JP5804342B2 - Therapeutic agent for hepatitis including adipose tissue-derived stromal cells - Google Patents
Therapeutic agent for hepatitis including adipose tissue-derived stromal cells Download PDFInfo
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Description
本発明は、脂肪組織由来間質細胞群を含む肝炎治療剤に関するものである。 The present invention relates to a therapeutic agent for hepatitis including a group of adipose tissue-derived stromal cells.
（肝臓及びその疾患）
肝臓は、身体に必要な物質を合成し、老廃物を排泄するなど、生命活動にとって重要な臓器である。
しかし、肝臓は、ウイルス、薬物、アルコール等の原因により、急性及び慢性的に障害を受け、肝炎、肝硬変、肝癌等の肝疾患を起こす。
(Liver and its diseases)
The liver is an important organ for life activity, such as synthesizing substances necessary for the body and excreting waste products.
However, the liver is acutely and chronically damaged due to causes such as viruses, drugs, and alcohol, and causes liver diseases such as hepatitis, cirrhosis, and liver cancer.
（急性肝炎）
急性肝炎は、ほとんどがウイルス（特に肝炎ウイルス）の感染により起こる。感染ウイルスの種類により、Ａ型肝炎、Ｂ型肝炎、Ｃ型肝炎、Ｄ型肝炎、Ｅ型肝炎、及びその他のウイルスによる肝炎に分類される。
日本における急性肝炎の年間発生は、約35万人といわれており、その内訳はＡ型肝炎10万人、Ｂ型肝炎10万人、Ｃ型肝炎14万人であり、Ｄ型肝炎とＥ型肝炎は僅かである。
(Acute hepatitis)
Acute hepatitis is mostly caused by infection with a virus (particularly hepatitis virus). According to the type of infectious virus, it is classified into hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, and hepatitis due to other viruses.
The annual incidence of acute hepatitis in Japan is said to be about 350,000. The breakdown is 100,000 hepatitis A, 100,000 hepatitis B, 140,000 hepatitis C, hepatitis D and E Hepatitis is slight.
（劇症肝炎）
劇症肝炎は、肝臓の肝細胞が急激かつ大量に壊れることによって、肝機能が低下していく疾患である。なお、肝機能が低下すると、血液を固めるために必要な凝固因子の産出が低下し、また、老廃物の蓄積により意識障害（肝性脳症）が出現する。
劇症肝炎の症状は、発熱、全身のだるさ、吐き気、食欲不振等の急性肝炎と同じ症状である。さらに、該症状が現れてから約8週間以内に、肝性脳症が見られ、血液中の凝固因子濃度がある一定値以下になった場合には、劇症肝炎と診断する。劇症肝炎では、肝臓の組織が大量に壊れるために、肝細胞の増殖が遅れて肝再生が十分生じない場合がある。
日本における劇症肝炎の年間発生は、約400人と推定される。
(Fulminant hepatitis)
Fulminant hepatitis is a disease in which liver function declines due to rapid and massive destruction of hepatocytes in the liver. In addition, when liver function falls, the production of coagulation factors necessary for consolidating blood falls, and disturbance of consciousness (hepatic encephalopathy) appears due to accumulation of waste products.
Symptoms of fulminant hepatitis are the same as acute hepatitis such as fever, general dullness, nausea, and loss of appetite. Furthermore, hepatic encephalopathy is observed within about 8 weeks after the appearance of the symptom, and fulminant hepatitis is diagnosed when the blood coagulation factor concentration falls below a certain value. In fulminant hepatitis, a large amount of liver tissue is destroyed, and hepatocyte proliferation may be delayed, resulting in insufficient liver regeneration.
The annual incidence of fulminant hepatitis in Japan is estimated to be about 400.
先行特許文献１では、「脂肪組織由来幹細胞とヘパリンを組み合わせた組織再生用組成物」を開示している。該文献では、脂肪細胞組織由来幹細胞を単独で含む組織再生組成物を開示している（参照：段落［0023］）。また、実施例での四塩化炭素投与による急性肝炎マウスでの実験結果では、肝障害マーカー（ALT、AST）値は、無治療群と比較して、約70〜54％を示した。加えて、脂肪細胞組織由来幹細胞のみを投与した群の生存率は33％であり、該細胞にヘパリンを併用投与した群の生存率は100％であった（参照：段落「0004」及び「0044」）。
さらに、本発明の肝炎治療剤を四塩化炭素投与による急性肝炎マウスに投与しても、該組織再生用組成物のような結果が見られなかった。
以上により、本発明の脂肪組織由来間質細胞群を含む肝炎治療剤は、該組織再生用組成物とは、組成内容、治療効果及び治療機構が明らかに異なる。
Prior Patent Document 1 discloses “a composition for tissue regeneration combining adipose tissue-derived stem cells and heparin”. This document discloses a tissue regeneration composition containing an adipocyte tissue-derived stem cell alone (see paragraph [0023]). Moreover, in the experimental result in the acute hepatitis mouse | mouth by carbon tetrachloride administration in an Example, the liver damage marker (ALT, AST) value showed about 70 to 54% compared with the no-treatment group. In addition, the survival rate of the group in which only the adipocyte tissue-derived stem cells were administered was 33%, and the survival rate of the group in which heparin was administered to the cells was 100% (see paragraphs “0004” and “0044”). ").
Further, even when the therapeutic agent for hepatitis of the present invention was administered to acute hepatitis mice by carbon tetrachloride administration, the results as in the tissue regeneration composition were not observed.
As described above, the therapeutic agent for hepatitis containing the adipose tissue-derived stromal cell group of the present invention is clearly different from the composition for tissue regeneration in composition content, therapeutic effect, and therapeutic mechanism.
急性肝炎・劇症肝炎は、治療奏功まで時間を要する場合があり、また、速やかな治療開始にもかかわらず急速な肝機能悪化を防ぐことができない場合がある。重篤な肝機能障害症状の場合には、血漿交換、さらには肝移植が必要である。しかし、肝移植のためのドナーが不足している状況である。
そこで、本発明者らは、新規な肝炎治療剤を開発することを目的とした。
For acute hepatitis and fulminant hepatitis, it may take time until the treatment is successful, and it may not be possible to prevent rapid liver function deterioration despite rapid start of treatment. In severe liver dysfunction symptoms, plasma exchange and even liver transplantation are necessary. However, there is a shortage of donors for liver transplantation.
Therefore, the present inventors have aimed to develop a novel therapeutic agent for hepatitis.
本発明者らは、上記課題を解決するために、脂肪組織由来幹細胞ではなく、脂肪組織由来間質細胞群に注目した。
そして、本発明者らは、脂肪組織由来間質細胞群が優れた安全性、肝細胞壊死抑制効果及び肝障害マーカー値の改善効果を有することを確認し、該細胞群を含む肝炎治療剤を完成した。
In order to solve the above-mentioned problems, the present inventors paid attention to a group of adipose tissue-derived stromal cells instead of adipose tissue-derived stem cells.
Then, the present inventors confirmed that the adipose tissue-derived stromal cell group has excellent safety, hepatocyte necrosis inhibitory effect and liver disorder marker value improving effect, and a hepatitis therapeutic agent containing the cell group completed.
本発明は以下からなる。
「１．内皮細胞、血球系細胞を含む脂肪組織由来間質細胞群を含む肝炎治療剤。
２．内皮細胞、血球系細胞、線維芽細胞、血管周囲細胞、平滑筋細胞を含む脂肪組織由来間質細胞群を含む前項１に記載の肝炎治療剤。
３．前記肝炎が、急性肝炎、劇症肝炎、慢性肝炎、劇症肝不全、又は急性肝不全である前項１又は２に記載の肝炎治療剤。」
The present invention comprises the following.
“1. A therapeutic agent for hepatitis including a group of adipose tissue-derived stromal cells including endothelial cells and blood cells.
2. 2. The therapeutic agent for hepatitis according to item 1 above, comprising a group of adipose tissue-derived stromal cells including endothelial cells, blood cells, fibroblasts, perivascular cells, and smooth muscle cells.
3. 3. The therapeutic agent for hepatitis according to item 1 or 2, wherein the hepatitis is acute hepatitis, fulminant hepatitis, chronic hepatitis, fulminant liver failure, or acute liver failure. "
本発明の肝炎治療剤は、優れた安全性、肝細胞壊死抑制効果及び肝障害マーカー値の改善効果を示す。 The therapeutic agent for hepatitis of the present invention exhibits excellent safety, hepatocyte necrosis inhibitory effect, and liver disorder marker value improving effect.
（肝炎治療剤）
本発明の肝炎治療剤は、少なくとも、内皮細胞、血球系細胞を含む脂肪組織由来間質細胞群、より好ましくは、内皮細胞、血球系細胞、線維芽細胞、血管周囲細胞、平滑筋細胞を含む脂肪組織由来間質細胞群を含む。
なお、脂肪組織由来間質細胞群は、好ましくは、自家性又は同系である。自家性脂肪組織由来間質細胞群とは、投与対象から得られた脂肪組織由来間質細胞群を意味する。また、同系脂肪組織由来間質細胞群とは、投与対象と同じ種から得られた脂肪組織由来間質細胞群を意味する。例えば、投与対象がヒトであるなら、同系脂肪組織由来間質細胞群は投与対象以外のヒトから得られる。
(Hepatitis treatment agent)
The therapeutic agent for hepatitis of the present invention includes at least an adipose tissue-derived stromal cell group including endothelial cells and blood cells, more preferably endothelial cells, blood cells, fibroblasts, perivascular cells, and smooth muscle cells. Includes adipose tissue-derived stromal cells.
The adipose tissue-derived stromal cell group is preferably autologous or syngeneic. The autologous adipose tissue-derived stromal cell group means the adipose tissue-derived stromal cell group obtained from the administration subject. The syngeneic adipose tissue-derived stromal cell group means an adipose tissue-derived stromal cell group obtained from the same species as the administration subject. For example, if the administration subject is a human, the syngeneic adipose tissue-derived stromal cell group is obtained from a human other than the administration subject.
（脂肪組織由来間質細胞群）
本発明の脂肪組織由来間質細胞群とは、分取した脂肪組織を酵素処理（好ましくは、コラゲナーゼ処理）して得られた細胞群を意味する。
脂肪組織由来間質細胞群は、少なくとも内皮細胞、血球系細胞を含み、好ましくは、内皮細胞、血球系細胞、線維芽細胞、血管周囲細胞、平滑筋細胞を含む。
(Adipose tissue-derived stromal cell group)
The adipose tissue-derived stromal cell group of the present invention means a cell group obtained by subjecting a sorted adipose tissue to an enzyme treatment (preferably collagenase treatment).
The adipose tissue-derived stromal cell group includes at least endothelial cells and blood cells, and preferably includes endothelial cells, blood cells, fibroblasts, perivascular cells, and smooth muscle cells.
（肝炎治療剤の適用対象）
本発明の肝炎治療剤の適用対象は、急性肝炎、劇症肝炎、慢性肝炎、劇症肝不全、又は急性肝不全等である。
さらに、本発明の肝炎治療剤は、肝炎予防にも利用できる。
(Application target for hepatitis treatment agents)
The application target of the therapeutic agent for hepatitis of the present invention is acute hepatitis, fulminant hepatitis, chronic hepatitis, fulminant liver failure, acute liver failure, or the like.
Furthermore, the therapeutic agent for hepatitis of the present invention can be used for preventing hepatitis.
（肝炎治療剤の治療対象）
本発明の肝炎治療剤の治療対象は、哺乳動物を包括的に含む。哺乳動物の例として、ヒト、ウシ、ウマ、ヒツジ、ブタ、ネコ、イヌ、マウス、ラット、ウサギ、モルモット、サル等であるが、特に限定されない。
(Health treatment agent)
The treatment target of the therapeutic agent for hepatitis of the present invention comprehensively includes mammals. Examples of mammals include, but are not limited to, humans, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and the like.
（肝炎治療剤に含まれる生理活性物質）
本発明の肝炎治療剤には、生理活性物質が含まれていても良い。生理活性物質は、例えば、タンパク質、ポリペプチド、多糖（例えばヘパリン）、オリゴ糖、単糖、二糖、有機化合物、有機金属化合物、又は無機化合物等が挙げられるが特に限定されない。より詳しくは、生物学的に活性な分子、例えば、ホルモン、増殖因子、増殖因子産生ウイルス、増殖因子阻害薬、増殖因子受容体、抗炎症薬、代謝拮抗薬、インテグリン遮断薬、又はセンス遺伝子若しくはアンチセンス遺伝子等が挙げられる。
さらにより詳しくは、増殖因子の例としては、白血病阻害因子（ＬＩＦ）、上皮増殖因子（ＥＧＦ）、線維芽細胞増殖因子（ＦＧＦ）、トランスフォーミング増殖因子−ベータ（ＴＧＦ−β）、インスリン様増殖因子（ＩＧＦ）、および血管内皮細胞増殖因子（ＶＥＧＦ）、ヒト成長ホルモン、血小板誘発増殖因子（ＰＤＧＦ）、インターロイキン、サイトカイン、又はそれらの組み合わせ等が挙げられる。
(Bioactive substances contained in hepatitis treatment agents)
The therapeutic agent for hepatitis of the present invention may contain a physiologically active substance. Examples of the physiologically active substance include, but are not limited to, proteins, polypeptides, polysaccharides (eg, heparin), oligosaccharides, monosaccharides, disaccharides, organic compounds, organometallic compounds, and inorganic compounds. More particularly, biologically active molecules such as hormones, growth factors, growth factor producing viruses, growth factor inhibitors, growth factor receptors, anti-inflammatory agents, antimetabolites, integrin blockers, or sense genes or An antisense gene etc. are mentioned.
More particularly, examples of growth factors include leukemia inhibitory factor (LIF), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-β), insulin-like growth. Factor (IGF), and vascular endothelial growth factor (VEGF), human growth hormone, platelet-induced growth factor (PDGF), interleukin, cytokine, or combinations thereof.
（肝炎治療剤に含まれる免疫抑制剤）
本発明の肝炎治療剤には、免疫抑制剤が含まれていても良い。例えば、自家性ではない脂肪組織由来間質細胞群を含む肝炎治療剤の場合には、免疫拒絶反応を抑えるために、好ましくは免疫抑制剤を含める。
(Immunosuppressant contained in hepatitis treatment agent)
The therapeutic agent for hepatitis of the present invention may contain an immunosuppressant. For example, in the case of a therapeutic agent for hepatitis including a non-autologous adipose tissue-derived stromal cell group, an immunosuppressive agent is preferably included in order to suppress immune rejection.
（肝炎治療剤に含まれる担体）
本発明の肝炎治療剤には、担体が含まれていても良い。担体の例としては、生理食塩水、溶媒、分散培地、細胞培養液、水溶性緩衝、抗酸化剤等が挙げられるが特に限定されない。
(Carriers included in hepatitis treatment agents)
The therapeutic agent for hepatitis of the present invention may contain a carrier. Examples of the carrier include, but are not limited to, physiological saline, a solvent, a dispersion medium, a cell culture solution, a water-soluble buffer, and an antioxidant.
（肝炎治療剤の投与形態）
本発明の肝炎治療剤の投与経路は特に限定されない。例えば、静脈内注射、動脈内注射、門脈内注射、皮内注射、皮下注射、筋肉内注射、又は腹腔内注射等を利用することができる。
また、本発明の肝炎治療剤の投与量は、例えば、１回の１治療部位当たり約1×102〜1×1015個の脂肪由来幹細胞を該治療部位に投与できるが、特に限定されない。
(Administration form of hepatitis treatment agent)
The administration route of the therapeutic agent for hepatitis of the present invention is not particularly limited. For example, intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or the like can be used.
The dose of the therapeutic agent for hepatitis of the present invention is not particularly limited, for example, although about 1 × 10 2 to 1 × 10 15 fat-derived stem cells per treatment site can be administered to the treatment site.
（脂肪組織由来間質細胞群の調製方法）
脂肪組織由来間質細胞群の調製方法を以下に記載するが、特に限定されない。
脂肪組織（皮下脂肪、内臓脂肪、筋肉内脂肪、筋肉間脂肪）を動物から切除、吸引などの手段で採取する。免疫拒絶を回避するため、治療対象と同一の個体から脂肪組織を採取することが好ましい。
採取した脂肪組織は、必要に応じて、付着した血液成分の除去及び細片化を経た後、酵素処理に供される。酵素処理は、脂肪組織をコラゲナーゼ、トリプシン、ディスパーゼ等の酵素によって消化する。
なお、脂肪組織を適当な緩衝液や培養液中で洗浄することによって血液成分等を除去することができる。
(Method for preparing adipose tissue-derived stromal cells)
Although the preparation method of an adipose tissue origin stromal cell group is described below, it is not specifically limited.
Adipose tissue (subcutaneous fat, visceral fat, intramuscular fat, intermuscular fat) is collected from the animal by means such as excision and suction. In order to avoid immune rejection, it is preferable to collect adipose tissue from the same individual as the treatment target.
The collected adipose tissue is subjected to enzyme treatment after removal of adhering blood components and fragmentation as necessary. In the enzyme treatment, adipose tissue is digested with enzymes such as collagenase, trypsin, and dispase.
In addition, a blood component etc. can be removed by wash | cleaning a fat tissue in a suitable buffer solution or a culture solution.
（脂肪組織由来間質細胞群の調製方法の例）
脂肪組織由来間質細胞群の調製方法の具体例を以下に示すが、特に限定されない。
ヒトから採取した脂肪を、緩衝液を含有する培養皿に置き、洗浄し、さらに別の緩衝液を含有する培養皿に移す。該脂肪を微細な小片に刻み、2mg/mLコラゲナーゼＩを用いて数時間、約30〜39℃で消化する。消化済みの脂肪の懸濁液をＤＭＥＭ−１０（ＤＭＥＭ＋１０％ＦＢＳ＋１％ペニシリン/アンピリシン）中で洗浄する。そして、消化されなかった脂肪組織を除去し、細胞ペレットを５ｍＬのＤＭＥＭ−１０中に懸濁させる。さらに、脂肪細胞懸濁液を、フィルターメッシュに通し、大きな粒子および凝集塊を除去して、脂肪組織由来間質細胞群を得る。
(Example of method for preparing adipose tissue-derived stromal cells)
Although the specific example of the preparation method of an adipose tissue origin stromal cell group is shown below, it is not specifically limited.
Fat collected from humans is placed in a culture dish containing a buffer, washed, and transferred to a culture dish containing another buffer. The fat is minced into small pieces and digested with 2 mg / mL collagenase I for several hours at about 30-39 ° C. The digested fat suspension is washed in DMEM-10 (DMEM + 10% FBS + 1% penicillin / ampicillin). Undigested adipose tissue is then removed and the cell pellet is suspended in 5 mL of DMEM-10. Furthermore, the fat cell suspension is passed through a filter mesh to remove large particles and aggregates, thereby obtaining a group of adipose tissue-derived stromal cells.
以下、実施例を挙げて本発明を詳細に説明するが、本発明の範囲はこれらの実施例により限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, the scope of the present invention is not limited by these Examples.
（脂肪組織由来間質細胞群の調製方法）
マウスの脂肪より脂肪組織由来間質細胞群を調製した。詳細は、以下の通りである。
C57Bl/6マウスを安楽死させ、鼡径部皮下脂肪組織を鈍的に剥離採取し、緩衝液を含有する培養皿に浸し洗浄し、さらに別の緩衝液を含有する培養皿に移した。該脂肪組織を細切し、0.15%コラゲナーゼIにて最大30分、37℃にて酵素処理し、抗真菌および抗生物質を含む10% FBS 添加DMEM/F12(1:1) 溶液へ懸濁し、フィルターメッシュ(100μm)に通した後、遠心分離にて沈降させ、初代間質細胞群を得た。
さらに、該初代間質細胞群を、抗真菌および抗生物質を含む10%FBS添加DMEM/F12(1:1)培養液で培養し、非選択的に継代培養増殖を行った。
(Method for preparing adipose tissue-derived stromal cells)
Adipose tissue-derived stromal cells were prepared from mouse fat. Details are as follows.
C57Bl / 6 mice were euthanized, and the inguinal subcutaneous adipose tissue was bluntly peeled, washed by immersion in a culture dish containing a buffer solution, and further transferred to a culture dish containing another buffer solution. The adipose tissue is minced, treated with 0.15% collagenase I for up to 30 minutes at 37 ° C., and suspended in a 10% FBS-added DMEM / F12 (1: 1) solution containing antifungal and antibiotics. After passing through a filter mesh (100 μm), sedimentation was performed by centrifugation to obtain a primary stromal cell group.
Further, the primary stromal cell group was cultured in a DMEM / F12 (1: 1) culture solution supplemented with 10% FBS containing antifungal and antibiotics, and subcultured and proliferated non-selectively.
（肝細胞壊死抑制効果の確認）
本発明の肝炎治療剤が肝細胞壊死抑制効果を有することを確認した。詳細は、以下の通りである。
(Confirmation of hepatocyte necrosis suppression effect)
It was confirmed that the therapeutic agent for hepatitis of the present invention has an effect of suppressing hepatocyte necrosis. Details are as follows.
（マウスの準備）
C57Bl/6マウス（メス、12週齢）を使用し、コンカナバリンA（Concanavalin A）投与群、並びに、コンカナバリンA及び脂肪組織由来間質細胞群投与群を準備した。各群の条件は、以下の通りである。
＜コンカナバリンA投与群＞
0.3mg コンカナバリンAをすべてのマウスの尾静脈投与した。そして、各マウスを、それぞれ、投与0時間後（投与直後）、投与3時間後、6時間後、12時間後、24時間後に、麻酔下で開腹して、肝臓を確認した。
＜コンカナバリンA及び脂肪組織由来間質細胞群投与群＞
0.3mg コンカナバリンAをすべてのマウスの尾静脈投与し、さらに該投与３時間後に実施例１で調製した約100,000個の脂肪組織由来間質細胞を尾静脈投与した。そして、各マウスを、それぞれ、コンカナバリンA投与前無処置マウス（脂肪組織由来間質細胞投与なし）、3時間後（脂肪組織由来間質細胞投与直後）、6時間後（脂肪組織由来間質細胞投与3時間後）、12時間後（脂肪組織由来間質細胞投与9時間後）、24時間後（脂肪組織由来間質細胞投与21時間後）に、麻酔下で開腹して、肝臓を確認した。
(Mouse preparation)
C57Bl / 6 mice (female, 12 weeks old) were used to prepare a group administered with concanavalin A (Concanavalin A) and a group administered with concanavalin A and adipose tissue-derived stromal cells. The conditions for each group are as follows.
<Concanavalin A administration group>
0.3 mg concanavalin A was administered to the tail vein of all mice. Each mouse was laparotomized under anesthesia 0 hours after administration (immediately after administration), 3 hours, 6 hours, 12 hours, and 24 hours after administration, and the liver was confirmed.
<Concanavalin A and adipose tissue-derived stromal cell group administration group>
0.3 mg of concanavalin A was administered to the tail vein of all mice, and about 100,000 adipose tissue-derived stromal cells prepared in Example 1 were administered to the tail vein 3 hours after the administration. Then, each mouse was treated with untreated mice before administration of concanavalin A (without administration of adipose tissue-derived stromal cells), after 3 hours (immediately after administration of adipose tissue-derived stromal cells), and after 6 hours (adipose tissue-derived stromal cells). 3 hours after administration), 12 hours after administration (9 hours after administration of adipose tissue-derived stromal cells), and 24 hours after administration (21 hours after administration of adipose tissue-derived stromal cells), the abdomen was anesthetized and the liver was confirmed. .
（肝臓の肝細胞壊死の確認）
上記コンカナバリンA投与群の肝細胞の画像を図１に示す。また、上記コンカナバリンA及び脂肪組織由来間質細胞群投与群の肝細胞の画像を図２に示す。
図１の画像より、コンカナバリンA投与後3時間、6時間では炎症を示唆する暗赤色調が経時的に強くなり、12時間より壊死部が明瞭となり、24時間でさらに拡大していることを確認できた。
一方、図２の画像により、コンカナバリンA投与後6時間（脂肪組織由来間質細胞投与3時間後）のマウスの肝臓では、炎症を示唆する暗赤色変化がみられるが、コンカナバリンA投与後12及び24時間の各マウスの肝臓では、色調は正常肝とほぼ同様、炎症の消褪が観察された。
以上により、本発明の脂肪組織由来間質細胞を含む肝炎治療剤は、迅速な肝細胞壊死抑制効果を有することを確認した。
(Confirmation of hepatocyte necrosis in the liver)
An image of hepatocytes in the concanavalin A administration group is shown in FIG. Moreover, the image of the hepatocyte of the said concanavalin A and adipose tissue origin stromal cell group administration group is shown in FIG.
From the image in Fig. 1, it was confirmed that the dark red tone suggesting inflammation became stronger with time at 3 and 6 hours after concanavalin A administration, and the necrotic part became clearer at 12 hours and expanded further at 24 hours. did it.
On the other hand, in the image of FIG. 2, in the liver of a mouse 6 hours after administration of concanavalin A (3 hours after administration of adipose tissue-derived stromal cells), a dark red change suggesting inflammation is observed. In the liver of each mouse for 24 hours, the disappearance of inflammation was observed in the same color tone as in the normal liver.
From the above, it was confirmed that the therapeutic agent for hepatitis containing adipose tissue-derived stromal cells of the present invention has a rapid hepatocyte necrosis inhibitory effect.
（肝障害マーカーの改善の確認）
本発明の肝炎治療剤が肝障害マーカー値改善効果を有することを確認した。詳細は、以下の通りである。
(Confirmation of improvement of liver injury marker)
It was confirmed that the therapeutic agent for hepatitis of the present invention has an effect of improving the liver marker value. Details are as follows.
（マウスの準備）
C57Bl/6マウス（メス、12週齢）を合計36匹用意した。
肝障害マーカーであるAST（アスパラギン酸アミノトランスフェラーゼ）測定用のコンカナバリンA投与群（4匹）、コンカナバリンA及び脂肪組織由来間質細胞群投与群（3匹）、コンカナバリンA及び大腸癌細胞株MC38投与群（3匹）、コンカナバリンA及び初代培養マウス肝細胞投与群（3匹）並びに正常群（5匹）を用意した。
肝障害マーカーであるALT（アラニンアミノトランスフェラーゼ）測定用のコンカナバリンA投与群（4匹）、コンカナバリンA及び脂肪組織由来間質細胞群投与群（3匹）、コンカナバリンA及び大腸癌細胞株MC38投与群（3匹）、コンカナバリンA及び初代培養マウス肝細胞投与群（3匹）並びに正常群（５匹）を用意した。
各群の条件は、以下の通りである。
＜コンカナバリンA投与群＞
0.3mg コンカナバリンAをマウスの尾静脈投与した。
＜コンカナバリンA及び脂肪組織由来間質細胞群投与群＞
コンカナバリンAをマウスの尾静脈投与し、さらに該投与３時間後に実施例１で調製した約100,000個の脂肪組織由来間質細胞を尾静脈投与した。
＜コンカナバリンA及び大腸癌細胞株MC38投与群＞
コンカナバリンAをマウスの尾静脈投与し、さらに該投与３時間後に約100,000個の大腸癌細胞株MC38を尾静脈投与した。
＜コンカナバリンA及び初代培養マウス肝細胞投与群＞
コンカナバリンAをマウスの尾静脈投与し、さらに該投与３時間後に約100,000個の初代培養マウス肝細胞を尾静脈投与した。
＜正常群＞
コンカナバリンAを投与していない。
(Mouse preparation)
A total of 36 C57Bl / 6 mice (female, 12 weeks old) were prepared.
Concanavalin A administration group for AST (aspartate aminotransferase) measurement (4 mice), concanavalin A and adipose tissue-derived stromal cell group administration group (3 mice), concanavalin A and colon cancer cell line MC38 administration A group (3 mice), a concanavalin A and primary culture mouse hepatocyte administration group (3 mice), and a normal group (5 mice) were prepared.
Concanavalin A administration group for ALT (alanine aminotransferase) measurement (4 mice), concanavalin A and adipose tissue-derived stromal cell group administration group (3 mice), concanavalin A and colon cancer cell line MC38 administration group (3 mice), concanavalin A and primary cultured mouse hepatocyte administration group (3 mice) and normal group (5 mice) were prepared.
The conditions for each group are as follows.
<Concanavalin A administration group>
0.3 mg concanavalin A was administered to the tail vein of mice.
<Concanavalin A and adipose tissue-derived stromal cell group administration group>
Concanavalin A was administered to the tail vein of mice, and about 100,000 adipose tissue-derived stromal cells prepared in Example 1 were administered to the tail vein 3 hours after the administration.
<Concanavalin A and colon cancer cell line MC38 administration group>
Concanavalin A was administered to the tail vein of mice, and about 100,000 colon cancer cell lines MC38 were further administered to the tail vein 3 hours after the administration.
<Concanavalin A and primary cultured mouse hepatocyte administration group>
Concanavalin A was administered to the tail vein of mice, and about 100,000 primary cultured mouse hepatocytes were further administered to the tail vein 3 hours after the administration.
<Normal group>
Concanavalin A is not administered.
（肝障害マーカー値の測定）
上記コンカナバリンA投与24時間後の各群の血清中のAST及びALTを測定した。
AST測定は、Ｌタイプワコー ＧOＴ・Ｊ２（和光純薬工業株式会社製）のキット及び自動分析装置（日立７１８０／日立ハイテクノロジーズ社製）を用いて行なった。
ALT測定は、Ｌタイプワコー ＧＰＴ・Ｊ２（和光純薬工業株式会社製）のキット及び自動分析装置（日立７１８０／日立ハイテクノロジーズ社製）を用いて行なった。
(Measurement of liver injury marker value)
AST and ALT in the serum of each group 24 hours after administration of the above concanavalin A were measured.
AST measurement was performed using a kit of L type Wako GOT · J2 (manufactured by Wako Pure Chemical Industries, Ltd.) and an automatic analyzer (manufactured by Hitachi 7180 / manufactured by Hitachi High-Technologies Corporation).
ALT measurement was performed using a kit of L type Wako GPT • J2 (manufactured by Wako Pure Chemical Industries, Ltd.) and an automatic analyzer (manufactured by Hitachi 7180 / manufactured by Hitachi High-Technologies Corporation).
（肝障害マーカー値の測定結果）
AST及びALTの測定結果を、それぞれ、図３及び図４に示す。
図３の結果より、コンカナバリンA及び脂肪組織由来間質細胞群投与群のASTは、コンカナバリンA投与群のASTと比較して、顕著に減少していることを確認した。
さらに、コンカナバリンA及び脂肪組織由来間質細胞群投与群のAST値は、コンカナバリンA投与群（無治療群）のAST値と比較して、約11.5％（750／6500）にまで減少した。
図４の結果より、コンカナバリンA及び脂肪組織由来間質細胞群投与群のALTは、コンカナバリンA投与群のALTと比較して、顕著に減少していることを確認した。
さらに、コンカナバリンA及び脂肪組織由来間質細胞群投与群のALT値は、コンカナバリンA投与群（無治療群）のALT値と比較して、約5.5％（300／5500）にまで減少した。
加えて、本発明の肝炎治療剤の効果を、先行特許文献１に記載の「組織再生用組成物」と比較すると、AST効果では約6倍（70／11.5×100）、ALT効果では約9倍（50／5.5×100）であった。
すなわち、本発明の肝炎治療剤の肝障害マーカー改善効果は、先行技術に記載の組織再生用組成物の肝障害マーカーの改善効果と比較して、6倍以上である。
以上により、本発明の脂肪組織由来間質細胞を含む肝炎治療剤は、肝障害マーカーを顕著に改善させることができることを確認した。
(Measurement result of liver injury marker value)
The measurement results of AST and ALT are shown in FIGS. 3 and 4, respectively.
From the results of FIG. 3, it was confirmed that the AST of the group administered with concanavalin A and the adipose tissue-derived stromal cell group was significantly reduced as compared with the AST of the group administered with concanavalin A.
Furthermore, the AST value of the group administered with concanavalin A and the adipose tissue-derived stromal cell group was reduced to about 11.5% (750/6500) compared to the AST value of the group administered with concanavalin A (non-treated group).
From the results of FIG. 4, it was confirmed that the ALT in the group administered with concanavalin A and the adipose tissue-derived stromal cell group was significantly reduced as compared with the ALT in the group administered with concanavalin A.
Furthermore, the ALT value of the group administered with concanavalin A and the adipose tissue-derived stromal cell group was reduced to about 5.5% (300/5500) as compared with the ALT value of the group treated with concanavalin A (non-treated group).
In addition, when the effect of the therapeutic agent for hepatitis of the present invention is compared with the “composition for tissue regeneration” described in Patent Document 1, the AST effect is about 6 times (70 / 11.5 × 100), and the ALT effect is about 9 times. Double (50 / 5.5 × 100).
That is, the liver injury marker improving effect of the therapeutic agent for hepatitis of the present invention is 6 times or more compared with the improving effect of the liver regeneration marker of the composition for tissue regeneration described in the prior art.
From the above, it was confirmed that the therapeutic agent for hepatitis containing adipose tissue-derived stromal cells of the present invention can remarkably improve the liver injury marker.
（安全性の確認）
本発明の肝炎治療剤が安全であることを確認するために、該治療剤を投与されたマウスの生存率を確認した。
極めて強い肝炎誘発マウスで安全性（生存率）を確認するために、マウスに投与するコンカナバリンAを0.6mgとした。詳細は、以下の通りである。
(Confirmation of safety)
In order to confirm that the therapeutic agent for hepatitis of the present invention is safe, the survival rate of mice administered with the therapeutic agent was confirmed.
In order to confirm safety (survival rate) in extremely strong hepatitis-induced mice, the concentration of concanavalin A administered to the mice was 0.6 mg. Details are as follows.
（マウスの準備）
C57Bl/6マウス（メス、12週齢）を使用し、コンカナバリンA（Concanavalin A）投与群、並びに、コンカナバリンA及び脂肪組織由来間質細胞群投与群を準備した。各群の条件は、以下の通りである。
＜コンカナバリンA投与群＞
0.6mg コンカナバリンAをすべてのマウス（４匹）の尾静脈投与した。そして、マウスの生存率を算出した。
＜コンカナバリンA及び脂肪組織由来間質細胞群投与群＞
0.6mg コンカナバリンAをすべてのマウス（３匹）の尾静脈投与し、さらに該投与直後に実施例１で調製した約100,000個の脂肪組織由来間質細胞を尾静脈投与した。そして、マウスの生存率を算出した。
(Mouse preparation)
C57Bl / 6 mice (female, 12 weeks old) were used to prepare a group administered with concanavalin A (Concanavalin A) and a group administered with concanavalin A and adipose tissue-derived stromal cells. The conditions for each group are as follows.
<Concanavalin A administration group>
0.6 mg concanavalin A was administered to all mice (4 animals) via the tail vein. Then, the survival rate of the mice was calculated.
<Concanavalin A and adipose tissue-derived stromal cell group administration group>
0.6 mg concanavalin A was administered to the tail vein of all mice (3 mice), and immediately after the administration, about 100,000 adipose tissue-derived stromal cells prepared in Example 1 were administered to the tail vein. Then, the survival rate of the mice was calculated.
（マウスの生存率の確認）
上記マウスの生存率を図５に示す。
図５に示すように、コンカナバリンA投与72時間経過後のコンカナバリンA及び脂肪組織由来間質細胞群投与群の生存率は100％であり、一方コンカナバリンA投与群の生存率は25％であった。
すなわち、本発明の肝炎治療剤は、先行技術に記載の組織再生用組成物とは異なり、ヘパリンを併用投与することなく、コンカナバリンA投与72時間経過後の生存率100％を達成することができた。
以上により、本発明の脂肪組織由来間質細胞を含む肝炎治療剤は、極めて強い肝炎（劇症肝不全類似）でも生存率100％を達成することができ、安全性が高いことを確認した。
(Confirmation of mouse survival rate)
The survival rate of the mice is shown in FIG.
As shown in FIG. 5, the survival rate of the concanavalin A and adipose tissue-derived stromal cell group administration group after 72 hours of concanavalin A administration was 100%, while the survival rate of the concanavalin A administration group was 25%. .
That is, unlike the composition for tissue regeneration described in the prior art, the therapeutic agent for hepatitis of the present invention can achieve a survival rate of 100% after 72 hours of concanavalin A administration, without concomitantly administering heparin. It was.
From the above, it was confirmed that the therapeutic agent for hepatitis containing adipose tissue-derived stromal cells of the present invention can achieve a survival rate of 100% even with extremely strong hepatitis (similar to fulminant liver failure) and is highly safe.
（総論）
実施例２、３及び４の結果より、本発明の肝炎治療剤は、安全性が高く、急激な肝炎に対する即効性の治療効果を有することを確認した。
(General)
From the results of Examples 2, 3 and 4, it was confirmed that the therapeutic agent for hepatitis of the present invention is highly safe and has an immediate therapeutic effect on acute hepatitis.
本発明では、新規な肝炎治療剤を提供できる。 In the present invention, a novel therapeutic agent for hepatitis can be provided.
Claims (2)
Including a group of adipose tissue-derived stromal cells including endothelial cells, blood cells, fibroblasts, perivascular cells and smooth muscle cells , and intravenously, intraarterial, intraportal injection of the cells into the treatment target, A therapeutic agent for acute hepatitis , characterized by suppressing inflammation by administration by intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection .
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