Kazutomo Kachi

Scanning electron microscopy of the hepatocyte cytoskeleton in human liver tissue

Portions of eight human livers taken by wedge biopsy or needle biopsy were extracted with 0.5% Triton X-100 and then studied by scanning electron microscopy and immunoelectron microscopy. The wedge-biopsy specimens were perfused with the detergent solution. Needle-biopsy specimens were quickly frozen and cracked and then the cracked tissue was immersed in the detergent solution. The three-dimensional filament network of hepatocytes was visualized. A dense network which consisted of intermediate filaments and microfilaments was observed within the cytoplasm of hepatocytes. These filaments were better preserved in the needle biopsies which were quick-frozen and cracked before extraction than in the wedge biopsies. Variation in the amount of the cytoskeletal filaments was less prominent in the hepatocytes treated by rapid freezing. It is concluded that freeze-cracking is the most favorable method for the study of cytoskeletal pathology in various liver diseases in man.

Mouse model of hepatocellular hyperplastic nodule formation characterization of mRNA expression

Abstract In previous studies a mouse model of hepatocellular tumorigenesis was developed. In this model of chronic griseofulvin feeding, preneoplastic foci developed over 5 months and numerous hyperplastic nodules with a few hepatocellular carcinomas developed after 10 months. In a subsequent study where 5- and 16-month livers were tested, the immediate early gene c-fos mRNA and transcription factor AP-1 were activated as well as was NFκB at both time intervals. However, the PPARα and RXRα genes were down-regulated. The evidence indicated that immediate early genes were involved in the promotion of tumor formation and that the direct hyperplasia pathway of regeneration was suppressed. To further characterize the involvement of the immediate early gene expression as well as other genes involved in the preneoplastic process we measured mRNA for c-jun, c-myc, hepatocyte growth factor activator (HGF-A), TGFβRII, γ-glutamyl transpeptidase (GGT), cytokeratin (CK8), ubiquitin (UB) and cellular transglutaminase (TG). The data indicated that c-jun an immediate early gene and c-myc, a delayed early gene were up-regulated at 5 and 16 months of feeding, both when preneoplastic foci appeared and when hyperplastic nodules developed. However, HGF-A was down-regulated at both time intervals. TGFβRII was up-regulated, as was GGT, CK8, TG and UB. GGT up-regulation was the only progression seen in gene expression at 16 months. It is concluded that a complex of cell proliferation and cell maintenance genes are involved in tumorigenesis in the mouse model of tumor promotion.

Immunoelectron microscopic study of intermediate filaments of hepatocytes in human liver using three kinds of cytokeratin antibodies.

ヒト正常肝生検組織の凍結切片をTriton X-100, nucleaseで処理し,3種の異なる分子量を持つCKに対する抗CK抗体,(1) MA-902; CK No. 8, (2) RPN. 1160; CK No. 18, (3) CAM5.2; CK No. 8, 18 & 19を用い免疫電顕を施行し,ヒト正常肝細胞におけるIFの異質性について,免疫組織化学的に検討した.この方法により,TEMで肝CSが明瞭に観察でき,細胞境界部および毛細胆管周囲に密な網目構造が存在した.細胞質内には直径約10nmの均一かつ平滑なIFが多数存在していた.免疫電顕の検討で,RPN. 1160では一部のIFにのみ,またMA-902やCAM5.2ではほぼ全てのIFに金粒子が標識され,MA-902とRPN. 1160との二重染色免疫電顕で2種の抗体の両方と反応するIFが観察された.以上の成績から,ヒト正常肝細胞にはCK No. 18を持たないIFも存在し,免疫学的に異なる少なくとも2種類のIFが存在することが示唆され,また単一のIFに2種のCKポリペプチドが存在することが証明された.

The effects of ethanol on the sinusoidal endothelial fenestration of rat liver.

走査電顕を用いて急性,慢性エタノール投与および断酒後のラット肝の類洞内皮細胞小孔(SEF)の変化を観察し,正常ラットのSEFと比較検討した.10cmH2Oの灌流圧で門脈より灌流し,SEFの直径,数,有孔度の定量的解析を行った.正常ラットの肝小葉内においてheterogeneityが存在しており,zone 1のSEFの直径はzone 3より有意(p<0.001)に大きく,その数はzone 3の方が有意(p<0.001)に多かった. SEFの有孔度はzone 1よりzone 3の方が有意(p<0.05)に大きかった.慢性エタノール投与によりzone 3でSEFの直径,有孔度は有意(p<0.001, p<0.01)に増大し,数は有意(p<0.001)に減少していた.断酒後,これらのSEFの変化は比較的短期間で正常ラットの状態に復した.今回の成績はアルコール性肝障害の初期病変であるアルコール性脂肪肝の成因にSEFの変化が関与する可能性を示唆するものである.

Transmission and scanning electron microscopy of the human liver cell cytoskeleton.

光顕的に正常肝組織像を呈するヒト肝組織片6個を対象とした.試料採取直後に細切し,凍結割断の後にdetergentで処理する方法と,採取した肝組織片に静注針を刺入しdetergentで潅流する2つの方法を用いた.得られた試料を透過電顕,走査電顕で観察し,肝細胞細胞骨格(H-CS)の二次元的,三次元的存在様式を明らかにした.細胞質全体に亘って中間径フィラメント(IFs)が多数存在し,IFsは接着・吻合を示し,微小管やマイクロフィラメントも観察できた.肝細胞と肝細胞の間で割断された試料では,毛細胆管周囲や細胞境界部が明瞭で,多くのCSが存在し,マイクロフィラメントも多数残存していた.今回の研究成績は,今後ヒトの肝細胞細胞骨格病理の研究に有用であると考えた.

Three dimensional organization of cytoskeleton of hepatocytes in mouse. Comparative study with rat cytoskeleton.

Triton X-100を含む灌流液にて,in situでマウス肝とラット肝を門脈より灌流し,透過電子顕微鏡と走査電子顕微鏡で肝細胞サイトスケレトンを観察した.マウス肝でもラット肝と同様,肝細胞サイトスケレトンを透過および走査電子顕微鏡で明瞭に観察しえた.走査電子顕微鏡による観察では,マウス,ラットともに,主として中間径フィラメントからなるサイトスケレトンの三次元的存在様式を明瞭に捉えることができた.走査電子顕微鏡により,中間径フィラメントの分枝,接触や核および細胞内小器官との接着様式を明瞭に表示しえた.マウスとラットの肝細胞サイトスケレトンには構造的差異はなかった.今回のマウス肝細胞サイトスケレトンの研究は,グリセオフルビン投与マウスでのマロリー体形成機序や,マロリー体とサイトスケレトンとの関係などを研究する上に重要な基礎的情報を与えてくれるものである.