Objective To investigate the effect of human hepatocellular carcinoma HepG2 cell-derived Exosome on the differentiation of mesenchymal stem cells(MSC)into cancer-associated myofibroblasts(CAF)and the impacts of CAF on liver cancer cell proliferation,migration,and invasion. Methods The protein expression of HepG2 cell-derived Exosome was detected by Western blotting. MSCs were separated from human adipose tissue and cultured with HepG2 cell-derived Exosome(100 ng/nl)to initiate differentiation. The expressions of mesenchymal markers and several interleukins were also detected by Western blotting. HepG2 cells were co-cultured with the conditioned media(CM),in which HepG2 Exosome induced the differentiation of MSC into CAF. The expressions of epithelial and mesenchymal markers were detected by real-time polymerase chain reaction(PCR)and Western blotting. Cell proliferation was assessed using MTS assay. Transwell chambers were used in the in vitro migration and invasion assay. Results HepG2 cell-derived particles expressed CD63,70 kilodalton heat shock proteins,and 90 kilodalton heat shock proteins. With the treatment of HepG2 cell-derived Exosome,the expressions of mesenchymal marker α-smooth muscle actin,fibroblast activation protein α,interleukin(IL)-6,IL-8,and IL-1β were up-regulated,while vascular endothelial growth factor had no significant change. The conditioned media which HepG2 Exosome induced MSC differentiation CAF(CAF-CM)could significantly promote HepG2 cells proliferation(1.075±0.104),compared to BSA control(0.874±0.066,P=0.023)and MSC-CM(0.649±0.034,P=0.0005). CAF-CM could significantly enhance cell migration [(42.5±9.1) cells vs.(18.5±3.1) cells,P=0.001] and invasion [(29.0±3.5) cells vs.(13.1±3.7) cells,P=0.009] compared to its control group. Moreover the conditioned medium which HepG2 Exosome induced MSC to differentiate into CAF could also promote the expressions of mesenchyme-related genes Smad interacting protein 1(P=0.040),β-catenin(P=0.038),fibronectin(P=0.029),and Vimentin(P=0.013)and inhibit the expression of epithelial related genes zonula ocdudens-1(P=0.010).Conclusions Exosome extracted from HepG2 cells can induce human adipose-derived MSC to differentiate into cancer-associated myofibroblasts. CAF-like cells can promote the migration of the liver cancer cell line HepG2.

Objective To investigate early renal damage of chronic hepatitis B (CHB) patients and the risk factors related to their renal function. Methods CHB patients who visited the second people’s hospital but did not receive systemic treatment were enrolled in our study. Those who visited for general check-up with no hepatic findings during the same period were selected as control group. Glomerular filtration rate (GFR) of all the participants were estimated by simplified MDRD equation and CKD-EPI equation (designated as M-eGFR and C-eGFR respectively). Influence factors of eGFR were statistically analyzed. Results In the total 528 cases in CHB group, 88 (16.67%) and 62 (11.74%) suffered declined M-eGFR and C-eGFR respectively. By contrast, 10 (8.77%) and 6 (5.26%) cases in the total 114 cases in control group present declined M-eGFR and C-eGFR ac?cordingly. Percentages of renal function impairment, estimated by both M-eGFR and C-eGFR, were higher in the CHB group than those in control group. The difference was statistically significant (χ2=4.518, P<0.05;χ2=4.156, P<0.05). Multiple linear regression analysis indicated that age, HBsAg and body mass index (BMI) were risk factors of M-eGFR while age, HBsAg, gender and serum albumin were risk factors of C-eGFR. On the other hand, HBV-DNA and HBeAg were not risk factors for M-eGFR or C-eGFR. Conclusion HBV infection can lead to early renal damage. Age and HBsAg are main risk factors of renal function impairment. Therefore, renal function should be scrutinized in CHB patients.

Objective To evaluate the effects of sevoflurane on proteome in the cortices of neonatal rats.Methods Thirty neonatal rats at postnatal day 7 (6 rats each litter,5 litters in total) were randomly assigned into 2 groups (n =15 each):control group (C group) and sevoflurane group (S group).The rats were exposed to air and 1.8 ％ sevoflurane for 4 h in C and S groups,respectively.One rat from each litter was chosen in each group at the end of anesthesia and the puncture needle was inserted into the left ventricle via the chest wall.Arterial blood samples were then collected for blood gas analysis and for determination of blood glucose.One rat from each litter was sacrificed in each group at 3 and 72 h after the end of anesthesia,and their cortices were then dissected.Two-dimensional differential in-gel electrophoresis (2-D DIGE) was used to identify patterns of protein expression in cortices cross-labeled with different CyDyes.The differentially expressed proteins were analyzed by using matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).Results Acid-base imbalance,anoxia or lycopenia were not found at 3 h after the end of anesthesia in both groups.The analysis showed there were 6 differentially expressed proteins at 3 h after the end of anesthesia in S group compared with C group.Among the 6 proteins,the expression of 4 proteins (class 2 c beta-tubulin,neuron-specific class Ⅲ beta-tubulin,CRMP-1 and CRMP-4) which belonged to cytoskeleton/neuronal growth proteins was down-regulated,the expression of 1 protein (ATP synthase beta subunit) which belonged to hydrolyses and transferases was down-regulated,and the expression of 1 protein (guanine nucleotide binding protein beta1) which belonged to signal transduction proteins was up-regulated (P ＜ 0.05).No significant changes in protein expression were identified at 72 h after 1.8％ sevoflurane anesthesia (P ＞ 0.05).Conclusion 1.8％ sevoflurane-induced 4 h anesthesia can induce short-time changes in the expression of proteins which are related to neuronal migration,differentiation,energy metabolism and signal transduction in cortices of neonatal rats,which may contribute to its neurodegenerative effects in brains of rats during the development period.

AIMTo construct underexpression HSP90alpha and overexpression HSP90beta human hepatoma cell line HepG2.

METHODSThe combined plasimid pSilencerHSP90alpha and pSmycHSP90beta were introduced into HepG2 by electroporation, respectively. The result of transfection was identified by Western-blotting and the curve of cell growth was drew by MTT. Observe the cell vitality and expression of HSP90.

RESULTSExpression of HSP90 in transfected cell line was shown by Western-blotting: Compared with control, expression of HSP90 in the cells transfected with pSilencerHSP90alpha decreased, whereas that in the cells transfected with pSmycHSP90beta increased.The growth curves of the two groups of transfected cells was as the same as that of the control group.

CONCLUSIONThe stable overexpression HSP90beta and underexpression HSP90alpha HepG2 cell lines were established.

Base Sequence ; Electroporation ; HSP90 Heat-Shock Proteins ; genetics ; metabolism ; Hep G2 Cells ; metabolism ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

A total of 152 strains of endophytic fungi were isolated from the barks of Eucommia ulmoides in three regions (Lueyang country, Zunyi country, Cili country). Based on morphological characteristics and analysis of ITS sequences, these strains were identified into 8 genera. Thereinto Phomopsis, Diaporthe and Alternaria were common genera to Eucommia barks from different sites. But the dominant genus was different: Alternaria was the dominant genus in the barks from Cili country, and Phomopsis was the dominant genus from Zunyi country, then Diaporthe was the one from Lueyang country. According to the similarity coefficient, the composition of the endophytic fungi was distinctly different between the barks from three sites. The diversity and species richness in Lueyang country and Cili country were found higher than those in Zunyi country. The evenness of endophytic fungi was 0.936 5 in Lueyang county, which was higher than 0.737 1 or 0.641 0 in Cili county or Zunyi county, respectively. After phylogenic analysis and calculating the genetic distances of typical strains belong to Phomopsis and its perfect stage--Diaporthe, there was very high genetic diversity in the two genera from our study. In conclusion, the community structure and diversity of endophytic fungi were significant different in Eucommia barks from the three habitats.
DNA, Fungal ; genetics ; DNA, Intergenic ; genetics ; Ecosystem ; Endophytes ; classification ; physiology ; Eucommiaceae ; microbiology ; Fungi ; classification ; genetics ; physiology ; Phylogeny ; Plant Bark ; microbiology

OBJECTIVETo investigate the effects of formaldehyde inhalation on the morphological damage, and Glu, GABA and NOS contents in olfactory bulb and hippocampus of rats.

METHODSTwenty SD rats were equally divided into two groups: rats in the control group inhaled fresh air, while the animals in experimental group were exposed to the air containing formaldehyde (12.5 mg/m(3), 4 h/d) for 7 days. Then rats were sacrificed and frozen sections of olfactory bulb and hippocampus were prepared. The morphological changes were examined and the Glu, GABA and NOS contents were detected using Nissl-staining, immunohistochemistry and Western blot, respectively.

RESULTCompared with the control group, there was a significant confusion and shrink of neuron morphology in experimental group, the number and staining intensity of Glu and NOS positive cells and protein contents were reduced. The protein expression of GABA was also decreased in the formaldehyde group.

CONCLUSIONFormaldehyde inhalation can cause a severe morphological damage of olfactory bulb and hippocampus in SD rats,which may further impair memory and learning ability through the reduction of Glu, GABA and NOS expression.

Animals ; Formaldehyde ; toxicity ; Glutamic Acid ; metabolism ; Hippocampus ; drug effects ; metabolism ; pathology ; Inhalation Exposure ; Learning ; drug effects ; Neurons ; drug effects ; metabolism ; pathology ; Nitric Oxide Synthase ; metabolism ; Olfactory Bulb ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; metabolism

OBJECTIVETo explore the expression of GSK-3beta, CDK-5 and PP2A and the regulation of them by Abeta(25-35) and ginsenoside Rb1 after neural stem cells (NSCs) are transformed into neurons.

METHODSTo culture NSCs from the dentate gyrus of newborn rats(24 h) hippocampus in vitro. NSCs of the third passage were induced towards neurons; the expressions of GSK-3beta(pTyr279,216), PP2A and the regulation of them by Abeta(25-35) and ginsenoside Rb1 were tested by the immunofluorescence cytochemical staining after NSCs had been induced for one week; The expressions of GSK-3beta, CDK-5, PP2A and the regulation of them by Abeta(25-35) and ginsenoside Rb1 were detected with RT-PCR.

RESULTSImmunofluorescence cytochemisty showed that neural cells from NSCs which had been differentiated after one week could express GSK-3j (pTyr279,216)and PP2A. Abeta(25-35) could enhance the expression of GSK-3beta(pTyr279,216), meanwhile it also restrained the expression of PP2A. Moreover ginsenoside Rb1 could reverse the affect of Abeta(25-35). RT-PCR found that neural stem cells which had been differentiated after one week could express GSK-3beta, CDK-5, PP2A . The expression of GSK-3beta and CDK-5 rose up and the expression of PP2A weakened when they were treated by Abeta(25-35). However, the effect of Abeta(25-35) was restrained when they were pretreated by ginsenoside Rb1.

CONCLUSIONThese observations indicated that NSCs which were cultured and induced in vitro can express GSK-3beta, CDK-5 and PP2A; moreover Abeta(25-35) and ginsenoside Rb1 can regulate the expressions of GSK-3beta, CDK-5 and PP2A. It hints that cells which differentiated from neural stem cells in vitro have protein phosphorylation regulation system of normal cells.

Amyloid beta-Peptides ; toxicity ; Animals ; Animals, Newborn ; Cell Differentiation ; Cells, Cultured ; Cyclin-Dependent Kinase 5 ; metabolism ; Female ; Ginsenosides ; pharmacology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Hippocampus ; cytology ; Male ; Neural Stem Cells ; cytology ; metabolism ; Peptide Fragments ; toxicity ; Protein Phosphatase 2 ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe MET-associated alteration during the trans-differentiation from MSCs to neuron-like cells, and to explore the possible molecular mechanism.

METHODSBone marrow MSCs were isolated from rat femur and purified in continuous cell culture. After induced differentiation to neuron-like cells by the combination of butylated hydroxyanisole (BHA) and dimethyl sulfoxide (DMSO), cells were tested by comparative polymerase chain reaction (PCR) for the relative expression of MET biomarkers and transcription factors, and for cell cycle by flow cytometry. Meanwhile, target genes of Wnt/β-catenin pathway were also analyzed by comparative PCR to determine the possible involvement.

RESULTSIn MSC-induced neuron-like cells, MET-associated transcription factors such as Snail, Slug, ZEB1, ZEB2, and Twist were significantly attenuated in expression level. The Mesenchymal marker Vimentin expression level was increased. Membrane protein E-cad was slightly down-regulated, while N-cad level was marginally elevated. Percentage of proliferating cells (S phase in cell cycle) markedly shrank from 40.42% for MSCs to 6.76% for MSC-derived neuron. Additionally, Wnt/β-catenin target genes β-catenin and c-myc were decreasingly expressed.

CONCLUSIONChemically induced trans-differentiation from MSC to neuron caused similar MET-featured alteration in gene expression and proliferation to known MET, which might be underlied by deactivation of Wnt/β-catenin pathway.

Animals ; Epithelial-Mesenchymal Transition ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Rats ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo study the autophagy activity between rat bone marrow stem cells (BMSCs) neural differentiation in order to explore the mechanism involve in this process.

METHODSBMSCs were passed by 3 generation, then was induced with the revulsant 2% (DMSO) + 200 µmol/L (BHA), NSE expression was detected by immunocytochemical stain, the mRNA expression of autophagy associated genes L3B, Beclinl, Atg5, Atg7, Atg10 were detected by RT-PCR, the autophagy protein LC3B was examined by Western blot and flow cytometry analysis.

RESULTSBMSCs were passed by 3 generation, the purity of BMSCs could reach more than 90%, the morphology of cells were like fibroblasts, after the revulsant 2% DMSO + 200 µmol/L BRA induced, cells were extended long neurites, like nerve cells, positive rate of NSE staining was (83±5) %, RT-PCR results showed that the expression of autophagy associated genes LC3B, Beclinl, Atg5, Atg7 Atg0 were rised after BMSCs neural differentiation, Western blot analysis showed that the LC3B-II protein expression was increased after neural differentiation and the MFI of L3B was highten by flow cytometry.

CONCLUSIONAutophagy is increased after rat BMSC neural differentiation.

Animals ; Autophagy ; Cell Differentiation ; Cells, Cultured ; Flow Cytometry ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; Rats

OBJECTIVETo evaluate a new type of diatomite-based machinable ceramic biocompatibility by studying its induced apoptosis on L929 cell in contrasted with other prosthodontics materials.

METHODSCell line was treated with extracting liquid containing different concentrations of diatomite-based machinable ceramic and other materials. Flow cytometry tested cell cycle progression and induced cell apoptosis. Annexin V-FITC/PI apoptosis staining kit quantitative detected cell death patterns. The expression of Bcl-2 and Bax mRNA were determined by reverse transcription-polymerase chain reaction.

RESULTSThe experimental groups had no special influence on cell cycle. Apoptosis rates of the new ceramic closed to the negative group (P > 0.05). The apoptosis rate of resin was the highest, and the cell necrosis level of resin was increased, which had significant difference to the new ceramic (P < 0.05). The Bcl-2 and Bax mRNA levels of the new ceramic and the negative group were closed to each other, which had no statistical significance (P > 0.05).

CONCLUSIONThe new diatomite-based machinable ceramic has no apparent cytotoxicity, which is consistent with the clinical application of the basic requirements of biocompatibility.

Animals ; Annexin A5 ; Apoptosis ; Cell Line ; Dental Materials ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Mice ; Necrosis