Makoto Nishikawa

Does a porcine hepatocyte hybrid artificial liver prolong the survival time of anhepatic rabbits

To examine cultured porcine hepatocytes as a bioreactor of a hybrid artificial liver (HAL) in rabbits, a small version of a multiplated HAL was manufactured. In vitro and ex vivo studies were performed to evaluate the small HAL. The HAL consisted of 50 glass plates (10 x 5 x 0.04 cm each) on which porcine hepatocytes were cultured in a monolayer at confluent cell density. After 2 days of standard cultivation, the glass plates with hepatocytes were placed in the module and were used for studies. The module contains about 5 grams of hepatocytes (1/10 of 2 kg.bw rabbit liver). After undergoing perfusion culture for 5 days, the HAL showed satisfactory hepatic function. Glucose and urea were synthesized at the rate of 4.06 +/- 0.7 mg/module/hr and 0.62 +/- 0.09 mg/module/hr, respectively, and loaded ammonia was metabolized at the rate of 1.29 +/- 0.26 mg/module/hr. Ex vivo extracorporeal plasma perfusion studies revealed that the module with cultured porcine hepatocytes, which were heterogeneous to rabbits, showed a tendency to prolong the survival time of anhepatic rabbits. These results indicate that the HAL system, using multiplated porcine hepatocyte monolayers, is a promising artificial liver support system for clinical cases.

Injuriousness of glycochenodeoxycholate and taurochenodeoxycholate to cultured hepatocytes

Abstract Among bile acids whose levels increase greatly during hepatic failure, glycocholate (GC), taurocholate (TC), glycochenodeoxycholate (GCDC), and taurochenodeoxycholate (TCDC) are potentially hepatotoxic. Most bioartificial liver systems utilize isolated or cultured hepatocytes, and therefore the injuriousness of these bile acids might constitute a clinical drawback. To assess the possible hepatocellular injuriousness of these bile acids, changes in gluconeogenesis, ureagenesis, alanine aminotransferase (ALT) in medium, DNA contents, and morphological changes in hepatocytes were analyzed. The measurements were made 1, 3, and 5 days after cultured hepatocytes were exposed to the bile acids. The concentration of each bile acid was 2 mM, which is lower than their reported critical micelle concentrations (CMCs). TCDC and GCDC showed significantly high elevation of ALT, significantly low gluconeogenesis and ureagenesis activities, and low DNA contents. They also caused serious injury to hepatocytes on morphological study. TCDC showed heavier hepatocyte toxicity than did GCDC. Although the DNA contents in the GCDC group were relatively preserved throughout the experimental period, marked necrosis and deformity of hepatocytes were observed, whereas GC and TC were not seen to cause any injury to hepatocytes. The CMCs of TCDC and GCDC were reported to be 2.5 mM. However, our experiment showed that 2 mM of TCDC or GCDC has strong toxicity to hepatocytes. This raises the possibility that the CMCs of TCDC and GCDC could be 2 mM or even lower. In the clinical use of a bioartificial liver, removal of elevated TCDC and GCDC must be considered. Even if the levels of individual bile acids are lower than these CMCs, their cumulative hepatotoxic effect must be considered and requires further investigation.

Isolation and culture of human hepatocytes from resected liver tissue.

手術時切除肝を用いてヒト肝細胞の分離培養を行い,単離肝細胞の収量,viability,培養肝細胞機能が入手肝組織の温阻血時間といかなる関係にあるかを検討した.肝細胞の分散はコラゲナーゼとディスパーゼの混合液を灌流することにより行い,分散された肝細胞を10日間培養し,糖新生能,尿素合成能,DNA量を測定した.入手肝組織の温阻血時間は,0-300分であった.その結果,単離肝細胞のviabilityは肝温阻血時間と関係がなく87.9%であったが,収量との間には有意の相関関係を認めた(p<0.05).温阻血時間が120分以内における収量は180分以上と比較し有意に高かった(p<0.05).培養肝細胞機能は10日間維持され,機能は温阻血の影響を受けなかった.以上から,肝細胞の分離培養で肝組織の温阻血時間が長くなると単離肝細胞の収量は減少するが,120分までは,収量およびviabilityともに良好である.また,温阻血障害を受けた肝細胞を培養することによりその機能が完全に回復するものと考えられた.

New thrombolytic modality — regional administration of tissue plasminogen activator for hepatic artery and portal vein thromboses after liver transplantation: Case report

A 2 1/2-year-old boy with biliary atresia underwent orthotopic living-related liver transplantation. On the 7th postoperative day, he had an episode of hepatic arterial thrombosis following disseminated intravascular coagulation (DIC) due to severe intraabdominal sepsis. Tissue plasminogen activator was administered regionally and the hepatic arterial flow recovered promptly. On postoperative day 33, portal vein thrombosis occurred and direct tissue plasminogen activator injections into the portal vein improved portal blood flow. However, the patient eventually died of poorly controlled DIC. Throughout the course, color Doppler ultrasonogram and arterial ketone body ratio were good indicators of hepatic arterial and portal blood flow. When hepatic arterial thrombosis and portal vein thrombosis occur, retransplantation is often inevitable. Thus, while the patient is awaiting a suitable donor, it could be possible to maintain blood flow to the graft with this new thrombolytic therapy.

Nutritional assessment of the patients with hepatocellular carcinoma before operation.

肝癌患者の栄養状態を明らかにするために, 良性腫瘍患者46名と肝癌患者69名について身体計測, 生化学的検査を栄養指標とし, 栄養評価を行った.理想体重比, 上腕三頭筋部皮下脂肪厚比, 上腕筋囲比などの身体計測では, 良性群と硬変併存肝癌群の間に有意の差がみられ, 後者は栄養状態の低下を示していた.生化学的指標でも硬変併存肝癌群が良性群に比べ有意に低い値を示し, 前老は肝の合成障害と臓器蛋白量の低下を示した.α1-マイクログロブリンは硬変併存肝癌群が良性群に比べ有意に低い値を示し, rapid turnover proteinの1つとして, 栄養指標となりうると考えられた.