背景/目的：これまで、急性肝不全の病態について遺伝子レベルからシグナルトランスダクションに至るまで様々な研究がなされてきたが、単一の遺伝子やシグナルの制御だけでは複雑な生体反応の制御は困難であることが明らかとなり、どれも臨床応用として成功はしていない。一方、近年、組織/器官障害に対する新しい治療法として幹細胞の応用が期待されている。本研究課題では、ラット急性肝不全モデルを用いて、高い幹細胞活性と多様な生理活性（抗炎症作用、組織保護作用、過剰な免疫抑制作用）等を有する間葉系幹細胞／間質細胞株（ASCL: Adipose-derived Mesenchymal Stem/stromal Cell Line, Tozawa K, Matsubara Y. Blood. 2019 Feb 14;133(7):633-643.）の有用性を検証する。
結果：Wistarラットから皮下脂肪組織を採取し、IV型コラゲナーゼを用いて組織から間質血管細胞群(Stromal Vascular Fraction, SVF)の分離を行った。分化培地を用いて継代培養しSVFから血管周辺細胞、白血球や赤血球の除去を行いASC(Adipose stem/stromal cell)の樹立を行った。このラット由来ASCにインスリン・インドメタシン・デキサメタゾンを添加して脂肪細胞への分化誘導を行った。分化誘導後、脂肪細胞を回収し、再びASC培地にて培養を行い幹細胞分化誘導用培地によりASCLの樹立に成功した。最終的にASCL(Adipose-derived Mesenchymal Stem/stromal Cell Line)から巨核球を経てASCL-PLC(人工血小板)を得る予定である。
薬剤誘導性の急性肝不全(acute liver failure, ALF)モデルの作製では、D-ガラクトサミン(D-Galactosamine, D-GalN)の最適な投与濃度を検証するため1.2g/kg, 2.0g/kg, 2.5g/kg濃度でWistarラットを用いて、腹腔内投与を行い生存率の検証を行った。結果として、2.0g/kgの投与における48時間の生存率が60%であり、この濃度をALFモデルのための投与量とした。D-GalN腹腔内投与から48時間後の生化学検査および血中サイトカインの測定の結果、ASTでは、コントロール群が88.25±5.44ng/mL (means ± SD)であるのに対しD-GalN投与群では9820.00±3553.76 (means ± SD)であり111倍上昇であった。ALTでは、コントロール群が45.25±8.61ng/mL (means ± SD)であるのに対しD-GalN投与群では9916.00±3529.25ng/mL (means ± SD)であり219倍上昇であった。IL-6では、コントロール群が10.78±6.10ng/mL (means ± SD)であるのに対しD-GalN投与群では57.14±25.92ng/mL (means ± SD)であり5.3倍上昇であった。また、病理所見において、D-GalN投与後48時間では、肝細胞は浮腫し、炎症細胞浸潤もみとめられた。
今後、ASLCから人工血小板（ASCL-PLC）を誘導し、ラットALFモデルを用いて有効性の評価を行う。
Background and Aim: Variety of reports have demonstrated the pathology of acute liver failure (ALF) in molecular level range from gene expression to signal pathway. However, ALF, known for its complex pathological mechanism, is too difficult to show positive evidence in molecular targeted treatment when it comes to clinical application. On the other hand, stem cell therapy is considered to be a new treatment in degenerative diseases or organic disorder. In our study, we established rat ALF model to assess therapeutic potential of a specific mesenchymal stem cell line named ASCL（Adipose-derived Mesenchymal Stem/stromal Cell Line, Tozawa K, Matsubara Y. Blood. 2019 Feb 14;133(7):633-643. ）, which is expected to alleviate ALF by the function of anti-inflammation, cytoprotection and anti-oxidative stress participation.
Results: Subcutaneous fat tissues harvested from Wistar rat were dissolved in type IV collagenase to obtain stromal vascular fraction (SVF). During incubation of SVF culture, non-adherent cells such as blood cells and vascular pericytes were removed by several passages so as to purified SVF into adipose stem cell (ASC).
ASCL was transformed from ASC by further process of differentiation. In brief, purified ASC was treated with insulin, indomethacin and dexamethasone to induce adipocyte differentiation. Those differentiated adipocytes were again de-differentiated into a one type stem cell line using mesenchymal stem cell specific differentiation medium so as to establish ASCL. ASCL is expected to secrete platelet-like cells (ACSL-PLC) by further differentiation to megakaryocytes.
Rat ALF model is induced by D-galactosamine (D-GalN). To determine the optimize D-GalN induced ALF model, D-GalN was injected into rats respectively at doses of 1.2g/kg, 2.0g/kg and 2.5g/kg intraperitoneally. As a result, we observed survival rate in each group and analyzed hepatic biomarker and IL-6. In model of 2.0g/kg D-GalN, the survival rate of 48 hours after toxic inducement turned out to be 60%. Aspartate aminotransferase (AST) was up to 9820.00±3553.76 (means ± SD) in inducement group, which increased 111 folds compared with normal rats (88.25±5.44ng/mL). Alanine aminotransferase (ALT) was up to 9916.00±3529.25ng/mL (means ± SD) in inducement group, which increased 219 folds compared with normal rats (45.25±8.61ng/mL). Interluekin-6 (IL-6) was up to 57.14±25.92ng/mL (means ± SD) in inducement group, which increased 5.3 folds compared with normal rats (10.78±6.10ng/mL). In addition, hepatic tissues of ALF model obviously changed in morphological level. Compared with normal liver by H.E. staining, toxic induced hepatocytes were observed to be suffered from edema and inflammation cells infiltration at the point of 48 hours after inducement.
In next step, we are going to transform ASCL into ASCL-PLC for assessing therapeutic potential of both ASCL and ASCL-PLC in the rat ALF model we have established.