Adult ; China ; epidemiology ; Circoviridae Infections ; epidemiology ; virology ; DNA, Viral ; analysis ; Female ; Genotype ; Hepatitis, Viral, Human ; virology ; Humans ; Male ; Substance Abuse, Intravenous ; virology ; Torque teno virus ; genetics ; isolation & purification

Hepatocyte growth factor (HGF) pretreatment could protect multiple cell types from apoptosis induced by various damages including oxidative stress. The present study was designed to investigate the protective effect of HGF on rat cortical neurons against apoptosis induced by hydrogen peroxide (H2O2) in culture, and then to explore whether HGF could influence the mitochondrial pathway of apoptosis. Primary rat cortical neurons were isolated from Sprague-Dawley rats and cultured in serum free medium containing 2% B27 and Neurobasal-A. To mimic the oxidative stress damage, cortical neurons were exposed to 100 mumol/L H2O2 for 4 h. To explore the effects of HGF on the neurons subjected to H2O2 injury, cells were pretreated with HGF 15, 30, 60 ng/mL for 24 h, respectively, and then exposed to 100 mumol/L H2O2 for 4 h. The cell viability was measured by MTT colorimetric assay and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. Apoptotic cells were detected by Hoechst 33258 staining and Annexin V-FITC/PI double labeled flow cytometry. The caspase-3 activity was assessed by colorimetry. The alteration of transmembrane potential of mitochondria was determined by confocal laser scanning microscopy. The expression of cytochrome C protein was measured by Western blot analysis. The results showed that H2O2 treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apoptotic cells. Pretreatment of HGF at different concentrations (15-60 ng/mL) could remarkably increase the cell viability of neurons. Compared with that of H2O2 group (53.4%+/-7.4%), the cell viabilities of neurons treated with 15, 30, and 60 ng/mL HGF significantly increased to (69.3+/-6.4)%, (77.5+/-6.1)% and (82.9+/-9.3)% (P<0.05), respectively. HGF preincubation also evidently decreased the LDH leakage rate in cortical neurons damaged by H2O2. The results of Hoechst staining revealed that HGF pretreatment could significantly reduce the apoptotic rate of neurons. The apoptotic rate of H2O2 group was (62.8+/-7.1)%, while that of HGF groups decreased significantly to (34.8+/-8.4)%, (23.5+/-3.2)% and (18.6+/-4.5)% (P<0.05), respectively. The data from caspase-3 activity assay indicated that HGF preconditioning could also remarkably decrease the caspase-3 activity of neurons. In addition, in the presence of various concentrations of HGF, the decrease of transmembrane potential of mitochondria in neurons caused by H2O2 injury could be reversed. Moreover, as detected by Western blot analysis, HGF downregulated the expression of cytochrome C protein in neurons. These results suggest that HGF has a protective effect on rat cortical neurons against apoptosis induced by H2O2, which might be related to the inhibition of the mitochondrial apoptotic pathway and the suppression of the caspase-3 activity.
Animals ; Apoptosis ; Brain ; cytology ; Caspase 3 ; metabolism ; Cell Survival ; Cells, Cultured ; Cytochromes c ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; Hydrogen Peroxide ; pharmacology ; Mitochondria ; physiology ; Neurons ; cytology ; drug effects ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley

Objective To build an intelligent field medical station based on new information technologies such as IOT.Methods The milieu exterieur and deficiencies of the existing field medical station were analyzed,and the overall architecture,main functions and technology implementation of the intelligent field medical station were proposed based on IOT and other new information technologies.Results The intelligent medical station was modified from the existing one,and enhanced its medical support ability greatly.Conclusion The intelligent field medical station adapts itself to future battlefield support as well as intelligent management and emergency support at peacetime,and thus can be applied in multi medical support mission.

OBJECTIVETo cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles.

METHODSFree of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSHygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium.

CONCLUSIONAfter the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

Cell Line ; Drug Resistance, Viral ; Genetic Vectors ; Genome, Viral ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Hygromycin B ; pharmacology ; Mutation ; Plasmids ; Retroviridae ; genetics ; Transfection ; Virus Assembly

BACKGROUNDTo explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein.

METHODSTwo kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSThe mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP.

CONCLUSIONDominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

Cell Line, Tumor ; Genetic Engineering ; Genetic Therapy ; Genetic Vectors ; Genome, Viral ; Hepatitis B Core Antigens ; biosynthesis ; physiology ; Hepatitis B virus ; genetics ; Humans ; Point Mutation ; Recombinant Fusion Proteins ; biosynthesis ; physiology ; Virus Replication

OBJECTIVETo evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.

METHODSA fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot.

RESULTSThe HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon.

CONCLUSIONIt is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

Cell Transformation, Viral ; Cells, Cultured ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; cytology ; virology ; Humans ; Liver ; cytology ; virology ; Recombinant Proteins ; genetics ; Transfection ; Virus Replication

OBJECTIVETo investigate the mechanism and the accelerating effect of rhEGF and rhbFGF on wound healing.

METHODSTwelve New Zealand rabbits with 72 incised wounds on ventral side of 24 ears were randomly divided into two therapeutic groups (rhEGF of 10 ug/cm(2) and rhbFGF of 100 AU/cm(2)) and a control group (1% silver sulfadiazine cream, SD-Ag). The general conditions of the wound healing was observed grossly. Biopsies were harvested at different time points for the pathomorphological examination, the electron microscopic examination, and for assessment of integrin beta1 mRNA expression by in situ hybridization.

RESULTSThe expressions of integrin beta 1 mRNA in two therapeutic groups were significantly higher than that of control group. The quality of the wound healing was improved in therapeutic group with its healing time shortened when compared with that in control group (P < 0.05). There was an obvious difference in the number of fibroblasts and capillary gemmules between the therapeutic and control groups (P < 0.05).

CONCLUSIONThe wound healing and quality could be improved by both rhEGF and rhbFGF, but rhbFGF seemed better to be employed during the early and middle stages of the wound repair for the growth of granulation tissue, while rhEGF should be applied at the late stage of wound repair to accelerate the re-epithelialization of the wound. Combined application of rhEGF with rhbFGF according to time effect could be more beneficial to the wound repair.

Animals ; Epidermal Growth Factor ; pharmacology ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; Integrin beta1 ; genetics ; Male ; Microscopy, Electron ; RNA, Messenger ; analysis ; Rabbits ; Recombinant Proteins ; pharmacology ; Wound Healing ; drug effects

To investigate the relationship between the expression of RASSFIA protein and promoter hypermethylation of RASSFIA gene, RASSFIA protein expression was measured by Western blot- ting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carci- noma (BTCC). The promoter methylation in BTCC and normal bladder tissues was detected by me- thylation-specific PCR (MSP). The results showed that the expression level of RASSFIA protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not corre- lated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF1A protein. It is con- cluded that RASSFIA gene promoter methylation may contribute to the low level or loss of RASSFIA protein expression, the inactivation of RASSFIA gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.

Objective To observe the effect of risperidone combined with clozapine in the treatment of vagrants with schizophrenia and its influence on social function of the patients.Methods Eighty patients with vagrant schizophrenia were selected as the study objects by a prospective randomized controlled trial,and were randomly divided into control group(40 cases)and observation group(40 cases).The control group was treated with risperidone,and the observation group was treated with risperidone and clozapine.Both groups received continuous treatment for 8 weeks.The clinical effects of the two groups were observed.The activities of daily living,rehabilitation[Inpatient Psychiatric Rehabilitation Efficacy Rating Scale(IPROS)score],social function,myocardial enzyme profile[lactate dehydrogenase(LDH),creatine kinase(CK),creatine kinase isoenzyme(CK-MB)]in the two groups before treatment and eight weeks after treatment were compared.The adverse reactions of the two groups were statistically analyzed.Results The total effective rate of treatment in the obser-vation group was higher than that of the control group(90.00％versus 72.50％,P＜0.05).After eight weeks of treatment,the abilities of daily living and social function of the two groups were stron-ger than before treatment,and the observation group was stronger than the control group.At 8 weeks of treatment,IPROS scores in two groups were lower than before treatment,and observation group was lower than control group(P＜0.05).After 8 weeks of treatment,LDH,CK and CK-MB levels in two groups were higher than before treatment(P＜0.05),but there was no statistical significant between-group difference(P＞0.05).Conclusion Risperidone combined with clozapine has a significant effect in thetreatment of vagrants with schizophrenia,which can enhance the patients'daily living ability and social ability,improve the rehabilitation status,and does not increase the adverse reactions.

Objective To observe the effect of risperidone combined with clozapine in the treatment of vagrants with schizophrenia and its influence on social function of the patients.Methods Eighty patients with vagrant schizophrenia were selected as the study objects by a prospective randomized controlled trial,and were randomly divided into control group(40 cases)and observation group(40 cases).The control group was treated with risperidone,and the observation group was treated with risperidone and clozapine.Both groups received continuous treatment for 8 weeks.The clinical effects of the two groups were observed.The activities of daily living,rehabilitation[Inpatient Psychiatric Rehabilitation Efficacy Rating Scale(IPROS)score],social function,myocardial enzyme profile[lactate dehydrogenase(LDH),creatine kinase(CK),creatine kinase isoenzyme(CK-MB)]in the two groups before treatment and eight weeks after treatment were compared.The adverse reactions of the two groups were statistically analyzed.Results The total effective rate of treatment in the obser-vation group was higher than that of the control group(90.00％versus 72.50％,P＜0.05).After eight weeks of treatment,the abilities of daily living and social function of the two groups were stron-ger than before treatment,and the observation group was stronger than the control group.At 8 weeks of treatment,IPROS scores in two groups were lower than before treatment,and observation group was lower than control group(P＜0.05).After 8 weeks of treatment,LDH,CK and CK-MB levels in two groups were higher than before treatment(P＜0.05),but there was no statistical significant between-group difference(P＞0.05).Conclusion Risperidone combined with clozapine has a significant effect in thetreatment of vagrants with schizophrenia,which can enhance the patients'daily living ability and social ability,improve the rehabilitation status,and does not increase the adverse reactions.