Selection of scFvs specific for the HepG2 cell line using ribosome display.

The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.

[Influence of cadmium on structure and functions of mitochondria in hepatic cells].

OBJECTIVE: To study the effects of cadmium on the structure and functions of mitochondria in hepatocytes. METHODS: Mitochondria were isolated from cultured human hepatocytes of the line WRL-68 and co-cultured with cadmium chloride (CdCl2) of the concentration of 1, 5, and 10 micromol/L, and WRL-68 cells not treated with CdCl2 (0 micromol/L) was used as control group. Cyclosporin A (CsA) was added into the culture medium. Mitochondrial permeability transition pore (MPTP) opening degree was tested by spectrophotometer. Morphologic changes of mitochondria were observed under transmission electron microscope. The activities of Na+ -K+ -ATPase, Ca2+ -Mg2+ -ATPase, LDH, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the contents of malondialdehyde (MDA) were measured Mitochondria membrane potential (MMP) was monitored by spectrofluorimeter with fluorescence dye Rh-123. RESULTS: CdCl2 reduced the absorbance of mitochondria, signifying the opening of MPTP, concentration-dependently. The absorbance of mitochondria co-cultured with CsA and CdCl2 10 micromol/L was higher than that of the. CdCl2, 10 micromol/L group. Mild swelling was seen in the mitochondria treated with CdCl2. The MMP values of the CdCl2 5 and 10 micromol/L groups were significantly lower than that of the control group (P < 0.05, P < 0.01). The activity levels of ATPase, LDH, SOD, and GSH-Px in mitochondria decreased in the CdCl2 groups (all P < 0.05), and the contents of MDA increased in the CdCl2 groups compared with the control group. CONCLUSION: CdCl2 causes destruction of mitochondria structure, opening of MPTP, decrease of MMP, and changes of vitality of mitochondria enzymes that all play important roles in apoptosis of hepatocytes.

Cadmium-induced apoptosis in human normal liver L-02 cells by acting on mitochondria and regulating Ca(2+) signals.

Cadmium is a well-known toxic compound for the liver. It has been demonstrated to induce hepatotoxicity partly via apoptosis, but no uniform mechanism of apoptosis has so far been proposed. This study was first to determine whether cadmium-induced apoptosis in L-02 cells, second to observe the mechanism of cadmium-induced apoptosis. Studies of morphology, DNA fragmentation and apoptotic rate demonstrated that 60µM cadmium induced apoptosis with strong effects on cell viability. A concomitant time-dependent decrease of Bcl-2 and mitochondrial transmembrane potential (ΔΨ(m)) was observed. Subsequently, increase of caspase-3 activity and release of mitochondrial AIF were detected. However, cell pretreatment with a broad-specificity caspase inhibitor (Z-Asp) did not abolish apoptosis. These data demonstrated that the apoptotic events involved a mitochondria-mediated apoptotic pathway but not necessarily caspase-dependent signaling. On the other hand, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of cadmium-exposed cells had significant increases and the Bapta-AM, a well-known calcium chelator, pretreatment partially blocked cadmium-induced apoptosis, indicating that the elevation of [Ca(2+)](i) may play an important role in the apoptosis. Together, these results support the notion that cadmium-induced hepatotoxicity is comparable to effects in L-02 by inducing apoptotic pathways on the basis of acting on mitochondria and regulating Ca(2+) signals.