JPWO2006112365A1 - Treatment of emphysema using mesenchymal stem cells - Google Patents
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Abstract
間葉系幹細胞（ＭＳＣ）を含有する，肺気腫を治療するための医薬組成物が開示される。間葉系幹細胞とは，種々の中胚葉細胞，例えば，骨芽細胞，軟骨細胞，骨格筋細胞，心筋細胞，および血管内皮細胞に分化することができる多能性成人幹細胞である。本発明の医薬組成物は，肺気腫における肺胞の再生を促進し，毛管の血管新生を促進することができる。ＭＳＣを成熟肺胞上皮細胞とともに共培養すると肺胞に分化し，肺胞形成を誘導することができる。また，静脈内投与したＭＳＣが肺に定着して，肺胞および血管系を再生し，このことによりラットのエラスターゼ誘起性肺気腫において肺構造および機能を改善することができる。Disclosed is a pharmaceutical composition for treating pulmonary emphysema comprising mesenchymal stem cells (MSC). Mesenchymal stem cells are pluripotent adult stem cells that can differentiate into various mesodermal cells such as osteoblasts, chondrocytes, skeletal muscle cells, cardiomyocytes, and vascular endothelial cells. The pharmaceutical composition of the present invention can promote alveolar regeneration in emphysema and promote capillary angiogenesis. When MSC is co-cultured with mature alveolar epithelial cells, it can differentiate into alveoli and induce alveolar formation. In addition, intravenously administered MSCs settle in the lung and regenerate alveoli and vasculature, which can improve lung structure and function in rat elastase-induced emphysema.
Description
本発明は肺気腫を治療するための医薬組成物に関する。 The present invention relates to a pharmaceutical composition for treating emphysema.
肺気腫は，世界中で呼吸不全および死亡の主要な原因である。肺気腫は，末端細気管支から離れた気腔の異常な永久拡大により特徴づけられる（Lopez AD, Murray CC, 1990-2020. Nat Med. 1998;4:1241-1243; Michaud CM, Murray CJ, JAMA. 2001;285:535-539; American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1995;152:S77-S121）。肺気腫は，進行性の気流制限を呈する疾患であり，労作性呼吸困難を生じる。末梢気道や肺胞壁の破壊や肺胞細胞のアポトーシスによって病態が進行し，いったん生じたこれらの病理学的変化は非可逆的であるといわれている。したがって現時点では，肺気腫の治療は生活の質や運動耐容能の改善を目的とする対症療法が中心である。肺胞壁の破壊や肺胞細胞のアポトーシス抑制に対して有効な治療法はなく，また肺組織の再生に有効な薬剤はない。また，肺気腫には重複する作用を有する多くのメディエータが関与しているため，この疾病の進行を予防するための有効な治療法はない（Barnes PJ, N Engl J Med 2000;343:269-280）。 Emphysema is the leading cause of respiratory failure and death worldwide. Emphysema is characterized by an abnormal permanent enlargement of the airspace away from the terminal bronchiole (Lopez AD, Murray CC, 1990-2020. Nat Med. 1998; 4: 1241-1243; Michaud CM, Murray CJ, JAMA. 2001; 285: 535-539; American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1995; 152: S77-S121). Emphysema is a disease with progressive airflow limitation that causes exertional dyspnea. It is said that these pathological changes once caused by the destruction of peripheral airways and alveolar walls and apoptosis of alveolar cells are irreversible. Therefore, at present, emphysema treatment is mainly symptomatic treatment aimed at improving quality of life and exercise tolerance. There is no effective treatment for the destruction of alveolar walls or the suppression of alveolar cell apoptosis, and there is no drug effective for the regeneration of lung tissue. In addition, emphysema involves many mediators with overlapping effects, so there is no effective treatment to prevent the progression of this disease (Barnes PJ, N Engl J Med 2000; 343: 269-280 ).
肺気腫における病理学的変化の１つである肺胞壁の破壊は，非可逆的であると考えられてきた。しかし，最近の研究は，骨髄細胞が末梢血中に移動して，肺線維芽細胞および肺胞に分化することを示した（Ishizawa K, et al., FEBS Lett. 2004;556:249-252; Yamada M, et al., J Immunol. 2004;172:1266-1272）。すなわち，肺気腫の治療には，骨髄細胞による肺再生が望ましいと考えられる。 Alveolar wall destruction, one of the pathological changes in emphysema, has been considered irreversible. However, recent studies have shown that bone marrow cells migrate into peripheral blood and differentiate into lung fibroblasts and alveoli (Ishizawa K, et al., FEBS Lett. 2004; 556: 249-252 Yamada M, et al., J Immunol. 2004; 172: 1266-1272). In other words, lung regeneration by bone marrow cells is desirable for the treatment of emphysema.
間葉系幹細胞（ＭＳＣ）は，骨髄中に存在する多能性成人幹細胞である（Pittenger MF, et al., Science. 1999;284:143-147）。ＭＳＣは，造血性幹細胞とは異なり，接着性であり，培養により増殖させることができる。ＭＳＣは種々の中胚葉細胞，例えば，骨芽細胞，軟骨細胞，骨格筋細胞，心筋細胞，および血管内皮細胞に分化することができる（Reyes M, et al., J Clin Invest. 2002;109:337-346; Toma C, et al., 2002;105: 93-98; Nagaya N, et al, Am J Physiol Heart Circ Physiol. 2004;287:H2670-H2676）。しかし，ＭＳＣが，主として内胚葉に由来する肺組織の再生を誘導しうるか否かは不明である。最近の研究により，全身投与されたＭＳＣがブレオマイシン誘起性創傷に応答して肺に集積することが示された（Ortiz LA, et al., Proc Natl Acad Sci U S A. 2003;100:8407-8411）。また，ＭＳＣがインビトロで気道上皮細胞に分化することが示されている（Wang G, et al., Proc Natl Acad Sci U S A. 2005;102:186-191）。しかし，ＭＳＣを投与することで実際に肺気腫の肺機能が改善するか否かは知られていなかった。また，肺機能の改善には肺胞のみでなく同時に血管が必要であるが，肺に集積したＭＳＣは，肺胞と血管を同時に再生させることで肺組織を再生させることは知られていなかった。 Mesenchymal stem cells (MSC) are pluripotent adult stem cells present in the bone marrow (Pittenger MF, et al., Science. 1999; 284: 143-147). Unlike hematopoietic stem cells, MSCs are adherent and can be grown in culture. MSCs can differentiate into various mesodermal cells such as osteoblasts, chondrocytes, skeletal muscle cells, cardiomyocytes, and vascular endothelial cells (Reyes M, et al., J Clin Invest. 2002; 109: 337-346; Toma C, et al., 2002; 105: 93-98; Nagaya N, et al, Am J Physiol Heart Circ Physiol. 2004; 287: H2670-H2676). However, it is unclear whether MSCs can induce regeneration of lung tissue mainly derived from endoderm. Recent studies have shown that systemically administered MSCs accumulate in the lung in response to bleomycin-induced wounds (Ortiz LA, et al., Proc Natl Acad Sci US A. 2003; 100: 8407-8411 ). MSC has also been shown to differentiate into respiratory epithelial cells in vitro (Wang G, et al., Proc Natl Acad Sci USA 2005; 102: 186-191). However, it has not been known whether administration of MSC actually improves the lung function of emphysema. In addition, improvement of lung function requires not only alveoli but also blood vessels at the same time, but MSCs accumulated in the lung have not been known to regenerate lung tissue by simultaneously regenerating alveoli and blood vessels. .
本明細書において引用される参考文献は以下のとおりである。これらの文献に記載される内容はすべて本明細書の一部としてここに引用する。
本発明の目的は，肺気腫を治療する方法を提供することである。 An object of the present invention is to provide a method for treating emphysema.
本発明者らは，ＭＳＣを成熟肺胞上皮細胞とともに共培養すると肺胞に分化し，肺胞形成を誘導しうこと，ならびに，静脈内投与したＭＳＣが肺に定着して，肺胞および血管系を再生し，このことによりラットのエラスターゼ誘起性肺気腫において肺構造および機能を改善しうることを見出した。 When the present inventors co-cultured MSCs with mature alveolar epithelial cells, they differentiated into alveoli and induced alveolar formation, and intravenously administered MSCs settled in the lungs, and alveoli and blood vessels The system was regenerated and found to improve lung structure and function in rat elastase-induced emphysema.
すなわち，本発明は，患者に間葉系幹細胞（ＭＳＣ）を投与することにより肺気腫を治療する方法，ならびに間葉系幹細胞（ＭＳＣ）を含有する，肺気腫を治療するための医薬組成物を提供する。間葉系幹細胞とは，種々の中胚葉細胞，例えば，骨芽細胞，軟骨細胞，骨格筋細胞，心筋細胞，および血管内皮細胞に分化することができる多能性成人幹細胞である。 That is, the present invention provides a method for treating emphysema by administering mesenchymal stem cells (MSC) to a patient, and a pharmaceutical composition for treating emphysema containing mesenchymal stem cells (MSC). . Mesenchymal stem cells are pluripotent adult stem cells that can differentiate into various mesodermal cells such as osteoblasts, chondrocytes, skeletal muscle cells, cardiomyocytes, and vascular endothelial cells.
本発明はまた，肺気腫の患者に間葉系幹細胞を投与することにより肺胞の再生を促進する方法，ならびに，間葉系幹細胞を含有する，肺気腫における肺胞の再生を促進するための医薬組成物を提供する。本発明はまた，肺気腫の患者に間葉系幹細胞を投与することにより毛管の血管新生を促進する方法，ならびに，肺気腫における毛管の血管新生を促進するための医薬組成物を提供する。投与方法としては，静脈内投与が好ましいが，手術時に患部に局所投与することや気管支鏡らを用いた経気道的投与も可能である。 The present invention also provides a method for promoting alveolar regeneration by administering mesenchymal stem cells to a patient with emphysema, and a pharmaceutical composition for promoting alveolar regeneration in emphysema containing mesenchymal stem cells. Offer things. The present invention also provides a method for promoting capillary angiogenesis by administering mesenchymal stem cells to a patient with emphysema, and a pharmaceutical composition for promoting capillary angiogenesis in emphysema. Intravenous administration is preferable as the administration method, but local administration to the affected area at the time of surgery or transtracheal administration using a bronchoscope or the like is also possible.
別の観点においては，本発明は，患者から採取した間葉系幹細胞を培養することを含む，上述の本発明の医薬組成物を製造する方法を提供する。 In another aspect, the present invention provides a method for producing the above-described pharmaceutical composition of the present invention, comprising culturing mesenchymal stem cells collected from a patient.
以下の実施例において示されるように，ＭＳＣを成熟肺胞上皮細胞とともに共培養すると，ＭＳＣは上皮様の表現型を示し，マトリゲル中で成熟肺胞上皮細胞と一緒に肺胞様構造を形成した。また，エラスターゼ誘起性肺気腫のラットモデルにおいて，静脈内投与したＭＳＣは肺胞壁に取り込まれ，肺胞上皮細胞または血管内皮細胞に分化し，肺胞ネットワークおよび血管構造を形成し，このことにより，肺構造および機能が改善されることが明らかになった。 As demonstrated in the examples below, when MSCs were co-cultured with mature alveolar epithelial cells, MSCs exhibited an epithelial-like phenotype and formed alveolar-like structures with mature alveolar epithelial cells in Matrigel. . In a rat model of elastase-induced emphysema, intravenously administered MSCs are taken up by the alveolar wall and differentiated into alveolar epithelial cells or vascular endothelial cells, forming alveolar networks and vascular structures, It was found that lung structure and function were improved.
これらの結果は，ＭＳＣの移植が，肺において肺胞および血管系の再生により肺気腫に有益な効果を有することを示す。したがって，ＭＳＣの移植は，肺気腫の新たな治療方法として有用である。 These results indicate that transplantation of MSC has a beneficial effect on emphysema by regeneration of alveoli and vasculature in the lung. Therefore, MSC transplantation is useful as a new treatment method for emphysema.
間葉系幹細胞は，移植を受けるべき患者の骨髄，大腿骨，脛骨，脂肪組織，その他の組織から細胞を採取し，慣用の方法により培養することにより，容易に調製することができる。培養液としては，10〜15％の自己血清または牛胎児血清（FBS）及び抗生物質を補充したα−ＭＥＭやＤＭＥＭを用いることができる。必要に応じて塩基性線維芽細胞成長因子（bFGF）やアドレノメデユリンなどの成長因子を加えることがある。培養は，哺乳動物の細胞の培養に適する任意の条件で実施することができるが，一般的には３７℃，５％ＣＯ2で数日間培養し，必要に応じて培地を交換する。間葉系幹細胞は培養基材に接着して増殖する性質を有するため，浮遊して増殖する造血性幹細胞と容易に分離することができる。間葉系幹細胞は，ＣＤ２９，ＣＤ４４，ＣＤ７１，ＣＤ９０，ＣＤ１０５等の細胞表面マーカーにより容易に確認することができる。また，培養した間葉系幹細胞は，慣用の方法を用いて凍結保存することが可能である。Mesenchymal stem cells can be easily prepared by collecting cells from the bone marrow, femur, tibia, adipose tissue, and other tissues of patients to be transplanted and culturing them by conventional methods. As the culture solution, α-MEM or DMEM supplemented with 10-15% autologous serum or fetal bovine serum (FBS) and antibiotics can be used. Growth factors such as basic fibroblast growth factor (bFGF) and adrenomedullin may be added as needed. Culturing can be performed under any conditions suitable for culturing mammalian cells. Generally, culturing is carried out at 37 ° C. and 5% CO 2 for several days, and the medium is changed as necessary. Since mesenchymal stem cells adhere to the culture substrate and proliferate, they can be easily separated from hematopoietic stem cells that proliferate in suspension. Mesenchymal stem cells can be easily confirmed by cell surface markers such as CD29, CD44, CD71, CD90, CD105. In addition, cultured mesenchymal stem cells can be cryopreserved using conventional methods.
このようにして得られた間葉系幹細胞は，ＰＢＳ等の生理学的に許容しうる媒体に懸濁して，患者に投与することができる。投与方法としては，静脈内投与が好ましいが，手術時に患部に局所投与することや気管支鏡らを用いた経気道的投与も可能である。投与すべき間葉系幹細胞の数は，成人の場合，好ましくは1x107個〜１x109個である。細胞は，5〜10 mLのＰＢＳに懸濁して投与する。The mesenchymal stem cells thus obtained can be suspended in a physiologically acceptable medium such as PBS and administered to a patient. Intravenous administration is preferable as the administration method, but local administration to the affected area at the time of surgery or transtracheal administration using a bronchoscope or the like is also possible. The number of mesenchymal stem cells to be administered is preferably 1 × 10 7 to 1 × 10 9 in adults. Cells are administered suspended in 5-10 mL of PBS.
本明細書において明示的に引用される全ての特許および参考文献の内容は全て本明細書の一部としてここに引用する。また，本出願が有する優先権主張の基礎となる出願である日本特許出願2005−117431号の明細書および図面に記載の内容は全て本明細書の一部としてここに引用する。 The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2005-117431, which is the application on which the priority of the present application is based, are cited herein as a part of this specification.
以下に実施例により本発明をより詳細に説明するが，これらの実施例は本発明の範囲を制限するものではない。 The present invention will be described in more detail with reference to the following examples, but these examples do not limit the scope of the present invention.
＜方法＞
骨髄ＭＳＣの培養
ＭＳＣは先に記載されている方法にしたがって増殖させた（Pittenger MF, et al., Science. 1999;284:143-147）。簡単には，雄Ｌｅｗｉｓラットおよびグリーン蛍光蛋白質（ＧＦＰ）を発現するトランスジェニックラットを犠牲死させ，大腿腔および脛骨腔をリン酸緩衝化食塩水（ＰＢＳ）でフラッシングすることにより骨髄を回収した。骨髄細胞を１００ｍｍのディッシュに加え，１０％ＦＢＳおよび抗生物質を補充したα−ＭＥＭ中で培養した。培養第５−７日までに目に見える対称のコロニーが形成された。非接着性の造血細胞を除去し，培地を交換した。接着した紡錘形状のＭＳＣ集団は，細胞を最初に播種してから約４−５代以内の継代で５０００万個以上に増殖した。<Method>
Bone marrow MSC culture MSCs were grown according to the method described previously (Pittenger MF, et al., Science. 1999; 284: 143-147). Briefly, male Lewis rats and transgenic rats expressing green fluorescent protein (GFP) were sacrificed and bone marrow was harvested by flushing the femoral and tibial cavities with phosphate buffered saline (PBS). Bone marrow cells were added to 100 mm dishes and cultured in α-MEM supplemented with 10% FBS and antibiotics. Visible symmetrical colonies were formed by day 5-7 of culture. Nonadherent hematopoietic cells were removed and the medium was changed. The attached spindle-shaped MSC population grew to more than 50 million cells in about 4-5 passages after the initial seeding of cells.
肺気腫のモデル
この実験では，体重１８０−２１０ｇの雄Ｌｅｗｉｓラット（Charles River Japan Inc.）を用いた。これらと同系のラットをＭＳＣのドナーおよびレシピエントとして用いて，自己移植を模倣した。肺気腫は，ブタ膵臓エラスターゼ（６００ユニット／ｋｇ；Sigma, St.Louis, MO）の気管内注入により生成させた。この実験プロトコルはThe Animal Care Committee of the National Cardiovascular Centerにより承認されたものである。 Model of emphysema Male Lewis rats (Charles River Japan Inc.) weighing 180-210 g were used in this experiment. These syngeneic rats were used as MSC donors and recipients to mimic autografts. Emphysema was generated by intratracheal injection of porcine pancreatic elastase (600 units / kg; Sigma, St. Louis, MO). This experimental protocol was approved by the Animal Care Committee of the National Cardiovascular Center.
ＭＳＣ移植
エラスターゼ注入の７日後，１．５ｘ１０６ＭＳＣ／５００μＬのＰＢＳまたは５００μＬのＰＢＳのみをラットの左頸静脈から静脈内投与した。擬似ラットにも５００μＬのＰＢＳを静脈内投与した。Seven days after MSC transplanted elastase injection, 1.5 × 10 6 MSC / 500 μL PBS or 500 μL PBS alone was administered intravenously from the left jugular vein of rats. Sham rats also received 500 μL of PBS intravenously.
免疫蛍光染色
免疫蛍光染色は，マウス抗サイトケラチン５および８モノクローナル抗体（Chemicon）（上皮細胞のマーカー）；ヤギ抗ＳＰ−Ａポリクローナル抗体（Santa Cruz）（肺胞上皮細胞のマーカー）；およびウサギ抗フォンビルブラント因子（ｖＷＦ）ポリクローナル抗体（ＤＡＫＯ）（血管内皮細胞のマーカー）を用いて行った。フルオレセインイソチオシアネート（ＦＩＴＣ）コンジュゲート化ＩｇＧ抗体（BD Pharmingen）を二次抗体として用いた。 Immunofluorescence staining Immunofluorescence staining was performed using mouse anti-cytokeratin 5 and 8 monoclonal antibodies (Chemicon) (epithelial cell marker); goat anti-SP-A polyclonal antibody (Santa Cruz) (alveolar epithelial cell marker); Von Willebrand factor (vWF) polyclonal antibody (DAKO) (a marker for vascular endothelial cells) was used. Fluorescein isothiocyanate (FITC) conjugated IgG antibody (BD Pharmingen) was used as the secondary antibody.
統計学的分析
数値は特に記載しない限り平均±ｓｅｍ．で表す。３群間のパラメータの比較は，一元分散分析（ＡＮＯＶＡ），続いてシェフ（Scheffe）多重比較検定により分析した。３群間のパラメータの変化の比較は，反復測定用二元ＡＮＯＶＡ，続いてシェフ多重比較検定により行った。Ｐ＜０．０５を有意であると考えた。 Statistical analysis values are mean ± sem unless otherwise stated. Represented by Comparison of parameters between the three groups was analyzed by one-way analysis of variance (ANOVA), followed by a Scheffe multiple comparison test. Comparison of parameter changes between the three groups was performed by two-way ANOVA for repeated measurements followed by chef multiple comparison test. P <0.05 was considered significant.
＜結果＞
肺胞上皮細胞との共培養における上皮分化および肺胞形成
ＭＳＣが上皮様表現型に分化するか否かを調べるために，ＧＦＰを発現するＭＳＣを，成熟肺胞上皮細胞とともに４日間共培養した。最初に，ヒト成熟肺胞上皮細胞（Ａ５４９）を播種し，１０％ＦＢＳおよび抗生物質を補充したＦ−１２Ｋ中で培養した。１日間培養した後，ＧＦＰを発現するＭＳＣを肺胞上皮細胞上に１：１の比率で播種した。細胞は，１０％ＦＢＳおよび抗生物質を補充したＦ−１２Ｋ中で４日間共培養した。次に，細胞を４％パラホルムアルデヒドで１５分間固定した。<Result>
To examine whether epithelial differentiation and alveolar MSCs co-cultured with alveolar epithelial cells differentiate into epithelial phenotype, MSCs expressing GFP were co-cultured with mature alveolar epithelial cells for 4 days . Initially, human mature alveolar epithelial cells (A549) were seeded and cultured in F-12K supplemented with 10% FBS and antibiotics. After culturing for 1 day, MSCs expressing GFP were seeded on alveolar epithelial cells at a ratio of 1: 1. Cells were co-cultured for 4 days in F-12K supplemented with 10% FBS and antibiotics. Cells were then fixed with 4% paraformaldehyde for 15 minutes.
結果を図１に示す。免疫細胞化学的分析により，培養した肺胞上皮細胞の大部分は，サイトケラチン（上皮細胞のマーカー）陽性であることが示された（図１Ａ）。肺胞上皮細胞中に取り込まれたＧＦＰ発現ＭＳＣの一部はサイトケラチンを発現していた。 The results are shown in FIG. Immunocytochemical analysis showed that the majority of cultured alveolar epithelial cells were positive for cytokeratin (epithelial cell marker) (FIG. 1A). Some of the GFP-expressing MSCs incorporated into alveolar epithelial cells expressed cytokeratin.
次に，ＭＳＣがインビトロで成熟肺胞上皮細胞とともに肺胞形成を誘導するか否かを調べるために，ＭＳＣと肺胞上皮細胞をマトリゲル中で６時間共培養した。結果を図１Bに示す。ＧＦＰ発現ＭＳＣは成熟肺胞上皮細胞とともに取り込まれ，これらの細胞とともに肺胞様構造を形成した。 Next, to examine whether MSCs induce alveolar formation with mature alveolar epithelial cells in vitro, MSCs and alveolar epithelial cells were co-cultured in Matrigel for 6 hours. The results are shown in FIG. 1B. GFP-expressing MSCs were taken up with mature alveolar epithelial cells and formed alveolar-like structures with these cells.
ＭＳＣの取り込みおよび分化の評価
５匹のエラスターゼ処置ラットを用いて，移植されたＭＳＣの肺における取り込みおよび分化を調べた。先に報告されているようにして（Messina LM, et al, Proc Natl Acad Sci USA. 1992;89:12018-12022），移植前に，懸濁したＭＳＣをPKH26 Red Fluorescent Cell Linker Kit（SigmaChemical Co.）を用いて蛍光色素で標識した。エラスターゼ注入の７日後に蛍光標識ＭＳＣを静脈内投与し，エラスターゼ注入の４週間後にラットを犠牲死させた。肺を摘出し，サンプルをＯＣＴ化合物中に埋包し，液体窒素中で急速に凍結し，切片を作成した。 Evaluation of MSC uptake and differentiation Five elastase-treated rats were used to examine the uptake and differentiation of transplanted MSCs in the lung. As previously reported (Messina LM, et al, Proc Natl Acad Sci USA. 1992; 89: 12018-12022), suspended MSCs were transferred to PKH26 Red Fluorescent Cell Linker Kit (Sigma Chemical Co. ) With a fluorescent dye. Fluorescently labeled MSCs were administered intravenously 7 days after elastase injection, and rats were sacrificed 4 weeks after elastase injection. The lungs were removed and samples were embedded in OCT compound and rapidly frozen in liquid nitrogen to produce sections.
結果を図２に示す。移植された細胞の一部は肺組織に集積し，肺胞壁に取り込まれた（図２Ａ）。移植された細胞は，ホスト肺胞上皮細胞と一緒に肺胞形成に関与し，サイトケラチン陽性であった。移植されホスト肺胞細胞とともに取り込まれた細胞の一部はＳＰ−Ａ陽性であり，肺胞形成に寄与した（図２Ｂ）。このインビボの結果は，ＭＳＣが肺胞上皮表現型を有する細胞に分化することを示唆しており，共培養系においてＭＳＣが成熟肺胞上皮細胞と一緒に肺胞形成を誘導したというインビトロの知見と一致する。すなわち，エラスターゼ処置肺において，移植されたＭＳＣがホスト細胞と一緒に肺胞の再生を誘導する可能性が示唆された。 The results are shown in FIG. Part of the transplanted cells accumulated in the lung tissue and taken up into the alveolar wall (FIG. 2A). The transplanted cells, together with host alveolar epithelial cells, were involved in alveolar formation and were positive for cytokeratin. Some of the cells transplanted and taken up with the host alveolar cells were SP-A positive and contributed to alveolar formation (FIG. 2B). This in vivo result suggests that MSCs differentiate into cells with an alveolar epithelial phenotype, and in vitro findings that MSCs induced alveolar formation together with mature alveolar epithelial cells in a co-culture system. Matches. That is, it was suggested that transplanted MSCs may induce alveolar regeneration together with host cells in elastase-treated lungs.
また，移植された細胞の少数が肺胞のインターセプトに認められ，これらの細胞はｖＷＦ陽性であった（図２Ｃ）。このことは，定着したＭＳＣの一部が肺において血管内皮細胞に分化したことを示唆する。 In addition, a small number of transplanted cells were observed in the alveolar intercept, and these cells were vWF positive (FIG. 2C). This suggests that some of the established MSCs differentiated into vascular endothelial cells in the lung.
肺血管系の再生
エラスターゼ処置ラットにＭＳＣを静脈内移植し，エラスターゼ注入の４週間後にラットを犠牲死させ，右肺（各ｎ＝５）からパラフィン切片を調製した。ＭＳＣの移植がエラスターゼ処置肺において血管新生を誘導するか否かを調べるため，組織切片をｖＷＦ（ＤＡＫＯ）について染色して，血管密度を評価した。肺胞数およびｖＷＦ陽性毛細管（直径１００μｍ未満）の数は，無作為に選択した１０高出力視野（ｘ２００）で計数した。毛細管密度は，１００個の肺胞あたりの毛細管の数として表した。MSCs were intravenously transplanted into regenerated elastase-treated rats of the pulmonary vasculature , the rats were sacrificed 4 weeks after elastase injection, and paraffin sections were prepared from the right lung (each n = 5). To examine whether MSC transplantation induces angiogenesis in elastase-treated lungs, tissue sections were stained for vWF (DAKO) to assess vessel density. The number of alveoli and the number of vWF positive capillaries (diameter <100 μm) were counted in 10 randomly selected high power fields (x200). Capillary density was expressed as the number of capillaries per 100 alveoli.
図３に肺におけるＭＳＣ誘起性血管新生を示す。図中，スケールバー：１００μｍ；データは平均±ＳＥＭである；＊Ｐ＜０．０５ｖｓ．擬似群；†Ｐ＜０．０５ｖｓ．エラスターゼ群。図３Ａは，擬似ラット（擬似群），ベヒクルを与えたエラスターゼラット（エラスターゼ群），およびＭＳＣを与えたエラスターゼラット（エラスターゼ／ＭＳＣ群）の肺におけるｖＷＦの免疫組織化学実験を示す。ＭＳＣの移植は肺における毛細管密度を増加させた。図３Ｂは，毛細血管密度の半定量的分析の結果を示す。 FIG. 3 shows MSC-induced angiogenesis in the lung. In the figure, scale bar: 100 μm; data are mean ± SEM; * P <0.05 vs. Pseudogroup; † P <0.05 vs. Elastase group. FIG. 3A shows immunohistochemical experiments of vWF in the lungs of sham rats (sham group), elastase rats given vehicle (elastase group), and elastase rats given MSC (elastase / MSC group). MSC transplantation increased the capillary density in the lung. FIG. 3B shows the results of a semi-quantitative analysis of capillary density.
全体として，ｖＷＦ陽性の肺血管数はエラスターゼ注入の４週間後に減少した。ＭＳＣを移植すると，エラスターゼ処置肺においてｖＷＦ−ポジティブ肺血管の数が有意に増加した。 Overall, the number of vWF-positive pulmonary vessels decreased 4 weeks after elastase infusion. Implantation of MSCs significantly increased the number of vWF-positive pulmonary vessels in elastase-treated lungs.
毛細管は肺胞壁の体積の実質的な部分を形成するため，肺気腫の進行にともなって毛細管ネットワークが破壊することは避けられない（Voelkel NF, Cool CD., Eur Respir J Suppl. 2003;46:28s-32s. Review）。したがって，肺気腫による変化を修復するためには，肺胞のみならず血管系の再生を同時に達成することが重要である。本発明においては，移植されたＭＳＣの少数がｖＷＦ陽性であり，肺において血管形成に関与することが見いだされた。さらに，ＭＳＣ移植は，エラスターゼ処置肺において減少したｖＷＦ陽性の肺血管数を有意に増加させた。これらの結果は，ＭＳＣがエラスターゼ処置ラットの肺において血管新生を誘導したことを示唆する。 Since capillaries form a substantial part of the alveolar wall volume, it is inevitable that the capillary network is destroyed as emphysema progresses (Voelkel NF, Cool CD., Eur Respir J Suppl. 2003; 46: 28s-32s. Review). Therefore, in order to repair changes caused by emphysema, it is important to simultaneously achieve regeneration of the vascular system as well as the alveoli. In the present invention, it has been found that a small number of transplanted MSCs are vWF positive and are involved in angiogenesis in the lung. Furthermore, MSC transplantation significantly increased the number of vWF-positive pulmonary blood vessels decreased in elastase-treated lungs. These results suggest that MSC induced angiogenesis in the lungs of elastase-treated rats.
ＭＳＣにおける血管新生，抗アポトーシス，および有糸***促進因子の産生
ＭＳＣが血管新生，抗アポトーシス，および成長因子を産生するか否かを調べるため，培地交換の２４時間後にコンディションド培地中のＶＥＧＦおよび幹細胞成長因子（ＨＧＦ）を測定した。ＶＥＧＦおよびＨＧＦは，酵素イムノアッセイ（VEGF Immunoassay R&D Systems Inc.; rat HGF EIA,Institute of Immunology Co.,Ltd.）により測定した。ＭＳＣまたはベヒクルの投与の前および２４時間後にＶＥＧＦおよびＨＧＦのインビボでの循環レベルを測定した（各群ｎ＝６）。 Angiogenesis, anti-apoptosis, and mitogenic factor production in MSCs To examine whether MSCs produce angiogenesis, anti-apoptosis, and growth factors, VEGF in conditioned media and 24 hours after media change Stem cell growth factor (HGF) was measured. VEGF and HGF were measured by enzyme immunoassay (VEGF Immunoassay R & D Systems Inc .; rat HGF EIA, Institute of Immunology Co., Ltd.). In vivo circulating levels of VEGF and HGF were measured before and 24 hours after administration of MSC or vehicle (n = 6 for each group).
結果を図４に示す。図中，データは平均±ＳＥＭである；＊Ｐ＜０．０５，ｖｓ．移植前。インビトロでは，２４時間培養後，ＭＳＣは線維芽細胞と比較して大量の血管内皮成長因子（ＶＥＧＦ）および幹細胞成長因子（ＨＧＦ）を分泌していた（図４ＡおよびＢ）。インビボでは，ＭＳＣの移植は，移植の２４時間後にＶＥＧＦの血中濃度を有意に増加させ，HGFも増加傾向を示したが，ベヒクル注入はこれらのパラメータを変化させなかった（図４ＣおよびＤ）。 The results are shown in FIG. In the figure, data are mean ± SEM; * P <0.05, vs. Before transplantation. In vitro, after 24 hours of culture, MSCs secreted larger amounts of vascular endothelial growth factor (VEGF) and stem cell growth factor (HGF) compared to fibroblasts (FIGS. 4A and B). In vivo, transplantation of MSC significantly increased blood levels of VEGF 24 hours after transplantation, and HGF also showed an increasing trend, but vehicle injection did not change these parameters (FIGS. 4C and D). .
移植されたＭＳＣがインビボで大量の血管新生，抗アポトーシス，および有糸***促進因子，例えば，ＶＥＧＦおよびＨＧＦを分泌したことは，定着したＭＳＣがパラクライン様式で血管新生を誘導した可能性を示唆する。先の血管新生の結果と合わせると，ＭＳＣは，毛細管様構造を生成する能力によるのみならず，成長因子媒介性パラクライン制御によっても，肺における新生血管形成に寄与しうると考えられる。 Transplanted MSCs secreted large amounts of angiogenesis, anti-apoptosis, and mitogenic factors such as VEGF and HGF in vivo, suggesting that established MSCs may have induced angiogenesis in a paracrine fashion To do. Combined with previous angiogenesis results, MSCs may contribute to neovascularization in the lung not only by the ability to generate capillary-like structures, but also by growth factor-mediated paracrine control.
肺上皮の再生
ラットにおいてエラスターゼ誘起性肺気腫に及ぼすＭＳＣの静脈内投与の効果を調べるために，以下の３つの実験群を作製した：ＭＳＣを与えたエラスターゼラット（エラスターゼ／ＭＳＣ群，ｎ＝１１）；ＰＢＳを与えたエラスターゼラット（エラスターゼ群，ｎ＝１１）；培地を与えた擬似ラット（擬似群，ｎ＝１１）。To investigate the effect of intravenous administration of MSC on elastase-induced emphysema in lung epithelial regeneration rats, the following three experimental groups were created: elastase rats given MSC (elastase / MSC group, n = 11) Elastase rats fed PBS (elastase group, n = 11); sham rats fed medium (pseudo group, n = 11).
エラスターゼ注射の４週間後，ラットを麻酔し，コンピュータ制御小動物換気装置（flexiVent; Scireq, Montreal, PQ, Canada）を用いて静的肺コンプライアンスを測定した。肺を除去し，２５ｃｍＨ2Ｏの一定の経肺圧で２４時間固定した。肺体積は，Ｓｃｈｅｒｌｅの方法（Scherle W. Mikroskopie 1970;26:57-60）により測定した。肺気腫の形態計測パラメータである平均肺胞径は，先に記載されているようにして（Thurlbeck WM. Thorax. 1967;22:483-496），光学顕微鏡により２０の無作為に選択した視野について計算した。Four weeks after elastase injection, rats were anesthetized and static lung compliance was measured using a computer controlled small animal ventilator (flexiVent; Scireq, Montreal, PQ, Canada). The lungs were removed and fixed for 24 hours at a constant transpulmonary pressure of 25 cm H 2 O. Lung volume was measured by Scherle's method (Scherle W. Mikroskopie 1970; 26: 57-60). The mean alveolar diameter, a morphometric parameter for emphysema, was calculated for 20 randomly selected fields of view with an optical microscope as described previously (Thurlbeck WM. Thorax. 1967; 22: 483-496). did.
結果を図５および図６に示す。図中，データは平均±ＳＥＭ；＊Ｐ＜０．０５ｖｓ．擬似群；†Ｐ＜０．０５ｖｓ．エラスターゼ群。エラスターゼ注入の４週間後，エラスターゼ群においては肺胞壁の破壊にともなう気腔拡大の発達が認められたが（図５Ａ，倍率ｘ１００），ＭＳＣの移植はエラスターゼ誘起性肺気腫の変化（エラスターゼ／ＭＳＣ群）を弱めた。エラスターゼ群における平均肺胞径は，擬似群と比較して有意に増加したが（図５Ｂ），この増加はＭＳＣにより有意に弱められた。また，エラスターゼ群の肺体積は，擬似群と比較して有意に増加していたが（図６Ａ），肺体積の増加は，ＭＳＣ移植により有意に弱められた。静的肺コンプライアンスはエラスターゼ群において有意に増加し，この変化はＭＳＣにより有意に弱められた（図６Ｂ）。 The results are shown in FIG. 5 and FIG. In the figure, data are mean ± SEM; * P <0.05 vs. Pseudogroup; † P <0.05 vs. Elastase group. Four weeks after elastase injection, in the elastase group, the development of air space enlargement with destruction of the alveolar wall was observed (Fig. 5A, magnification x100), but MSC transplantation changed elastase-induced emphysema (elastase / MSC Group) weakened. The average alveolar diameter in the elastase group increased significantly compared to the sham group (FIG. 5B), but this increase was significantly attenuated by MSC. In addition, the lung volume of the elastase group was significantly increased compared to the sham group (FIG. 6A), but the increase in lung volume was significantly attenuated by MSC transplantation. Static lung compliance was significantly increased in the elastase group and this change was significantly attenuated by MSC (FIG. 6B).
これらの結果は，ＭＳＣの移植が，エラスターゼ処置ラットにおいて，肺体積，静的肺コンプライアンス，および平均肺胞径の増加を有意に緩和したことを示す。したがって，ＭＳＣ誘起性肺胞再生の結果として，肺の構造および機能が改善されたと考えられる。 These results indicate that MSC implantation significantly mitigated increases in lung volume, static lung compliance, and mean alveolar diameter in elastase-treated rats. Thus, lung structure and function may have improved as a result of MSC-induced alveolar regeneration.
本発明は，肺気腫の治療に有用である。
The present invention is useful for the treatment of emphysema.
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