15m/分(20分)
3日目:15m/分(15分)→20m/分(15分)
4日目:15m/分(5分)→20m/分(15分)→25m/分(10分)
5日目:15m/分(5分)→20m/分(10分)→25m/分(15分)
Test Example 7 Measurement of the Expression Level of MYBPH Gene in Hyukku Muscle after Exercise Test in Mice
A 10-week-old male C57BL / 6J mouse (purchased from Japan Charles River) is pre-reared for 1 week to acclimate to the environment, and then the training for getting used to treadmill exercise is as follows: went.
Day 1: 10 m / min (15 minutes) → 15 m / min (15 minutes)
Day 2: 10 m / min (10 minutes) → 15 m / min (20 minutes)
Third day: 15 m / min (15 minutes) → 20 m / min (15 minutes)
Fourth day: 15 m / min (5 minutes) → 20 m / min (15 minutes) → 25 m / min (10 minutes)
Day 5: 15 m / min (5 minutes) → 20 m / min (10 minutes) → 25 m / min (15 minutes)
その後、体重が等しくなるようにマウスを3群に分けた(N=4/群)。このうち2群には走行速度25m/分の走行運動を30分間行った。残りの1群は安静群とした。
運動群は運動1時間後及び6時間後に解剖し、各群のヒフク筋を採取した。
Then, the mice were divided into 3 groups so that their weights would be equal (N = 4 / group). In the second group, the running speed of 25 m / min was performed for 30 minutes. The remaining group was the resting group.
The exercise group was dissected one hour and six hours after the exercise, and the muscle of each group was collected.
RNA抽出キット(商品名:RNeasy Fibrous Kit、QIAGEN社製)を用いて、ヒフク筋からTotal RNAを抽出した。Total RNAの品質チェックには、BioAnalyzer(Agilent technology社製)を用い、全てのサンプルのRIN(RNA integrity number)値が7.0以上でマイクロアレイに十分な品質であることを確認した。
品質チェック後のTotal RNAを用いて以下の要領でマイクロアレイを実施した。アレイスライドはAgilent社製Mouse SurePrint G3を用いた。ラベル化cRNAの調製は、Agilent社のプロトコルに従って実施した。ハイブリダイゼーション及び洗浄後のアレイスライドは、速やかに窒素充填して遮光し、北海道システム・サイエンス株式会社に依頼してスキャンを行った。
スキャン後、QC reportから全てのアレイスライドに技術的な問題がないことを確認し、データの解析を行った。解析ソフトウェアGeneSpringを用い、Agilent推奨の75%法(Scaling)で正規化を実施した。その後、運動後1時間群、運動後6時間群におけるMYBPHのシグナル値を安静群と比較した。その結果を表3に示す。
Total RNA was extracted from calf muscle using an RNA extraction kit (trade name: RNeasy Fibrous Kit, manufactured by QIAGEN). For quality check of total RNA, using a BioAnalyzer (manufactured by Agilent technology), it was confirmed that the RIN (RNA integrity number) value of all the samples is 7.0 or more and the quality is sufficient for the microarray.
The microarray was implemented as follows using Total RNA after quality check. The array slide used Mouse SurePrint G3 by Agilent. Preparation of labeled cRNA was performed according to the protocol of Agilent. The array slide after hybridization and washing was immediately filled with nitrogen and shielded from light, and requested to Hokkaido System Science Co., Ltd. for scanning.
After scanning, QC reports confirmed that all array slides had no technical problems, and analyzed the data. Normalization was performed by the 75% method (Scaling) recommended by Agilent using analysis software GeneSpring. Then, the signal value of MYBPH in the 1 hour group after exercise and the 6 hour group after exercise was compared with the resting group. The results are shown in Table 3.
表3に示すように、マウスにトレッドミル運動を負荷すると、MYBPH遺伝子の発現量が増加した。 As shown in Table 3, when mice were subjected to treadmill exercise, the expression level of MYBPH gene was increased.
試験例1〜7に示すように、MYBPH又はMYBPH遺伝子の発現レベルと、運動機能との間に相関性がある。よって、MYBPH又はMYBPH遺伝子を含む本発明のマーカーは、運動機能、筋量、骨格筋細胞の分化の程度、及び筋管肥大の程度の指標とすることができる。 As shown in Test Examples 1 to 7, there is a correlation between the expression level of MYBPH or MYBPH gene and motor function. Thus, the marker of the present invention containing the MYBPH or MYBPH gene can be used as an indicator of the motor function, muscle mass, the degree of differentiation of skeletal muscle cells, and the degree of myotube hypertrophy.
Claims (4)
測定した発現レベルに基づいて筋管直径の変化による筋量変化を評価する、
筋量の評価方法。 Measuring the expression level of skeletal muscle-derived myosin binding protein H or a gene encoding the same collected from an individual;
Evaluate changes in muscle mass due to changes in myotube diameter based on measured expression levels,
How to assess muscle mass.
測定した発現レベルに基づいて筋管肥大の程度を評価する、
筋管肥大の評価方法。 Measuring the expression level of skeletal muscle-derived myosin binding protein H or a gene encoding the same collected from an individual;
Assess the degree of myotube hypertrophy based on the measured expression levels,
Evaluation method of myotube hypertrophy.
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