氨基葡萄糖、N-乙酰氨基葡萄糖和小分子壳聚糖对C_2H_5OH和CCl_4所致肝细胞损伤的预防和修复作用

研究了氨基葡萄糖（D-glucosamine,Glc-NH2）,N-乙酰氨基葡萄糖（N-acetyl-D-glucosamine,GlcNAc）和小分子壳聚糖（chitosan,CS）对酒精（C2H5OH）或四氯化碳（CCl4）诱导的小鼠肝细胞损伤的预防和修复作用。采用组织块培养法获取小鼠肝细胞,通过对小鼠传代培养过程中肝细胞活性的测定,证实该细胞在第6d达到最佳生长状态。分别用无糖培养基,含500或1000μg/mL GlcNH2、GlcNAc或小分子CS的培养基对肝细胞培养3d后用C2H5OH或CCl4诱导损害不同的时间,继续培养6d。结果发现GlcNH2、GlcNAc和小分子CS都能预防CCl4诱导的肝细胞损伤;而只有小分子CS对C2H5OH诱导的肝细胞损伤具有较好的预防效果。2000μg/mL的Glc-NH2、GlcNAc和小分子量CS对C2H5OH诱导损伤小鼠肝细胞的修复正相性都很明显;而对于CCl4诱导的肝细胞损伤,只有GlcNH2对CCl4诱导损伤30min的肝细胞具有修复作用。     

聚砜膜生物反应器实验系统对重肝患者血浆的影响

目的：了解聚砜膜生物反应器实验系统对重肝患者血浆的影响,探索聚砜膜中空纤维反应器作为生物人工肝反应器的可行性。方法：用磁力搅动法将分离的新生实验小型猪肝细胞进行球形体培养,接种到聚砜膜中空纤维反应器外腔,观察聚砜膜生物反应器实验系统对重肝患者血浆的血氨、胆红素、凝血酶原时间、TNF-α、TGF-β1的影响。结果：该实验系统能降低重肝患者血浆的血氨、胆红素,缩短凝血酶原时间,降低TNF-α、TGF-β1水平。结论：聚砜膜生物反应器实验系统能够清除重肝患者血浆中的小分子有毒物质,补充有益成分,降低细胞因子水平。     

Methionine restriction up‐regulates the expression of the pi class of glutathione S‐transferase partially via the extracellular signal‐regulated kinase‐activator protein‐1 signaling pathway initiated by glutathione depletion

Understanding the molecular events underlying gene regulation by amino acids has attracted increasing attention. Here, we explored whether the mechanism by which methionine restriction affects the expression of the π class of glutathione S‐transferase (GSTP) is related to oxidative stress initiated by glutathione (GSH) depletion. Rat primary hepatocytes were cultured in an L‐15‐based medium in the absence or presence of 200 μM L ‐buthionine sulfoximine (BSO) or in a methionine‐restricted L‐15 medium supplemented with 20 μM L ‐methionine up to 72 h. BSO and methionine restriction time‐dependently induced GSTP mRNA and protein expression in a similar pattern accompanied by a decrease in the cellular GSH level. The phosphorylation of extracellular signal‐regulated kinase (ERK), but not of c‐Jun NH₂‐terminal kinase and p38, was stimulated by methionine restriction and BSO. Electromobility gel shift assay showed that the DNA‐binding activity of nuclear activator protein‐1 (AP‐1) increased in cells exposed to methionine restriction or BSO. With the ERK inhibitor FR180204, AP‐1 activation and GSTP expression were abolished. Moreover, the induction of GSTP by methionine restriction and BSO was reversed by GSH monoethyl ester and N‐acetylcysteine. Our results suggest that methionine restriction up‐regulates GSTP gene expression, which appears to be initiated by the ERK‐AP‐1 signaling pathway through GSH depletion in rat hepatocytes.     

Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface

A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Modeling Human Liver Biology Using Stem Cell-Derived Hepatocytes

Stem cell-derived hepatocytes represent promising models to study human liver biology and disease. This concise review discusses the recent progresses in the field, with a focus on human liver disease, drug metabolism and virus infection.     

The Effect of Physical and Chemical Cues on Hepatocellular Function and Morphology

Physical topographical features and/or chemical stimuli to the extracellular matrix (ECM) provide essential cues that manipulate cell functions. From the physical point of view, contoured nanostructures are very important for cell behavior in general, and for cellular functions. From the chemical point of view, ECM proteins containing an RGD sequence are known to alter cell functions. In this study, the influence of integrated physical and chemical cues on a liver cell line (HepG2) was investigated. To mimic the physical cues provided by the ECM, amorphous TiO₂ nanogratings with specific dimensional and geometrical characteristics (nanogratings 90 nm wide and 150 nm apart) were fabricated. To mimic the chemical cues provided by the ECM, the TiO₂ inorganic film was modified by immobilization of the RGD motif. The hepatic cell line morphological and functional changes induced by simultaneously combining these diversified cues were investigated, including cellular alignment and the expression of different functional proteins. The combination of nanopatterns and surface modification with RGD induced cellular alignment and expression of functional proteins, indicating that physical and chemical cues are important factors for optimizing hepatocyte function.     

便携式肝细胞功能测试仪的设计

设计一种便携式测试仪,在肝细胞培养的过程中对其功能进行快速的现场检测,并可配合上位机将数据处理、分析、统计等功能整合完成.选用光学比色法,将单色光源、集成光电传感器做为测量元件构建单片机控制系统,使用溴甲酚绿法和脲酶法,分别测量培养液中白蛋白和尿素的含量.实验中能够快速、准确的完成肝细胞功能的现场测试,白蛋白浓度测量范围10-100 g/L±5%,尿素浓度测量范围1-15 mmol/L±10%,拟和度均达99%以上.该仪器可作为判断生物人工肝治疗效果的重要依据,已在治疗现场得到了成功应用.     

Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes

Mycelia of Antrodia cinnamomea (AC), an edible fungus native to Taiwan, were produced by submerged fermentation with various fermentation times in 250 mL, 5 and 500 L fermentors and were evaluated for the effect of fermentation products on the viabilities of Hep3B and HepG2 hepatoma cells and normal primary rat hepatocytes. The results showed that the ethanolic extracts of AC mycelia (from 250 mL fermentation for 8 wk and 5 and 500 L fermentations for 4 wk) possessed high antihepatoma activity. The IC(50) of ethanolic extract of AC mycelia fermented for 8 wk in a 250 mL fermentor against Hep3B and HepG2 cells were 82.9 and 54.2 microg/mL, respectively. Furthermore, the IC(50) for Hep3B and HepG2, treated with ethanolic extract of AC mycelia fermented for 4 wk in the 5 L fermentor were 48.7 and 3.8 microg/mL, respectively. Those treated with ethanolic extract of AC mycelia fermented for 4 wk in the 500 L fermentor were 36.9 and 3.1 microg/mL, respectively. No adverse effects of all samples on normal primary rat hepatocytes were observed.