10.3969/j.issn.1007-3969.2013.04.005

Objective - （ ） To investigate the effects of nuclear factor erythroid 2 related factor 2 NRF2 on the oxidative stress （ ） Methods ） ，， induced by trichloromethane TCM in human normal hepatocyte L02 cells. i L02 cells were stimulated with 1 2 ， ， ， （ ）， 4 8 12 16 and 20 mmol/L TCM solution dissolved in dimethyl sulfoxide and the control group and blank group were set ， - ， up. After culturing for 24 hours the cell viability was detected by CCK 8 colorimetric method and the concentration of TCM ） -， - stimulation was screened. ii L02 cells in logarithmic growth phase were randomly divided into control group and low medium - ， ， ， and high dose groups. After 24 hours of exposure to 0 4 8 and 12 mmol/L TCM the cells were collected. The activity of （ ）， （ ）， （ - ） （ ） superoxide dismutase SOD catalase CAT glutathione peroxidase GSH Px and the level of malondialdehyde MDA NRF2， - （HO-1）， were detected by colorimetric analysis. The mRNA expression levels of heme oxygenase 1 glutamate cysteine （GCLC） （） （NQO1） - ligase catalytic subunit and NAD P H quinone dehydrogenase 1 were detected by real time fluorescence ， - ， polymerase chain reaction. The protein levels of NRF2 HO 1 GCLC and NQO1 were detected by Western blotting.Results ） ， ， ， ， i When the concentration of TCM was 4 8 12 16 and 20 mmol/L the survival rate of L02 cells decreased （ P ） ， ， significantly compared with the control group all <0.05 . The concentration of 0 4 8 and 12 mmol/L were selected as the ） ， - stimulation doses for subsequent experiments. ii Compared with the control group the activities of SOD and GSH Px in L02 （ P ） （ P ）， - cells in the three doses groups decreased all <0.05 and the levels of MAD increased all <0.05 with a dose effect - （P ）， relationship. The CAT activity of L02 cells in the medium dose group was lower than that in the control group <0.05 and the - （ P ） CAT activity of L02 cells in the high dose group was lower than that in the others three groups all <0.05 . Compared with the ， NRF2 - （P ），NRF2 control group the relative expression levels of mRNA in L02 cells in the low dose group decreased <0.05 - （P ）， NRF2 mRNA in L02 cells in the medium dose group increased <0.05 mRNA and NRF2 protein expression in L02 cells in （ P ） HO-1，GCLC， NQO1 ， the highdose group increased both <0.05 . The relative expression level of mRNA and GCLC NQO1 （ P ） protein expression in L02 cells in the three doses groups increased compared with the control group all <0.05 . The relative NRF2 - - - expression level of mRNA in L02 cells in the high dose group was higher than that in the low and medium dose groups （ P ）， - （P ）， both <0.05 and the relative expression of NRF2 protein was higher than that in the low dose group <0.05 but the HO-1 GCLC - - （ relative expression levels of and mRNA and HO 1 protein level were lower than those in the medium dose group all P ）Conclusion - <0.05 . TCM exposure can inhibit the proliferation of L02 cells by inducing oxidative stress with a dose effect ， relationship. In this process the antioxidant mechanism mediated by NRF2 was activated. The expression of antioxidant defense ， - ， and detoxification related target genes downstream of NRF2 signaling pathway was activated and the expression of HO 1 - GCLC and NQO1 was up regulated to alleviate the oxidative damage caused by TCM.

OBJECTIVETo obtain two-dimensional gel electrophoresis maps of the prefrontal cortex (PFC) proteins of normal and heroin-addicted rats for identifying the differentially expressed proteins in the addicted rats.

METHODSRat models of heroin addiction were established, and the proteins in the PFC underwent two-dimensional gel electrophoresis with immobiline pH gradient isoelectric focusing as the first and vertical SDS-PAGE as the second dimension. ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS as well as NCBInr database searching were performed to separate and identify the proteome in the rat PFC.

RESULTSSeven protein spots to represent 5 proteins were identified by PMF and MALDI-TOF-TOF-MS. Among those proteins, glucose-regulated protein (58 kD) and 26 S proteasome subunit p40.5 existed only in heroin-addicted rats, in which ATP synthase D chain was down-regulated. Ndufa10 and Eno1 were present in both groups, but their molecular mass and pI were different.

CONCLUSIONImmobilized pH gradient two-dimensional gel electrophoresis allows good reproducibility in separation and identification of the proteome in the PDF of heroin-addicted rats, which facilitates further investigation of pathogenic mechanisms of heroin addiction and central nerve injury.

Animals ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Heroin ; adverse effects ; Image Processing, Computer-Assisted ; Prefrontal Cortex ; drug effects ; metabolism ; Proteome ; metabolism ; Proteomics ; Rats ; Reproducibility of Results ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo investigate the protective effect of lycium barbarum polysaccharides (LBP) against heat stress-induced apoptosis of germ cells in rats and its action mechanism.

METHODSNinety male Sprague-Dawley rats were randomly divided into five groups of 18 each: control, heat stress (HS), high-dose LBP, median-dose LBP and low-dose LBP. The rats of the three LBP groups were given LBP by intragastric administration at 100 mg/(kg x d), 50 mg/(kg x d) and 10 mg/(kg x d) respectively for 14 days, and on the 15th day they, together with those of the HS group, were exposed to a heat of 43 degrees C for 30 minutes. At 24 h, 48 h and 7 d after heat stress, the animals were killed by cervical dislocation, followed by observation of the apoptotic germ cells by TUNEL, determination of the expression of Caspase-3 by immunohistochemistry and detection of cytochrome C in the cytosol by ELISA.

RESULTSCompared with the HS group, the three LBP groups showed statistically significant decreases in the apoptosis index (P<0.05), the expression level of Caspase-3 in germ cells (P<0.05) and the concentration of cytochrome C in the cytosol (P<0.05).

CONCLUSIONLBP can inhibit cytochrome C release from mitochondria, decrease the expression of Caspase-3 and hence reduce the apoptosis of germ cells. It thus can be deduced that LBP can protect germ cells against apoptosis via the mitochondrial pathway.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cytochromes c ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Germ Cells ; drug effects ; Male ; Mitochondria ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study apoptosis-related gene expression of human villous trophoblasts exposed to 50 Hz magnetic field and to investigate the possible mechanism of human reproductive health effects caused by 50 Hz magnetic field.

METHODSCultured human villous trophoblasts were exposed to 50 Hz magnetic field at 0.4 mT for 6, 48, 72 hours. Gene expressions of Bcl-2, Bax, Caspase-3, p53 and Fas were analyzed using real-time reverse transcription polymerase chain reaction (RT-PCR) assay.

RESULTSWithin 72 hours, the average fold change for each gene was near 1.00, and there was no significant difference on expression pattern in each gene between exposure and control groups (P > 0.05).

CONCLUSION0.4 mT 50 Hz magnetic field does not affect the apoptosis-related gene expression of human villous trophoblasts in vitro.

Caspase 3 ; metabolism ; Cells, Cultured ; Female ; Gene Expression Regulation ; Humans ; Magnetic Fields ; adverse effects ; Pregnancy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Trophoblasts ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the effects of specific RNA interference (RNAi) to Notch ligand Delta1 on proliferation and differentiation of human dental pulp stem cells (DPSC).

METHODSDPSC were infected by lentivirus vectors carrying Delta1-RNAi. DPSC were divided into three groups, DPSC/Delta1-RNAi group, DPSC/wt group and DPSC/vector group as control. Cell counting kit-8 (CCK-8) assay and flow cytometry were used to evaluate the proliferation of DPSC. Expression of proliferating cell nuclear antigen (PCNA) was examined with immunohistochemical staining. All groups were cultured in an odonto-inductive medium and were observed under microscope. The number of mineralization nodules was counted after Alizarin red staining. Alkaline phosphatase (ALP) activity and the expression of dentin sialophosphoprotein (DSPP) were detected by ALP activity assay and Western blotting.

RESULTSCompared with DPSC/wt or DPSC/vector separately, proliferating rate and S-cycle of DPSC/Delta1-RNAi was significantly lower. The S phase and proliferation index (PI) decreased markedly from 22.32 ± 2.35 and 33.68 ± 4.19 (DPSC/Delta1-RNAi) to 5.44 ± 0.91 and 16.00 ± 6.07 (DPSC/wt). The PCNA staining of DPSC/Delta1-RNAi was evidently weaker. DPSC/Delta1-RNAi group had more calcified cell nodules than the other two control groups, and ALP activity and DSPP expression of DPSC/Delta1-RNAi group increased markedly.

CONCLUSIONSDelta1-RNAi induced by the lentivirus vectors may inhibit DPSC proliferation and differentiation. Notch-Delta signal pathway plays an important role in self-renewal and differentiation.

Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dental Pulp ; cytology ; metabolism ; Epithelial Cells ; Extracellular Matrix Proteins ; biosynthesis ; Genetic Vectors ; Homeodomain Proteins ; Humans ; Lentivirus ; Phosphoproteins ; biosynthesis ; RNA Interference ; Sialoglycoproteins ; biosynthesis ; Signal Transduction ; Stem Cells ; metabolism

BACKGROUND: Embryonic stem cells are pluripotent stem cells that can differentiate into all cell types and propagate themselves indefinitely in vitro, which have been used widely in biological research. OBJECTIVE: To summarize the culture methods of embryonic stem cells in vitro, as well as the significance, methods, clinical applications of embryonic stem cells as genetic disease models. METHODS: Relevant articles were searched in PubMed using the key words of "embryonic stem cell, genetic diseases" which appeared in the titles and abstracts. The latest articles were preferred. Totally 43 eligible articles were obtained for result analysis. RESULTS AND CONCLUSION: In the genetics study, embryonic stem cells have been widely used to construct monogenic and chromosome disease models, which can make up the limitations of animal and cell models. Models constructed by embryonic stem cells are conducive to the investigations on pathological processes and molecular mechanisms of genetic diseases. But there are also some ethic disputes on the use of embryonic stem cells, which need further studies.

Objective To confirm the effect of cigarette smoking on β-cell function, and further investigate the mediation effect of abdominal obesity. Methods Participants would include 1440 Chinese smokers who had participated in a community-based chronic disease screening project in Guangzhou and Zhuhai from 2006 to 2007. They were interviewed with structured questionnaire on their socio-demographic status and smoking behaviors. Waist-to-hip ratio (WHR) and fasting serum C-peptide concentration were also measured. Results After adjustment for the potential confounding factors, when compared with smokers with consumption 1-10 cigarettes/day, smokers with consumption of 11-20 cigarettes/day (adjusted OR=1.53, 95%CI: 1.22-1.90) or ＞20 cigarettes/day (adjusted OR=1.92, 95%CI: 1.32-2.79) had significant higher risks to get C-peptide concentration larger than its median. Furthermore, 37.54% of the effect of cigarette smoking on C-peptide concentration was partially mediated by abdominal obesity. Conclusion Cigarette smoking might be a risk factor for β-cell dysfunction and abdominal obesity.

BACKGROUNDAnkylosing spondylitis (AS) is a common inflammatory rheumatic disease which lacks satisfactory treatment so far. Sinomenine (SIN) is an alkaloid and has recently been utilized in treating multiple rheumatic diseases including AS in China, but its exact mechanism remains to be explored. This study investigated the alteration of proteome in peripheral blood mononuclear cells (PBMCs) from AS patients.

METHODSThirty AS patients were enrolled in this study. PBMCs from each AS patient were cultured in medium with or without SIN respectively. Then PBMCs proteins from both groups were separated by two-dimensional electrophoresis (2-DE) and analyzed by mass spectrometry (MS). Two differentially expressed proteins were then chosen to be verified using Western blotting.

RESULTSSeven proteins, including a-synuclein (SNCA), calmodulin (CALM), acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A), chloride intracellular channel protein 1 (CLIC1), guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 (GNB1), gelsolin (GSN) and histone H2B type 1-M (HISTH2BM) were over-expressed, while coronin- 1A (CORO1A) was under-expressed in the SIN-treated PBMCs. Further bioinformatics search indicated that the changes of SNCA, ANP32A and CLIC1 pertained to apoptosis, while changes of GSN and CORO1A were associated with both apoptosis and inhibition of immunological function. Subsequently GSN and CORO1A were selected to validate by Western blotting and the results were consistent with those of 2-DE.

CONCLUSIONThere were 8 differentially expressed proteins in the SIN-treated PBMCs, which might shed some light on the mechanism of SIN in the treatment of AS.

Adolescent ; Adult ; Blood Proteins ; analysis ; Blotting, Western ; Cells, Cultured ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Leukocytes, Mononuclear ; chemistry ; Male ; Middle Aged ; Morphinans ; pharmacology ; Proteomics ; methods ; Reproducibility of Results ; Spondylitis, Ankylosing ; blood

Intestinal tract, which produces more than fifty kinds of gut peptides, is regarded as the largest endocrine organ. With regard to the gut peptides, a number of studies were focused on their structure, function and the roles in some diseases. The changes in output or distribution of gut peptides in the intestinal tract during development have been largely unknown. This study was aimed to investigate the changes of somatostatin (SST) and somatostatin receptor 2 (SSTR2) in small intestinal and hepatic tissues during the development of macaque. The tissue samples of small intestine, liver or blood samples from peripheral and portal vein of 4 macaques in 6-month fetus, 2-day neonate, 45-day neonate and adult were obtained after anesthetization. The concentrations of SST in blood or tissues of macaques were measured by radioimmunoassay. The distributions of SST in small intestinal or hepatic tissues were visualized by immunohistochemical staining. The expression of SSTR2 was detected by in situ hybridization. SST concentration of intestinal tissue in 6-month-old macaque was (27.3+/-16.6) ng /mg protein and light positive staining of SST was localized in mucosal crypts but negative in muscle layer. The intestinal concentration of SST increased gradually with macaque development and reached to the peak [(120.1+/-35.3) ng /mg protein] in adult. It was significantly higher than that in fetus (P<0.01). Strong positive staining of SST was found in both mucosal crypts and myenteric nerve plexus of adult animal. SSTR2 was obviously expressed in intestinal epithelium of fetus but its expression was greatly reduced in epithelium and was shifted to mucosal crypts when grown to adult. Negative staining of SSTR2 in muscle layer of fetal or neonatal macaque turned to be positive in myenteric nerve plexus of adult. The levels of SST or SSTR2 in liver decreased gradually during development. SST concentrations of small intestinal tissue kept significantly higher than those of hepatic tissues in the macaque developing stages. SST levels of portal vein were also maintained significantly higher than those of peripheral blood in the macaque developing stages. In conclusion, the level of SST and expression of SSTR2 in mucosal crypt increased gradually with macaque development. SST from intestinal tract was quickly degraded in portal vein before entering into liver. SST positive myenteric nerve plexus was visualized only in mature macaque.
Animals ; Animals, Newborn ; Fetus ; Intestine, Small ; metabolism ; Liver ; metabolism ; Macaca mulatta ; growth & development ; metabolism ; Male ; Receptors, Somatostatin ; metabolism ; Somatostatin ; metabolism