JP2016196440A - Cytoglobin expression enhancer - Google Patents
Cytoglobin expression enhancer Download PDFInfo
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- JP2016196440A JP2016196440A JP2015077604A JP2015077604A JP2016196440A JP 2016196440 A JP2016196440 A JP 2016196440A JP 2015077604 A JP2015077604 A JP 2015077604A JP 2015077604 A JP2015077604 A JP 2015077604A JP 2016196440 A JP2016196440 A JP 2016196440A
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- cytoglobin
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Abstract
Description
本発明は、サイトグロビン発現増強剤に関する。より具体的には、本発明は、サイトグロビンの発現を増強し、生体内でサイトグロビンによる有益な機能を効果的に発揮させるサイトグロビン発現増強剤。 The present invention relates to a cytoglobin expression enhancer. More specifically, the present invention relates to a cytoglobin expression enhancer that enhances cytoglobin expression and effectively exerts a beneficial function of cytoglobin in vivo.
近年、肝癌患者数の増大が問題となっている。２０１２年度国立がん研究センターの報告によると、国内の肝癌死亡者数は男女ともに全癌中４位であり、２０１０年には年間３３０００人にも及んでいる。肝癌は、Ｂ型、Ｃ型肝炎ウイルス感染、多量飲酒や糖尿病肥満と関連する非アルコール性脂肪性肝炎（ＮＡＳＨ）を土台として発生する。即ち、肝癌は慢性炎症と線維化肝を母地として生じ、病因の如何を問わず年率８％で発癌し、一旦肝癌が生じると再発と肝内転移を繰り返す。肝硬変とは、肝実質がＩ型コラーゲンなどの細胞外マトリックス蛋白で置換されて機能的肝細胞が減少する病態である。この線維性肝臓は、生理的状態ではビタミンＡ貯蔵を主機能とする肝星細胞（Ｈｅｐａｔｉｃ ｓｔｅｌｌａｔｅ ｃｅｌｌ、ＨＳＣ）が活性化して形質を変えた筋線維芽細胞（Ｍｙｏｆｉｂｒｏｂｌａｓｔ、ＭＦＢ）で肝実質が置換される病態である。この形質転換には、トランスフォーミング増殖因子−β（Ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ（ＴＧＦ）−β）や結合組織成長因子（ｃｏｎｎｅｃｔｉｖｅ ｔｉｓｓｕｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ、ＣＴＧＦ）が関与し、これらの因子が肝星細胞の持続活性化や実質での筋線維芽細胞の増加が肝細胞機能を低下させる要因であり、肝癌発症に寄与することが報告されている（非特許文献１及び２）。そのため、肝星細胞の活性化抑制と筋線維芽細胞の制御が肝線維化及び肝癌の治療法開発に繋がると考えられている。 In recent years, an increase in the number of liver cancer patients has become a problem. According to a report from the 2012 National Cancer Center, the number of deaths from liver cancer in Japan is 4th among all cancers, reaching 33,000 per year in 2010. Liver cancer develops based on non-alcoholic steatohepatitis (NASH) associated with hepatitis B and C virus infection, heavy drinking and obesity in diabetes. In other words, liver cancer is caused by chronic inflammation and fibrotic liver as the base, and it occurs at an annual rate of 8% regardless of the etiology. Once liver cancer occurs, recurrence and intrahepatic metastasis are repeated. Cirrhosis is a condition in which the liver parenchyma is replaced with an extracellular matrix protein such as type I collagen, resulting in a decrease in functional hepatocytes. This fibrotic liver is replaced with myofibroblasts (MFB), which are activated by hepatic stellate cells (HSC) whose main function is vitamin A storage in a physiological state, and whose characteristics are changed. Is a pathological condition. In this transformation, transforming growth factor-β (Transforming growth factor (TGF) -β) and connective tissue growth factor (CTGF) are involved, and these factors activate hepatic stellate cells continuously. It has been reported that an increase in myofibroblasts in the body and parenchyma is a factor that decreases the function of hepatocytes and contributes to the development of liver cancer (Non-patent Documents 1 and 2). Therefore, suppression of hepatic stellate cell activation and control of myofibroblasts are thought to lead to the development of treatment methods for liver fibrosis and liver cancer.
一方、本発明者等は、ラットＨＳＣのプロテオミクス解析で、Ｓｔｅｌｌａｔｅ ｃｅｌｌ ａｃｔｉｖａｔｉｏｎ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎを発見し、第１７番染色体のヒト遺伝子も同定した。このタンパク質は、今ではサイトグロビン（Ｃｙｔｏｇｌｏｂｉｎ、ＣＹＧＢ）と呼ばれており、ヘモグロビン、ミオグロビン、ニューログロビンに次ぐ哺乳類第４番目のグロビンとして位置付けられている（非特許文献３及び４）。 On the other hand, the inventors of the present invention have found a cell-activated-associated protein by proteomic analysis of rat HSC, and have also identified the human gene of chromosome 17. This protein is now called cytoglobin (CYGBin) and is positioned as the fourth mammalian globin following hemoglobin, myoglobin, and neuroglobin (Non-patent Documents 3 and 4).
サイトグロビンは、肝星細胞のみならず、膵臓（膵星細胞）や腎臓の尿細管上皮近傍の線維芽細胞等にも発現していることが知られており、これまでに、肝臓以外の生体組織でも、重要な役割を果たしていることが報告されている。 Cytoglobin is known to be expressed not only in hepatic stellate cells but also in pancreas (pancreatic stellate cells) and fibroblasts in the vicinity of renal tubular epithelium. Organizations have also been reported to play an important role.
例えば、特許文献１には、サイトグロビンを欠損させたマウスが肝発癌物質であるジエチルニトロサミン投与に対して易発癌性を示し、サイトグロビンが肝癌の予防乃至治療作用を発揮していることが報告されており、サイトグロビンは、肝癌の予防乃至治療に有効であると考えられている。また、非特許文献５には、サイトグロビンには腫瘍の抑制作用を示すことが報告されている。非特許文献６には、サイトグロビンが、酸素や一酸化窒素等と結合して生体内ガスのリザーバーとして機能していることが報告されている。また、非特許文献７には、サイトグロビンが低酸素誘導因子（Ｈｙｐｏｘｉａ−ｉｎｄｕｃｉｂｌｅ Ｆａｃｔｏｒ、ＨＩＦ）によって転写調節され、酸素センサーとなることも報告されている。更に、非特許文献８には、サイトグロビンには、ペルオキシダーゼ活性により過酸化水素を分解して細胞内酸化ストレス代謝を調節する役割を担っていることも報告されている。非特許文献９には、サイトグロビンは、低酸素状態や虚血状態の環境下において、亜硝酸を還元して一酸化窒素を発生させ、これにより可溶性グアニル酸シクラーゼを活性化し、血管の拡張をもたらし得ることが報告されている。更に、非特許文献１０には、虚血状態下での酸化ストレスに対して間質病変に対するサイトグロビンの治療応用効果が期待されることが開示されている。 For example, Patent Document 1 reports that a mouse deficient in cytoglobin exhibits carcinogenicity to administration of diethylnitrosamine, which is a hepatocarcinogen, and that cytoglobin exhibits a preventive or therapeutic action for liver cancer. Thus, cytoglobin is considered to be effective for the prevention or treatment of liver cancer. Non-Patent Document 5 reports that cytoglobin exhibits a tumor suppressive action. Non-Patent Document 6 reports that cytoglobin binds to oxygen, nitric oxide, or the like and functions as a reservoir for in vivo gas. Non-Patent Document 7 also reports that cytoglobin is transcriptionally regulated by a hypoxia-inducible factor (HIF) and becomes an oxygen sensor. Furthermore, Non-Patent Document 8 reports that cytoglobin plays a role in regulating intracellular oxidative stress metabolism by decomposing hydrogen peroxide by peroxidase activity. Non-Patent Document 9 discloses that cytoglobin reduces nitrous acid to generate nitric oxide in a hypoxic or ischemic environment, thereby activating soluble guanylate cyclase and dilating blood vessels. It has been reported that this can be brought about. Furthermore, Non-Patent Document 10 discloses that a therapeutic application effect of cytoglobin on interstitial lesions is expected against oxidative stress under ischemic conditions.
このようにサイトグロビンは様々な生体機能に関与しており、サイトグロビンの発現の減弱が一因となって発症している疾患、サイトグロビンの機能によって予防乃至治療効果が見込まれる疾患等に対して、生体内でのサイトグロビンの発現を増強させる方策が有効になると考えられている。 In this way, cytoglobin is involved in various biological functions, for diseases that develop due to decreased expression of cytoglobin, diseases that are expected to have preventive or therapeutic effects due to cytoglobin function, etc. Therefore, it is considered that a measure for enhancing the expression of cytoglobin in vivo becomes effective.
一方、従来、サイトグロビンを発現させる方法としては、細胞を低酸素条件下に晒す方法（非特許文献１１）、過酸化水素等によって細胞に酸化ストレスを与える方法（非特許文献１２）、カルシニューリンを細胞に導入して過剰発現させる方法（非特許文献１３）、アルンジン酸を細胞に暴露させる方法（非特許文献１４）等が知られている。これらの方法の内、通常の細胞条件や生体内（ｉｎ ｖｉｖｏ）において利用でき、臨床的に応用可能な技術は、アルンジン酸を用いる方法のみであり、必ずしも、汎用的な手法が確立されているとはいえず、他の手法の選択肢も存在していない。 On the other hand, conventional methods for expressing cytoglobin include a method of exposing cells to hypoxic conditions (Non-Patent Document 11), a method of applying oxidative stress to cells with hydrogen peroxide or the like (Non-Patent Document 12), and calcineurin. A method for introducing into cells and overexpressing the cells (Non-Patent Document 13), a method for exposing arundic acid to cells (Non-Patent Document 14), and the like are known. Among these methods, the only technique that can be used in normal cell conditions or in vivo and that can be applied clinically is a method using arundic acid, and a general-purpose method has always been established. However, there are no other method options.
このような従来技術を背景として、生体内でサイトグロビンの発現を増強させる新たな治療技術の開発が切望されている。 Against the background of such conventional technology, development of a new therapeutic technique that enhances the expression of cytoglobin in vivo is eagerly desired.
本発明の目的は、生体内でサイトグロビンの発現を増強させ得る新たなサイトクロビン発現増強剤を提供することである。 An object of the present invention is to provide a novel cytoclobin expression enhancer capable of enhancing cytoglobin expression in vivo.
本発明者らは、前記課題を解決するために鋭意研究を重ねたところ、線維芽細胞増殖因子（Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ Ｆａｃｔｏｒ）であるＦＧＦ−１及びＦＧＦ−２には、生体内でサイトグロビンの発現を増強させ得ることを見出した。本発明は、かかる知見に基づいて、更に検討を重ねることにより完成したものである。 The inventors of the present invention have made extensive studies in order to solve the above problems. As a result, FGF-1 and FGF-2, which are fibroblast growth factors, have cytoglobin expression in vivo. It has been found that it can be enhanced. The present invention has been completed by further studies based on this finding.
即ち、本発明は、下記に掲げる態様の発明を提供する。
項１． ＦＧＦ−１及びＦＧＦ−２からなる群より選択される少なくとも１種を有効成分とする、サイトグロビン発現増強剤。
項２． 肝癌、肝線維化、又は肝硬変の予防乃至治療に使用される、項１に記載のサイトグロビン発現増強剤。
項３． 肝癌以外の癌の治療予防乃至治療に使用される、項１に記載のサイトグロビン発現増強剤。
項４． 間質病変の予防乃至治療に使用される、項１に記載のサイトグロビン発現増強剤。
That is, this invention provides the invention of the aspect hung up below.
Item 1. A cytoglobin expression enhancer comprising at least one selected from the group consisting of FGF-1 and FGF-2 as an active ingredient.
Item 2. Item 2. The cytoglobin expression enhancer according to Item 1, which is used for prevention or treatment of liver cancer, liver fibrosis, or cirrhosis.
Item 3. Item 2. The cytoglobin expression enhancer according to Item 1, which is used for treatment prevention or treatment of cancers other than liver cancer.
Item 4. Item 2. The cytoglobin expression enhancer according to Item 1, which is used for prevention or treatment of stromal lesions.
本発明によれば、生体内でサイトグロビンの発現を増強させることができるので、生体内でサイトグロビンの発現を増強できるので、サイトグロビンの発現増強によって予防乃至治療効果が期待される疾患や症状の予防乃至治療、具体的には、正常な状態でサイトグロビンが発現している臓器（例えば、肝臓、消化管、腎臓、肺臓等）における線維化や癌、肝硬変、腎尿細管間質性腎炎等の予防乃至治療に有効であり、更には細胞内の酸化ストレス代謝の調節、低酸素状態や虚血状態における血管収縮の抑制等に有効である。その他、 According to the present invention, since the expression of cytoglobin can be enhanced in vivo, the expression of cytoglobin can be enhanced in vivo. Therefore, diseases and symptoms that are expected to have preventive or therapeutic effects due to enhanced expression of cytoglobin. Prevention, treatment, specifically, fibrosis and cancer, liver cirrhosis, renal tubulointerstitial nephritis in organs (eg, liver, gastrointestinal tract, kidney, lung, etc.) in which cytoglobin is expressed in a normal state It is effective for the prevention or treatment of the above, and further for the regulation of intracellular oxidative stress metabolism and the suppression of vasoconstriction in hypoxic and ischemic conditions. Other,
本発明のサイトグロビン発現増強剤は、ＦＧＦ−１及びＦＧＦ−２からなる群より選択される少なくとも１種を有効成分とすることを特徴とする。以下、本発明のサイトグロビン発現増強剤について詳述する。 The cytoglobin expression enhancer of the present invention comprises at least one selected from the group consisting of FGF-1 and FGF-2 as an active ingredient. Hereinafter, the cytoglobin expression enhancer of the present invention will be described in detail.
有効成分
本発明のサイトグロビン発現増強剤は、有効成分として、ＦＧＦ−１及びＦＧＦ−２からなる群より選択される少なくとも１種を使用する。
Active ingredient The cytoglobin expression enhancer of the present invention uses at least one selected from the group consisting of FGF-1 and FGF-2 as an active ingredient.
ＦＧＦ−１は、線維芽細胞増殖因子の１種として、生体内に存在しているタンパク質である。本発明で使用されるＦＧＦ−１としては、具体的には、ヒトＦＧＦ−１、そのオルソログ、及びそれらの変異体が挙げられる。 FGF-1 is a protein that exists in vivo as one type of fibroblast growth factor. Specific examples of FGF-1 used in the present invention include human FGF-1, orthologs thereof, and mutants thereof.
ヒトＦＧＦ−１は、配列番号１に示すアミノ酸配列からなることが知られている。また、ヒトＦＧＦ−１としては、配列番号１における第２〜１５位のアミノ酸が欠失したアミノ酸配列からなるもの（配列番号２）も見出されておる。本発明では、これらのいずれのヒトＦＧＦ−１を使用してもよい。 Human FGF-1 is known to have the amino acid sequence shown in SEQ ID NO: 1. Moreover, as human FGF-1, what consists of an amino acid sequence from which amino acids 2 to 15 in SEQ ID NO: 1 were deleted (SEQ ID NO: 2) has also been found. Any of these human FGF-1s may be used in the present invention.
ＦＧＦ−１のオルソログとしては、特に制限されないが、例えば、ラット、ハムスター、モルモット、マウス、ウシ、ヒツジ、ブタ、ヤギ、サル、ウサギ等の哺乳動物；ニワトリ、ダチョウ等の鳥類等に由来するものが挙げられる。ＦＧＦ−１は、投与対象となる生物種に応じて、その由来を適宜設定すればよい。 Although it does not restrict | limit especially as an ortholog of FGF-1, For example, mammals, such as a rat, a hamster, a guinea pig, a mouse, a cow, a sheep, a pig, a goat, a monkey, a rabbit; Birds, such as a chicken and an ostrich, etc. Is mentioned. The origin of FGF-1 may be appropriately set according to the species to be administered.
ＦＧＦ−１の変異体としては、ＦＧＦ−１が本来有する生物活性を保持していることを限度として特に制限されず、例えば、突然変異したＦＧＦ−１、遺伝子工学的手法によって改変したＦＧＦ−１等が挙げられる。 The mutant of FGF-1 is not particularly limited as long as it retains the biological activity inherent to FGF-1, and includes, for example, mutated FGF-1, modified FGF-1 by genetic engineering techniques Etc.
ＦＧＦ−１は、遺伝子工学的手法により製造された組換え体であってもよく、また生体から抽出、精製したものを使用してもよい。 FGF-1 may be a recombinant produced by a genetic engineering technique, or may be extracted and purified from a living body.
ＦＧＦ−２は、線維芽細胞増殖因子の１種として、生体内に存在しているタンパク質である。本発明で使用されるＦＧＦ−２としては、具体的には、ヒトＦＧＦ−２、そのオルソログ、及びそれらの変異体が挙げられる。 FGF-2 is a protein that exists in vivo as one type of fibroblast growth factor. Specific examples of FGF-2 used in the present invention include human FGF-2, orthologs thereof, and mutants thereof.
ヒトＦＧＦ−２は、配列番号３に示すアミノ酸配列からなることが知られている。また、ヒトＦＧＦ−２としては、配列番号１における第１〜１３４位のアミノ酸が欠失したアミノ酸配列からなるもの（配列番号４）も見出されておる。本発明では、これらのいずれのヒトＦＧＦ−２を使用してもよい。 Human FGF-2 is known to have the amino acid sequence shown in SEQ ID NO: 3. Moreover, as human FGF-2, the thing (sequence number 4) which consists of an amino acid sequence from which the amino acid of the 1st-134th position in sequence number 1 was deleted is also discovered. Any of these human FGF-2s may be used in the present invention.
ＦＧＦ−２のオルソログとしては、特に制限されないが、例えば、ラット、ハムスター、モルモット、マウス、ウシ、ヒツジ、ブタ、ヤギ、サル、ウサギ等の哺乳動物；ニワトリ、ダチョウ等の鳥類等に由来するものが挙げられる。ＦＧＦ−２は、投与対象となる生物種に応じて、その由来を適宜設定すればよい。 Although it does not restrict | limit especially as an ortholog of FGF-2, For example, mammals, such as a rat, a hamster, a guinea pig, a mouse, a cow, a sheep, a pig, a goat, a monkey, a rabbit; Birds, such as a chicken and an ostrich, etc. Is mentioned. The origin of FGF-2 may be appropriately set according to the species to be administered.
ＦＧＦ−２の変異体としては、ＦＧＦ−２が本来有する生物活性を保持していることを限度として特に制限されず、例えば、突然変異したＦＧＦ−２、遺伝子工学的手法によって改変したＦＧＦ−２等が挙げられる。 The variant of FGF-2 is not particularly limited as long as it retains the biological activity inherent to FGF-2. For example, mutated FGF-2, FGF-2 modified by genetic engineering techniques Etc.
ＦＧＦ−２は、遺伝子工学的手法により製造された組換え体であってもよく、また生体から抽出、精製したものを使用してもよい。 FGF-2 may be a recombinant produced by genetic engineering techniques, or may be extracted and purified from a living body.
本発明のサイトグロビン発現増強剤では、ＦＧＦ−１又はＦＧＦ−２のいずれか一方を単独で使用してもよく、またこれらを組み合わせて使用してもよい。 In the cytoglobin expression enhancer of the present invention, either FGF-1 or FGF-2 may be used alone or in combination.
他の成分
本発明のサイトグロビン発現増強剤は、前記有効成分の他に、治療対象となる疾患の種類に応じて、他の薬理活性成分を含んでいてもよい。
Other Components The cytoglobin expression enhancer of the present invention may contain other pharmacologically active ingredients in addition to the active ingredients, depending on the type of disease to be treated.
また、本発明のサイトグロビン発現増強剤は、前記有効成分の他に、所望の投与形態及び製剤形態に調製するために、必要に応じて、薬学的に許容される担体や添加剤を含んでいてもよい。このような担体や添加剤としては、希釈剤、賦形剤、結合剤、崩壊剤、滑沢剤、懸濁化剤、溶解補助剤、安定化剤、甘味剤、着色剤、矯味剤、矯臭剤、界面活性剤、保湿剤、保存剤、ｐＨ調整剤、緩衝剤、粘稠化剤等が挙げられる。 Further, the cytoglobin expression enhancer of the present invention contains, in addition to the above active ingredients, pharmaceutically acceptable carriers and additives as necessary in order to prepare a desired dosage form and preparation form. May be. Such carriers and additives include diluents, excipients, binders, disintegrants, lubricants, suspending agents, solubilizers, stabilizers, sweeteners, colorants, flavoring agents, flavoring agents. Agents, surfactants, humectants, preservatives, pH adjusters, buffers, thickeners and the like.
剤型
本発明のサイトグロビン発現増強剤の剤型については、特に制限されず、その投与形態等に応じて適宜設定すればよい。本発明のサイトグロビン発現増強剤の剤型として、具体的には、注射剤、シロップ剤、細胞懸濁液、リポソーム製剤等の液状製剤；錠剤、硬カプセル剤、軟カプセル剤、顆粒剤、散剤、丸剤等の固形状製剤等が挙げられる。また、注射剤にする場合には、使用前に生理食塩水等で溶解する用時調製用粉末（例えば凍結乾燥粉末）の形態であってもよい。
The dosage form of the dosage forms site globin expression enhancer of the present invention is not particularly limited, it may be appropriately set depending on the dosage forms and the like. Specific examples of the dosage form of the cytoglobin expression enhancer of the present invention include liquid preparations such as injections, syrups, cell suspensions, liposome preparations; tablets, hard capsules, soft capsules, granules, powders. And solid preparations such as pills. In addition, in the case of an injection, it may be in the form of a powder for preparation (for example, freeze-dried powder) that is dissolved in physiological saline before use.
投与対象
本発明のサイトグロビン発現増強剤は、生体内でサイトグロビンの発現を増強できるので、サイトグロビンの発現増強によって予防乃至治療効果が期待される疾患に適用して使用される。
Administration target Since the cytoglobin expression enhancer of the present invention can enhance the expression of cytoglobin in vivo, it is applied to a disease for which prevention or treatment effect is expected by enhancing the expression of cytoglobin.
例えば、サイトグロビンが肝癌の予防乃至治療作用を発揮し得ることが報告されている（特許文献１）。また、肝星細胞の活性化抑制は肝癌の予防や治療に有効であると考えられており（非特許文献１及び２）、その一方で後述する実施例に示すように、サイトグロビンの発現増強によって肝星細胞の活性化を抑制できることが確認されている。従って、本発明のサイトグロビン発現増強剤は、肝癌の予防乃至治療目的で使用することができる。 For example, it has been reported that cytoglobin can exert a preventive or therapeutic effect on liver cancer (Patent Document 1). In addition, suppression of hepatic stellate cell activation is considered to be effective for the prevention and treatment of liver cancer (Non-patent Documents 1 and 2), while on the other hand, as shown in Examples described later, enhanced expression of cytoglobin. It has been confirmed that the activation of hepatic stellate cells can be suppressed. Therefore, the cytoglobin expression enhancer of the present invention can be used for the purpose of preventing or treating liver cancer.
また、肝星細胞の活性化抑制は肝線維化や肝硬変の予防や治療に有効であると考えられている（非特許文献１及び２）。一方、前述するように、サイトグロビンの発現増強によって肝星細胞の活性化を抑制できるので、本発明のサイトグロビン発現増強剤は、肝線維化や肝硬変の予防乃至治療目的で使用することもできる。 In addition, suppression of hepatic stellate cell activation is considered to be effective in the prevention and treatment of liver fibrosis and cirrhosis (Non-Patent Documents 1 and 2). On the other hand, as described above, since the activation of hepatic stellate cells can be suppressed by enhancing the expression of cytoglobin, the cytoglobin expression enhancer of the present invention can also be used for the purpose of preventing or treating liver fibrosis and cirrhosis. .
更に、サイトグロビンには、肝癌に止まらず、他の腫瘍に対しても予防や治療に有効であることが知られているので（非特許文献５）、本発明のサイトグロビン発現増強剤は、肝癌以外の癌の予防乃至治療目的で使用することもできる。本発明のサイトグロビン発現増強剤の予防乃至治療対象となる癌（肝癌以外）としては、例えば、肺癌、乳癌、胃癌、大腸癌、舌癌、甲状腺癌、腎臓癌、肺癌、前立腺癌、子宮癌、卵巣癌、骨肉腫、軟骨肉腫、横紋筋肉腫等が挙げられる。特に、本発明のサイトグロビン発現増強剤は、正常な状態でサイトグロビンが発現している臓器（例えば、肝臓、消化管、腎臓、肺臓等）における線維化や癌の予防乃至治療目的で好適に使用できる。 Furthermore, since it is known that cytoglobin is effective not only for liver cancer but also for other tumors (Non-Patent Document 5), the cytoglobin expression enhancer of the present invention is It can also be used for the purpose of preventing or treating cancers other than liver cancer. Examples of cancers (other than liver cancer) to be prevented or treated by the cytoglobin expression enhancer of the present invention include lung cancer, breast cancer, stomach cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, lung cancer, prostate cancer, uterine cancer. Ovarian cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma and the like. In particular, the cytoglobin expression enhancer of the present invention is suitable for the purpose of preventing or treating fibrosis and cancer in organs (eg, liver, gastrointestinal tract, kidney, lung, etc.) in which cytoglobin is expressed in a normal state. Can be used.
また、サイトグロビンには、ペルオキシダーゼ活性により過酸化水素を分解して細胞内酸化ストレス代謝を調節する役割を担っていることも報告されているので（非特許文献８）、本発明のサイトグロビン発現増強剤は、細胞内の酸化ストレス代謝の異常をきたしている疾患（例えば、肺線維症、慢性膵炎、炎症性腸疾患、慢性腎障害等）の予防乃至治療目的で使用することもできる。 Cytoglobin has also been reported to play a role in regulating intracellular oxidative stress metabolism by degrading hydrogen peroxide by peroxidase activity (Non-patent Document 8). The enhancer can also be used for the purpose of prevention or treatment of diseases (for example, pulmonary fibrosis, chronic pancreatitis, inflammatory bowel disease, chronic kidney injury, etc.) causing abnormal oxidative stress metabolism in cells.
更に、サイトグロビンは、低酸素状態や虚血状態の環境下において、亜硝酸を還元して一酸化窒素を発生させ、これにより可溶性グアニル酸シクラーゼを活性化し、血管の拡張をもたらし得ることも報告されているので（非特許文献９）、本発明のサイトグロビン発現増強剤は、低酸素状態や虚血状態における血管収縮が生じている疾患（例えば、心筋梗塞、腎不全、虚血性脳疾患等）の予防乃至治療目的で使用することもできる。 In addition, it has been reported that cytoglobin can reduce nitrous acid and generate nitric oxide in hypoxic and ischemic environments, thereby activating soluble guanylate cyclase and causing vasodilation. (Non-patent Document 9), the cytoglobin expression enhancer of the present invention is a disease in which vasoconstriction occurs in a hypoxic state or an ischemic state (for example, myocardial infarction, renal failure, ischemic brain disease, etc.) ) Can also be used for prevention or treatment purposes.
また、サイトグロビンは、間質病変に対して治療効果を示し得ることが報告されているので（非特許文献１０）、発明のサイトグロビン発現増強剤は、腎間病変の予防乃至治療目的で使用することもできる。本発明のサイトグロビン発現増強剤の予防乃至治療対象となる腎間病変としては、具体的には、慢性腎炎、糖尿病性腎炎等の尿細管間質性腎炎、薬剤性腎障害、腎不全等が挙げられる。 Moreover, since it has been reported that cytoglobin can show a therapeutic effect on interstitial lesions (Non-patent Document 10), the cytoglobin expression enhancer of the invention is used for the purpose of prevention or treatment of interrenal lesions. You can also Examples of interrenal lesions to be prevented or treated by the cytoglobin expression enhancer of the present invention include tubulointerstitial nephritis such as chronic nephritis and diabetic nephritis, drug-induced nephropathy, renal failure and the like. Can be mentioned.
本発明のサイトグロビン発現増強剤において、投与対象となる生物は、サイトグロビン発現増強が求められる生物であればよく、ヒトの他、ラット、ハムスター、モルモット、マウス、ウシ、ヒツジ、ブタ、ヤギ、サル、ウサギ等の哺乳動物等が挙げられる。本発明で使用される有効成分の由来は、投与対象となる生物の種類に応じて設定すればよい。例えば、ヒトに対して適用する場合であれば、有効成分は、ヒトＦＧＦ−１及び／又はヒトＦＧＦ−２を使用すればよい。 In the cytoglobin expression enhancer of the present invention, the organism to be administered may be an organism for which cytoglobin expression enhancement is required, and in addition to humans, rats, hamsters, guinea pigs, mice, cows, sheep, pigs, goats, Examples thereof include mammals such as monkeys and rabbits. What is necessary is just to set the origin of the active ingredient used by this invention according to the kind of organism used as administration object. For example, in the case of application to humans, the active ingredient may be human FGF-1 and / or human FGF-2.
投与方法
本発明のサイトグロビン発現増強剤の投与形態としては、例えば、局所投与、皮下投与、腹腔内投与、筋肉内投与、静脈内投与、経直腸的、皮内投与等の非経口投与；経口投与が挙げられ、適用する疾患の種類等に応じて適宜設定すればよい。本発明のサイトグロビン発現増強剤の投与形態として、好ましくは非経口投与が挙げられる。
Administration method Examples of the administration form of the cytoglobin expression enhancer of the present invention include topical administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration, rectal administration, intradermal administration, and the like; oral administration Administration may be mentioned, and it may be appropriately set according to the type of disease to be applied. The administration form of the cytoglobin expression enhancer of the present invention is preferably parenteral administration.
本発明のサイトグロビン発現増強剤の投与量については、適用する疾患の種類、投与対象者の年齢、性別、体重、症状の程度、投与形態等に応じて適宜設定すればよいが、例えば、ＦＧＦ−１及び／又はＦＧＦ−２が１日当たり、３〜１００μｇ／ｋｇμｇ程度となる量を１又は数回に別けて投与すればよい。 The dosage of the cytoglobin expression enhancer of the present invention may be appropriately set according to the type of disease to be applied, the age, sex, body weight, symptom level, dosage form, etc. of the administration subject. For example, FGF -1 and / or FGF-2 may be administered in an amount of about 3 to 100 μg / kg μg per day separately in one or several times.
以下、本発明を実施例により更に詳細に説明するが、本発明はこれらの実施例によって何ら限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by these Examples.
実施例１：ＦＧＦ−２によるサイトグロビン（ＣＹＧＢ）の発現増強効果の確認（１）
６０ｍｍプレートに、ヒト肝星細胞株（ＨＨＳｔｅＣ）細胞を５×１０5ｃｅｌｌｓ／ｗｅｌｌ播種し、Ｓｕｐｐｌｅｍｅｎｔ（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）、２％ウシ胎児血清（ＦＢＳ）、ペニシリン、及びストレプトマイシンを添加したＳｔｅＣＭ培地（ＳｃｉｅｎＣｅｌｌ，Ｃａｔ．Ｎｏ．５３００）にて、約２４時間培養した。
Example 1: Confirmation of cytoglobin (CYGB) expression enhancing effect by FGF-2 (1)
A 60 mm plate was seeded with 5 × 10 5 cells / well of human hepatic stellate cell line (HHSteC), and supplemented (SteCGS, Cat. No. 5352), 2% fetal bovine serum (FBS), penicillin, and streptomycin were added. The cultured cells were cultured for about 24 hours in the SteCM medium (ScienCell, Cat. No. 5300).
培養後、Ｓｕｐｐｌｅｍｅｎｔ無添加ＳｔｅＣＭ培地に交換し、Ｓｕｐｐｌｅｍｅｎｔ（ｘ１００）（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）及びＦＧＦ−２中和抗体の無添加、Ｓｕｐｐｌｅｍｅｎｔ（ｘ１００）（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）のみ添加、又はＳｕｐｐｌｅｍｅｎｔ（ｘ１００）（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）とＦＧＦ２中和抗体(Anti-FGF2/basic FGF (neutralizing), clone bFM-1 (Monoclonal antibody, Cat. NO.05-117, Millipore)（２μｇ/ｍｌ)の添加の条件で培養を行った。 After culturing, the medium was replaced with Supplement-free SteCM medium, and Supplement (x100) (SteCGS, Cat. No. 5352) and FGF-2 neutralizing antibody were not added, and Supplement (x100) (SteCGS, Cat. No. 5352) only. Addition or Supplement (x100) (SteCGS, Cat. No. 5352) and FGF2 neutralizing antibody (Anti-FGF2 / basic FGF (neutralizing), clone bFM-1 (Monoclonal antibody, Cat. NO.05-117, Millipore) Cultivation was performed under the conditions of addition of (2 μg / ml).
培養７２時間後に細胞の形態観察を行った。また、培養７２時間後の細胞を回収し、ＲＩＰＡ（Ｒａｄｉｏ−Ｉｍｍｕｎｏｐｒｅｃｉｐｉｔａｔｉｏｎ Ａｓｓａｙ）バッファーを用いて細胞溶解液１００μｌを調製した。調製された細胞溶解液（２０μｇ相当のタンパク質含有）に５×ローディングＢｕｆｆｅｒ（２−メルカプトエタノール含有）を添加し、９５℃で５分間熱処理後、ＳＤＳ−ＰＡＧＥを行った。一次抗体として、マウス抗ヒトαＳＭＡ抗体（Ｃｌｏｎｅ １Ａ４、ＤＡＫＯ製、１／１００ ｉｎ ＰＢＳ）とウサギ抗ヒトＣＹＧＢ抗体（Ｒａｂｂｉｔ Ｐｏｌｙｃｌｏｎａｌ、ｉｎ ｈｏｕｓｅ）を使用して反応させ、次いで、二次抗体として、其々ＰＯＤ（ｐｅｒｏｘｙｄａｓｅ）標識ウサギ抗マウスＩｇＧ抗体（１：２００、Ｄａｋｏ製）とＰＯＤ標識ヤギ抗ウサギＩｇＧ抗体（１：２００、Ｄａｋｏ製）で反応させた後、化学発光基質ＥＣＬ（ＧＥ Ｈｅａｌｔｈｃａｒｅ、Ｂｕｃｋｉｎｇｈａｍｓｈｉｒｅ）で処理し、高感度ＣＣＤイメージアナライザー（ＬＡＳ １０００ ｄｅｖｉｃｅ、富士フィルム製）を用いて検出した。なお、タンパク質のローディングコントロールとしてＧＡＰＤＨを使用した。 Cell morphology was observed after 72 hours of culture. In addition, cells after 72 hours of culture were collected, and 100 μl of a cell lysate was prepared using RIPA (Radio-Immunoprecipitation Assay) buffer. 5 × loading Buffer (containing 2-mercaptoethanol) was added to the prepared cell lysate (containing 20 μg of protein), and after heat treatment at 95 ° C. for 5 minutes, SDS-PAGE was performed. As a primary antibody, a mouse anti-human αSMA antibody (Clone 1A4, manufactured by DAKO, 1/100 in PBS) and a rabbit anti-human CYGB antibody (Rabbit Polyclonal, in house) were used for reaction. After reaction with POD (peroxydase) -labeled rabbit anti-mouse IgG antibody (1: 200, manufactured by Dako) and POD-labeled goat anti-rabbit IgG antibody (1: 200, manufactured by Dako), chemiluminescent substrates ECL (GE Healthcare, Buckinghamshire) ) And detected using a high-sensitivity CCD image analyzer (LAS 1000 device, manufactured by Fuji Film). GAPDH was used as a protein loading control.
得られた結果を図１に示す。図１の左図において、「Ｓ−」はＳｕｐｐｌｅｍｅｎｔ及びＦＧＦ２中和抗体の無添加、「Ｓ＋」はＳｕｐｐｌｅｍｅｎｔのみ添加、「２μg/ml Ant-FGF2 + Supplement」は、ＳｕｐｐｌｅｍｅｎｔとＦＧＦ−２の添加の条件で培養した場合の形態観察の結果である。形態観察の結果、Ｓｕｐｐｌｅｍｅｎｔ添加によるヒト肝星細胞の形態変化はＦＧＦ−２中和抗体により阻害され、Ｓｕｐｐｌｅｍｅｎｔ無添加と同様の形態を示した。ウエスタンブロッティングの結果から、Ｓｕｐｐｌｅｍｅｎｔで誘導されるヒト肝星細胞におけるサイトグロビンの発現増強が、ＦＧＦ−２中和抗体で抑制されることが分かった。また、サイトグロビンの発現増強に伴って、肝星細胞の活性化マーカーであるα平滑筋アクチン（αＳＭＡ）の減少も確認され、サイトグロビンの発現量の増加は、肝星細胞の活性化抑制をもたらしていると考えられた。 The obtained results are shown in FIG. In the left diagram of FIG. 1, “S-” indicates that no supplement and FGF2 neutralizing antibody are added, “S +” indicates that only supplement is added, and “2 μg / ml Ant-FGF2 + Supplement” indicates that supplement and FGF-2 are added. It is the result of the form observation at the time of culture | cultivating on conditions. As a result of morphological observation, the morphological change of human hepatic stellate cells due to the addition of Supplement was inhibited by the FGF-2 neutralizing antibody, and showed the same morphology as when no supplement was added. From the results of Western blotting, it was found that the enhancement of cytoglobin expression in human hepatic stellate cells induced by Supplement was suppressed by FGF-2 neutralizing antibody. Along with the enhanced expression of cytoglobin, a decrease in α-smooth muscle actin (αSMA), an activation marker for hepatic stellate cells, was also confirmed. An increase in the expression level of cytoglobin suppressed the activation of hepatic stellate cells. It was thought to have brought.
実施例２：ＦＧＦ−２によるサイトグロビン（ＣＹＧＢ）の発現増強効果の確認（２）
６０ｍｍプレートに、ヒト肝星細胞株（ＨＨＳｔｅＣ）細胞を５×１０5ｃｅｌｌｓ／ｗｅｌｌ播種し、Ｓｕｐｐｌｅｍｅｎｔ（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）、２％ウシ胎児血清（ＦＢＳ）、ペニシリン、及びストレプトマイシンを添加したＳｔｅＣＭ培地（ＳｃｉｅｎＣｅｌｌ，Ｃａｔ．Ｎｏ．５３００）にて、約２４時間培養した。
Example 2: Confirmation of cytoglobin (CYGB) expression enhancing effect by FGF-2 (2)
A 60 mm plate was seeded with 5 × 10 5 cells / well of human hepatic stellate cell line (HHSteC), and supplemented (SteCGS, Cat. No. 5352), 2% fetal bovine serum (FBS), penicillin, and streptomycin were added. The cultured cells were cultured for about 24 hours in the SteCM medium (ScienCell, Cat. No. 5300).
培養後、Ｓｕｐｐｌｅｍｅｎｔ無添加ＳｔｅＣＭ培地に交換し、Ｓｕｐｐｌｅｍｅｎｔ（ｘ１００）、又は以下に示す条件でヒトＦＧＦ−２（配列番号３）を添加して細胞の処理を行った。時間依存性試験（図２の左図）のために、ＦＧＦ−２の添加濃度を４ｎｇ／ｍｌにして、０、８、２４、４８、及び７２時間、細胞を処理した後に、細胞を回収した。濃度依存性試験（図２の右図）のために、ＦＧＦ−２の添加濃度を０．５、１、２、及び４ｎｇ／ｍｌを処理し７２時間後に細胞を回収した。回収した各細胞について、前記実施例１と同様の方法でＳＤＳ−ＰＡＧＥを行い、サイトグロビン、αＳＭＡ、及びＧＡＰＤＨ（ローディングコントロール）の発現量の測定を行った。 After culturing, the cells were replaced with the Supplement-free SteCM medium, and the cells were treated with Supplement (x100) or human FGF-2 (SEQ ID NO: 3) under the conditions shown below. For the time-dependent test (left figure in FIG. 2), the cells were collected after treating the cells for 0, 8, 24, 48, and 72 hours with an added concentration of FGF-2 of 4 ng / ml. . For the concentration dependency test (the right diagram in FIG. 2), the addition concentrations of FGF-2 were treated with 0.5, 1, 2, and 4 ng / ml, and the cells were collected 72 hours later. About each collect | recovered cell, SDS-PAGE was performed by the method similar to the said Example 1, and the expression level of cytoglobin, (alpha) SMA, and GAPDH (loading control) was measured.
得られた結果を図２に示す。図２の右図において「Ｓ＋」は、Ｓｕｐｐｌｅｍｅｎｔ添加且つＦＧＦ−２未添加の場合の条件を指す。この結果から、ＦＧＦ−２の添加によって、ヒト肝星細胞におけるサイトグロビンの発現量が経時的及び濃度依存的に増加しており、ＦＧＦ−２にはサイトグロビンの発現を増強させる作用があることが明らかとなった。また、肝星細胞の活性化マーカーであるα平滑筋アクチン（αＳＭＡ）は、サイトグロビンの発現量が増加するのに伴って、その発現量が低下しており、サイトグロビンの発現量の増強は、肝星細胞の活性化抑制をもたらし得ることも明らかとなった。 The obtained results are shown in FIG. In the right diagram of FIG. 2, “S +” indicates a condition when Supplement is added and FGF-2 is not added. From this result, the addition of FGF-2 increases the amount of cytoglobin expression in human hepatic stellate over time and in a concentration-dependent manner, and FGF-2 has the effect of enhancing the expression of cytoglobin. Became clear. In addition, α-smooth muscle actin (αSMA), an activation marker for hepatic stellate cells, decreases as the expression level of cytoglobin increases. It was also revealed that hepatic stellate cell activation can be suppressed.
実施例３：ＦＧＦ−２によるサイトグロビン（ＣＹＧＢ）の発現増強効果の確認（３）
３５ｍｍプレートに、ヒト肝星細胞株（ＨＨＳｔｅＣ）細胞を５×１０5ｃｅｌｌｓ／ｗｅｌｌ播種し、Ｓｕｐｐｌｅｍｅｎｔ（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）、２％ウシ胎児血清（ＦＢＳ）、ペニシリン、及びストレプトマイシンを添加したＳｔｅＣＭ培地（ＳｃｉｅｎＣｅｌｌ，Ｃａｔ．Ｎｏ．５３００）にて、約２４時間培養した。
Example 3: Confirmation of cytoglobin (CYGB) expression enhancing effect by FGF-2 (3)
A 35 mm plate was seeded with 5 × 10 5 cells / well of human hepatic stellate cell line (HHSteC), and supplemented (SteCGS, Cat. No. 5352), 2% fetal bovine serum (FBS), penicillin, and streptomycin were added. The cultured cells were cultured for about 24 hours in the SteCM medium (ScienCell, Cat. No. 5300).
培養後、Ｓｕｐｐｌｅｍｅｎｔ無添加ＳｔｅＣＭ培地に交換し、Ｓｕｐｐｌｅｍｅｎｔ（ｘ１００）及びヒトＦＧＦ−２（配列番号３）（４ｎｇ／ｍｌ）を添加した。添加０、４、８、２４、及び４８時間後に、それぞれ、Ｔｒｉｚｏｌ５００μｌに溶解し、direct-Zol RNA miniPrep（ZYMO RESEARCH）キットでＲＮＡ抽出を行った。抽出されたRNA、１００ｎｇをSuperscript III Reverse Transcriptase (Invitrogen)でｃＤＮＡ合成し、Fast SYBR Green Master mixを用いてリアルタイム定量ＰＣＲ（Applied Biosystems 7500 Fast Real-time PCR system）を行った。なお、本試験では、内在性コントロールとして１８Ｓを使用した。 After culturing, the medium was replaced with Supplement-free SteCM medium, and Supplement (x100) and human FGF-2 (SEQ ID NO: 3) (4 ng / ml) were added. At 0, 4, 8, 24, and 48 hours after the addition, each was dissolved in 500 μl of Trizol, and RNA was extracted with a direct-Zol RNA miniPrep (ZYMO RESEARCH) kit. 100 ng of the extracted RNA was synthesized with cDNA using Superscript III Reverse Transcriptase (Invitrogen), and real-time quantitative PCR (Applied Biosystems 7500 Fast Real-time PCR system) was performed using Fast SYBR Green Master mix. In this test, 18S was used as an endogenous control.
得られた結果を図３に示す。この結果から、ＦＧＦ−２の添加によって、ヒト肝星細胞におけるサイトグロビンのｍＲＮＡの発現量が経時的に増加しており、ＦＧＦ−２にはサイトグロビンの発現を増強させる作用があることが明らかとなった。また、サイトグロビンの発現量が増加するのに伴いαＳＭＡ発現量が低下しており、ＦＧＦ−２によりサイトグロビンの転写が誘導され、肝星細胞の活性化が抑制された。 The obtained results are shown in FIG. From this result, it is clear that the amount of cytoglobin mRNA expression in human hepatic stellate cells increases over time by the addition of FGF-2, and that FGF-2 has the effect of enhancing the expression of cytoglobin. It became. Moreover, as the expression level of cytoglobin increased, the expression level of αSMA decreased. Cytoglobin transcription was induced by FGF-2, and activation of hepatic stellate cells was suppressed.
実施例４：ＦＧＦ−２によるサイトグロビン（ＣＹＧＢ）の発現増強効果の確認（４）
４ｗｅｌｌチャンバースライドに、ＨＨＳｔｅＣ細胞２×１０4ｃｅｌｌｓ／ｗｅｌｌとなるように播種し、約２４時間培養した。培養は、Ｓｕｐｐｌｅｍｅｎｔ、２％ＦＢＳ、ペニシリン、及びストレプトマイシンを添加したＳｔｅＣＭ培地（ＳｃｉｅｎＣｅｌｌ Ｒｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ製、Ｃａｔ．Ｎｏ．５３００）を用いて行った。
Example 4: Confirmation of cytoglobin (CYGB) expression enhancing effect by FGF-2 (4)
It seed | inoculated so that it might become HHSTEC cell 2 * 10 < 4 > cells / well to a 4- well chamber slide, and it cultured for about 24 hours. The culture was performed using SteCM medium (ScienCell Research Laboratories, Cat. No. 5300) supplemented with Supplement, 2% FBS, penicillin, and streptomycin.
その後、Ｓｕｐｐｌｅｍｅｎｔ無添加ＳｔｅＣＭ培地に交換し、Ｓｕｐｐｌｅｍｅｎｔ（×１００）及びヒトＦＧＦ−２（配列番号３）（４ｎｇ／ｍｌ）を添加して培養を継続し、７２時間後に４％パラフォルムアルデヒド／ＰＢＳＴで細胞を固定した。次いで、固定化した細胞に対して、一次抗体として、マウス抗ヒトＳＭＡ抗体（Ｃｌｏｎｅ １Ａ４、ＤＡＫＯ製、１／１００ ｉｎ ＰＢＳ）とウサギ抗ヒトＣＹＧＢ抗体（Ｒａｂｂｉｔ Ｐｏｌｙｃｌｏｎａｌ、ｉｎ ｈｏｕｓｅ）１時間反応させた後、ＰＢＳＴ（Ｐｈｓｏｐｈａｔｅ Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎｅ ｗｉｔｈ Ｔｗｅｅｎ ２０）溶液で洗浄した。次いで、二次抗体として、ＡｌｅｘａＦｌｕｏｒ ４８８標識ヤギ抗マウスＩｇＧ抗体（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ、ライフテクノロジーズ製）とＡｌｅｘａＦｏｕｒ ５９４標識ヤギ抗ウサギＩｇＧ抗体（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ、ライフテクノロジーズ製）をそれぞれ使用し、反応させた。反応後、ＰＢＳＴ溶液で洗浄した後、ＤＡＰＩ（ｄｉ−ａｍｉｎおーｐｈｅｎｙｌ−ｉｎｄｏｌｅ）で核染色し、蛍光顕微鏡（ＢＺ−８０００、キーエンス製）にて観察を行った。また、比較のために、Ｓｕｐｐｌｅｍｅｎｔ及びヒトＦＧＦ−２を添加しない条件、及びＳｕｐｐｌｅｍｅｎｔのみを添加した条件でも、前記と同様に試験を行った。 Thereafter, the medium was replaced with a supplement-free SteCM medium, and supplementation (× 100) and human FGF-2 (SEQ ID NO: 3) (4 ng / ml) were added to continue the culture. After 72 hours, 4% paraformaldehyde / PBST The cells were fixed with. Next, the immobilized cells were reacted as a primary antibody with mouse anti-human SMA antibody (Clone 1A4, DAKO, 1/100 in PBS) and rabbit anti-human CYGB antibody (Rabbit Polyclonal, in house) for 1 hour. Thereafter, the plate was washed with a PBST (Phosphate Buffered Saline with Tween 20) solution. Next, AlexaFluor 488-labeled goat anti-mouse IgG antibody (Molecular Probes, manufactured by Life Technologies) and AlexaFour 594-labeled goat anti-rabbit IgG antibody (Molecular Probes, manufactured by Life Technologies) were used and reacted as secondary antibodies. After the reaction, the plate was washed with a PBST solution, stained with DAPI (di-amine-phenyl-indole), and observed with a fluorescence microscope (BZ-8000, manufactured by Keyence). For comparison, the test was performed in the same manner as described above under the conditions in which Supplement and human FGF-2 were not added, and in the condition in which only Supplement was added.
得られた結果を図４に示す。図４中、Ｓ（−）はＳｕｐｐｌｅｍｅｎｔ及びヒトＦＧＦ−２を添加しなかった場合、Ｓ（＋）はＳｕｐｐｌｅｍｅｎｔのみを添加した場合、ＦＧＦ２はＳｕｐｐｌｅｍｅｎｔ及びヒトＦＧＦ−２を添加した場合の結果である。図４から明らかなように、ヒトＦＧＦ−２を添加した場合には、α−ＳＭＡが消失し、ＣＹＧＢが強く発現されていた。即ち、本試験結果からも、ＦＧＦ−２にはサイトグロビンの発現を増強させる作用があることが確認された。 The obtained results are shown in FIG. In FIG. 4, S (−) is the result when Supplement and human FGF-2 are not added, S (+) is the result when only Supplement is added, and FGF2 is the result when Supplement and human FGF-2 are added. . As apparent from FIG. 4, when human FGF-2 was added, α-SMA disappeared and CYGB was strongly expressed. That is, from this test result, it was also confirmed that FGF-2 has an action of enhancing the expression of cytoglobin.
実施例５：ＦＧＦ−１によるサイトグロビン（ＣＹＧＢ）の発現増強効果の確認（５）
６０ｍｍプレートに、ヒト肝星細胞株（ＨＨＳｔｅＣ）細胞を５×１０5ｃｅｌｌｓ／ｗｅｌｌ播種し、Ｓｕｐｐｌｅｍｅｎｔ（ＳｔｅＣＧＳ, Ｃａｔ. Ｎｏ. ５３５２）、２％ウシ胎児血清（ＦＢＳ）、ペニシリン、及びストレプトマイシンを添加したＳｔｅＣＭ培地（ＳｃｉｅｎＣｅｌｌ，Ｃａｔ．Ｎｏ．５３００）にて、約２４時間培養した。
Example 5: Confirmation of cytoglobin (CYGB) expression enhancing effect by FGF-1 (5)
A 60 mm plate was seeded with 5 × 10 5 cells / well of human hepatic stellate cell line (HHSteC), and supplemented (SteCGS, Cat. No. 5352), 2% fetal bovine serum (FBS), penicillin, and streptomycin were added. The cultured cells were cultured for about 24 hours in the SteCM medium (ScienCell, Cat. No. 5300).
培養後、Ｓｕｐｐｌｅｍｅｎｔ無添加ＳｔｅＣＭ培地に交換し、Ｓｕｐｐｌｅｍｅｎｔ（ｘ１００）、又は以下に示す条件でヒトＦＧＦ−１（配列番号１）を添加して細胞の処理を行った。時間依存性試験（図５の上図）のために、ＦＧＦ−１の添加濃度を４ｎｇ／ｍｌにして、０、８、２４、４８、及び７２時間、細胞を処理した後に、細胞を回収した。濃度依存性試験（図５の下図）のために、ＦＧＦ−１の添加濃度を０．５、１、２、及び４ｎｇ／ｍｌを処理し７２時間後に細胞を回収した。回収した各細胞について、前記実施例１と同様の方法でＳＤＳ−ＰＡＧＥを行い、サイトグロビン、αＳＭＡ、及びＧＡＰＤＨ（ローディングコントロール）の発現量の測定を行った。 After culturing, the cells were replaced with a supplement-free SteCM medium, and cells were treated with supplement (x100) or human FGF-1 (SEQ ID NO: 1) under the conditions shown below. For time-dependent testing (upper figure in FIG. 5), cells were harvested after treating the cells for 0, 8, 24, 48, and 72 hours with an added concentration of FGF-1 of 4 ng / ml. . For the concentration dependency test (bottom of FIG. 5), FGF-1 addition concentrations of 0.5, 1, 2, and 4 ng / ml were treated, and cells were collected 72 hours later. About each collect | recovered cell, SDS-PAGE was performed by the method similar to the said Example 1, and the expression level of cytoglobin, (alpha) SMA, and GAPDH (loading control) was measured.
得られた結果を図５に示す。図５の下図において、「Ｓ−」はＳｕｐｐｌｅｍｅｎｔ未添加且つＦＧＦ−２未添加の場合の条件を指し、「Ｓ＋」は、Ｓｕｐｐｌｅｍｅｎｔ添加且つＦＧＦ−２未添加の場合の条件を指す。この結果から、ＦＧＦ−１の添加によって、ヒト肝星細胞におけるサイトグロビンの発現量が、ＦＧＦ−２の場合と同様に、経時的及び濃度依存的に増加しており、ＦＧＦ−１にはサイトグロビンの発現を増強させる作用があることが明らかとなった。また、肝星細胞の活性化マーカーであるα平滑筋アクチン（αＳＭＡ）は、サイトグロビンの発現量が増加するのに伴って、その発現量が低下しており、サイトグロビンの発現量の増強は、肝星細胞の活性化抑制をもたらし得ることも明らかとなった。 The obtained results are shown in FIG. In the lower diagram of FIG. 5, “S−” indicates a condition when Supplement is not added and FGF-2 is not added, and “S +” indicates a condition when Supplement is added and FGF-2 is not added. From this result, the addition of FGF-1 increased the expression level of cytoglobin in human hepatic stellate cells over time and in a concentration-dependent manner as in the case of FGF-2. It has been clarified that there is an action to enhance the expression of globin. In addition, α-smooth muscle actin (αSMA), an activation marker for hepatic stellate cells, decreases as the expression level of cytoglobin increases. It was also revealed that hepatic stellate cell activation can be suppressed.
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