Synchronization isolation method for multiple types of cells from mouse liver / 中华肝脏病杂志

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Identification and characterization of microRNAs in normal equine tissues by Next Generation Sequencing.

The role of microRNAs (miRNAs) as a post-transcriptional gene regulator has been elucidated in a broad range of organisms including domestic animals. Characterization of miRNAs in normal tissues is an important step to investigate the functions of miRNAs in various physiological and pathological conditions. Using Illumina Next Generation Sequencing (NGS) technology, we identified a total of 292 known and 329 novel miRNAs in normal horse tissues including skeletal muscle, colon and liver. Distinct sets of miRNAs were differentially expressed in a tissue-specific manner. The miRNA genes were distributed across all the chromosomes except chromosomes 29 and 31 in the horse reference genome. In some chromosomes, multiple miRNAs were clustered and considered to be polycistronic transcript. A base composition analysis showed that equine miRNAs had a higher frequency of A+U than G+C. Furthermore, U tended to be more frequent at the 5' end of miRNA sequences. This is the first experimental study that identifies and characterizes the global miRNA expression profile in normal horse tissues. The present study enriches the horse miRNA database and provides useful information for further research dissecting biological functions of miRNAs in horse.

Hydrogen peroxide promotes epithelial to mesenchymal transition and stemness in human malignant mesothelioma cells.

Reactive oxygen species (ROS) are known to promote mesothelial carcinogenesis that is closely associated with asbestos fibers and inflammation. Epithelial to mesenchymal cell transition (EMT) is an important process involved in the progression of tumors, providing cancer cells with aggressiveness. The present study was performed to determine if EMT is induced by H2O2 in human malignant mesothelioma (HMM) cells. Cultured HMM cells were treated with H2O2, followed by measuring expression levels of EMT-related genes and proteins. Immunohistochemically, TWIST1 expression was confined to sarcomatous cells in HMM tissues, but not in epithelioid cells. Treatment of HMM cells with H2O2 promoted EMT, as indicated by increased expression levels of vimentin, SLUG and TWIST1, and decreased E-cadherin expression. Expression of stemness genes such as OCT4, SOX2 and NANOG was also significantly increased by treatment of HMM cells with H2O2. Alteration of these genes was mediated via activation of hypoxia inducible factor 1 alpha (HIF-1α) and transforming growth factor beta 1 (TGF-ß1). Considering that treatment with H2O2 results in excess ROS, the present study suggests that oxidative stress may play a critical role in HMM carcinogenesis by promoting EMT processes and enhancing the expression of stemness genes.

Regulation of NMDA receptor subunits after acute ethanol treatment in rat brain.

AIMS: Tolerance to ethanol-induced inhibition of N-methyl-D-aspartate receptors (NMDARs) is thought to underlie the acute adaptive mechanisms against ethanol. To explore these compensatory upregulating mechanisms of NMDARs, we investigated the expression and phosphorylation of NMDAR subunits in vivo following an acute ethanol treatment. METHODS: Male Sprague-Dawley rats were given 4 g/kg ethanol, and the phospho-S896-NR1, NR2A and NR2B subunits of NMDAR were immunoblotted from the cerebral cortex and hippocampus. We also examined the mRNAs and ubiquitinated forms of the NR2A and NR2B subunits. RESULTS: Acute ethanol treatment increased phospho-S896-NR1 at 30 min in the cerebral cortex and hippocampus, and the increase was maintained until 2 h in the hippocampus. Ethanol increased total NR2A and NR2B expression at 30 min in the cortex and hippocampus, and the NR2A increase was maintained until 2 h in the hippocampus. The increased expression of the NR2A and NR2B subunits was not associated with statistically significant alterations in mRNA expression or protein ubiquitination. CONCLUSION: Acute ethanol treatment increased NR1 subunit phosphorylation and NR2A and NR2B subunit expression in the cerebral cortex and hippocampus of rats. These effects of ethanol on the NMDAR subunits may underlie the mechanisms that compensate for ethanol-induced inhibition of NMDARs. However, the regulation of NR2A and NR2B in this paradigm is not dependent on transcriptional changes.

Effects of clozapine on behavioral sensitization induced by cocaine.

Using cocaine-sensitized mice as a model for psychosis, this study investigated whether subchronic treatment with clozapine could affect the sensitized state of the animals and examined the accompanying molecular changes in the brain. To induce sensitization, ICR mice (n=44) were treated with cocaine for 5 days. After 7 days of withdrawal, sensitization was confirmed by a cocaine challenge. Then, the sensitized animals were treated with clozapine for 5 days and rechallenged with cocaine. The frontal cortices were removed from the mice (n=16) 24 h after the last challenge, and the phosphorylation status of some key signaling molecules was investigated. Compared with the sensitized mice receiving the vehicle treatment, the sensitized mice receiving subchronic clozapine showed less locomotor activity, with an activity level similar to that of non-sensitized mice. However, clozapine did not directly affect the stimulatory effect of cocaine. Clozapine also reversed some of the sensitization-induced biochemical changes, including increased phosphorylation of GSK-3beta and CREB, in the frontal cortex. Subchronic treatment with clozapine apparently de-sensitized the sensitized mice. The long-term effect of clozapine on stimulant-induced sensitization may be related to the therapeutic effect of the drug as an antipsychotic agent.

Effects of electroconvulsive shock on the phosphorylation of DARPP-32 in rat striatum.

Dopamine- and cAMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP-32) is a key integrative molecule in the dopaminergic and glutamatergic signaling pathways in the striatum. Electroconvulsive shock (ECS), which induces massive neuronal depolarization, can activate various signaling pathways. In this study we investigated whether ECS could affect the phosphorylation status of DARPP-32. Male Sprague-Dawley rats underwent ECS and were sacrificed by decapitation at 0, 2, 10, 60, and 180 min after treatment. The phosphorylations of Thr34 and Thr75 residues of DARPP-32 and Ser159 residue of cyclin-dependent kinase 5 (CDK5) were investigated in the striatum. The activity of protein phosphatase 1 (PP1) and the binding between DARPP-32 and PP1 were also analyzed. Thr34 phosphorylation of DARPP-32 increased immediately after ECS and this state was maintained for more than 60 min. The activity of PP1 decreased and the binding between PP1 and DARPP-32 increased in accordance with this phosphorylation pattern. However, the phosphorylation at Thr75 showed no significant change except for an initial transient decrease. The phosphorylation of CDK5, which is responsible for Thr75 phosphorylation of DARPP-32, did not exhibit significant fluctuations. Our findings indicate that ECS increases Thr34 phosphorylation of DARPP-32, and thus inhibits the activity of PP1.

Up-regulation of cocaine- and amphetamine-regulated transcript (CART) in the rat nucleus accumbens after repeated electroconvulsive shock.

Cocaine- and amphetamine-regulated transcript (CART) peptide regulates appetite, reward, and mood. CART expression is regulated via the protein kinase A (PKA) pathway, and electroconvulsive shock (ECS), an efficient antipsychotic and antidepressant measure, activates PKA-related signaling. Thus, we hypothesized that ECS may regulate the expression of CART. ECS given daily for five consecutive days increased CART mRNA and protein in the rat nucleus accumbens (NAc), accompanied by an increase in CREB phosphorylation. Our results suggest that ECS-induced CART up-regulation might be associated with PKA-CREB signaling, but the causal direction remains to be elucidated in future studies.