Objective To investigate the effect of dichloroacetate on the expression of Kv1.5 in a rat model of pulmonary arterial hypertension (PAH) .Methods Thirty-two male SD rats weighing 200-250 g were randomly divided into 4 groups ( n = 8 each): normal control group (group C), dichloroacetate control group (group D),PAH group, and PAH + dichloroacetate group (group PD). PAH was induced by left lung resection combined with subcutaneous injection of monocrotaline 60 mg/kg in PAH and PD groups. In group PD, dichloroacetate 80 mg/kg was given through a gastric tube into stomach once a day for 28 consecutive days after monocrotaline injection,while the equal volume of normal saline was given instead of dichloroacetate in group PAH. Group D only received dichloroacetate 80 mg/kg through a gastric tube into stomach once a day for 28 consecutive days. Pulmonary arterial pressure (PAP) was measured at day 28 after monocrotaline injection. The rats were then sacrificed and lung tissues were removed to calculate the percentage of thickness of the tunica media of pulmonary artery and right venicular hypertrophy index and to determine the proliferating cell nuclear antigen (PCNA) and Kv1.5 protein expression (by Western blot) and Kv1.5 mRNA expression (by RT-PCR).Results Compared with group C, the PAP,percentage of thickness of the tunica media, right ventricular hypertrophy index were significantly increased, Kv1.5 mRNA and protein expression was down-regulated and PCNA expression was up-regulated in groups PAH and PD ( P ＜ 0.05). Compared with group PAH, the PAP, percentage of thickness of the tunica media, right ventricular hypertrophy index were significantly decreased, Kv1.5 mRNA and protein expression was up-regulated and PCNA expression was down-regulated in group PD (P ＜ 0.05). There was no significant difference in the indexes mentioned above between group C and group D ( P ＞ 0.05). Conclusion Dichloroacetat alleviates PAH through upregulating Kv1.5 expression in lung tissues and inhibiting pulmonary vascular remodeling in rats.

Objective To evaluate the changes in the expression of hypoxia-inducible factor 1 alpha (HIF-1α) in the proliferated intima of pulmonary arterioles in rats with pulmonary hypertension.Methods Thirty-two pathogen-free male Sprague-Dawley rats,aged 2 months,weighing 200-230 g,were used in the study.Thirty-two rats were randomly divided into 4 groups with 8 animals in each group using a random number table:shamoperation group (group S),left pneumonectomy group (group P),monocrotaline group (group M),and left pneumonectomy combined with monocrotaline group (group PM).The rats underwent left pneumonectomy in P and PM groups.In M and PM groups,monocrotaline 60 mg/kg was injected subcutaneously at 7 days after operation.On day 35 after operation,mean pulmonary artery pressure (mPAP) and right ventricle systolic pressure (RVSP)were measured via direct puncture of right ventricle outflow tract with gauge 24 iv catheter connected to pressure transducer.At the end of experiment,the hearts and right lobes of the lung were excised for measurement of right ventricle hypertrophy index (RVHI) and the thickness of the medial layer (tunica media) of pulmonary arterioles (MT).Vascular occlusion score (VOS) was performed.The expression of HIF-1α,α-smooth muscle actin (α-SMA),and proliferating cell nuclear antigen (PCNA) in the pulmonary arterioles was determined.Results Compared with S group,MT was significandy increased,and the expression of α-SMA was up-regulated in P group,mPAP,RVSP,RVHI and MT were increased in M group,mPAP,RVSP,RVHI,MT and VOS were increased in PM group,and the expression of HIF-1α,α-SMA,and PCNA was up-regulated in M and PM groups.Compared with P group,mPAP,RVSP,RVHI and MT were significantly increased,and the expression of HIF1α,α-SMA,and PCNA was up-regulated in M and PM groups.Compared with M group,RVSP,RVHI,MT and VOS were si gnificantly increased,and the expression of HIF-1α,α-SMA,and PCNA was up-regulated in PM group.Conclusion The mechanism of pulmonary arteriole intimal proliferation is related to up-regulated HIF-1α expression and promoted proliferation of vascular smooth muscle cells in rats with pulmonary hypertension.

Hepcidin are small cationic peptides with antibacterial activity expressed mainly in the liver of living organisms, and they play important roles in the host's immune response against microbial invasion and regulation of iron metabolism. Thus, they are considered to be good substitutes for traditional antibiotics. It is a good choice that the antimicrobial peptides are prepared by recombinant DNA expression. In the present study, two hepcidin mature peptide cDNAs from channel catfish (Ictalurus punctatus) (mCH) and tilapia (Oreochromis niloticus) (mTH) were connected by SOE-PCR in order to obtain more recombinant hepcidin with broad antimicrobial spectrum, and EcoR I and Not I sites were added to 5'- and 3'- ends of the fragment, respectively. The recombinant eukaryotic expression vector "pPIC9K-mCH-mTH" was successfully constructed, and transformed into Pichia pastoris GS115. The transformants containing multicopy gene insertion were selected by using different concentrations of G418 and other specific mediums, and identified by PCR for yeast genomic DNA. Expression was induced by adding 1% methanol at 30 degrees C for different times. Tricine-SDS-PAGE analysis demonstrated that the most appropriate expression time was 72 h, at which a high expression yield (77 mg/L) for the target protein was exhibited. The highly purified target protein was obtained from the fermentation supernatant by SP-Sepharose cation exchange chromatography. Bacteriostatic activity assay demonstrated that the fermentation supernatant containing the target protein and purified recombinant target protein had bacteriostatic activities against gram-positive and gram-negative bacterium. The present result provides the important initial value for industrial production of hepcidin antimicrobial peptide.
Animals ; Electrophoresis, Polyacrylamide Gel ; Fish Proteins ; biosynthesis ; genetics ; Fishes ; genetics ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Hepcidins ; biosynthesis ; genetics ; Pichia ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

Marginal liver donor,a way to expand the liver pool,has been maximized in the unique position due to the shortage of donors.But the definition of marginal donor liver varies from center to center and the standard is very complex.With the enhancement of organ perfusion solution,preservation methods and surgical techniques,the edge donor criteria are also gradually expanding.What decision should we make,facing such clinical controversies.This paper makes a review on the marginal liver donor in the donation after citizen deceased,so as to improve its clinical application.

Objective To evaluate the effect of phosphatidylinositol-3 kinase inhibitor LY294002 combined with dichloroacetate on apoptosis in human pulmonary arterial smooth muscle cells (SMCs) and AKT/GSK-3β/HK-2 signaling pathway.Methods Human pulmonary arterial SMCs were seeded into culture plates at a density of 2 x 104 cells/ml after 3-5 passages.After being incubated for 72 h,the SMCs were cultured in the medium supplemented with 0.2％ fetal bovine serum for 24 h to induce starvation prior to experiments.The cells were then randomly divided into 6 groups (n =6 each) using a random number table:control group (group C),positive control group (group F),LY294002 group (group L),different concentrations of dichloroacetate groups (D1 and D2 groups),and LY294002 combined with dichloroacetate group (group LD1).In group C,the cells were cultured in the medium supplemented with 0.2％ fetal bovine serum.In F,L,D1,D2 and LD1 groups,the cells were cultured in the medium supplemented with 10％ fetal bovine serum.LY294002 2 μmol/L was added to the medium in group L.Dichloroacetate l0 and 20 mmol/L were added to the medium in D1 and D2 groups,respectively.In group LD1,LY294002 (2 μmol/L) was added,and 30 min later dichloroacetate 10 mmol/L was added to the medium in LD1 group.The cells were incubated for 48 h.Flow cytometry was used to measure the cell apoptosis and mitochondrial membrane potential.The expression of phosphorylated AKT (p-Akt),phosphorylated glycogen synthase kinase 3β (p-GSK-3β),and hexokinase-2 (HK-2) was detected using Western blot.Apoptosis rate was calculated.Results Compared with group C,apoptosis rate was significantly increased,and mitochondrial membrane potential was decreased in D2 and LD1 groups,the expression of p-Akt,p-GSK-3β and HK-2 was up-regulated in group F,and no significant changes were found in apoptosis rate and mitochondrial membrane potential in F,L and D1 groups.Compared with group D2,apoptosis rate was significantly increased,mitochondrial membrane potential was decreased,and the expression of p-Akt,p-GSK-3β and HK-2 was down-regulated in LD1 group.The expression of p-Akt,p-GSK-3β and HK-2 was significantly lower in D2 and LD1 groups than in group F.Conclusion LY294002 combined with dichloroacetate can promote apoptosis in human pulmonary arterial SMCs possibly through blocking AKT/GSK-3β/HK-2 signaling pathway.

Objective To establish a new effective method by using real-time polymerase chain reaction (PCR) to detect single nucleotide polymorphism (SNP) typing for rapid identifying apolipoprotein E alleles.Methods To determine alleles of human apolipoprotein E genetic polymorphism at Cys112Arg locus was detected by PCR melting curve analysis with fluorophore SYBR Green I. In order to increase the speciality of SNP assays, high fidelity Taq polymerase was used. The reliability of SNP typing was validated by comparison with the results of direct DNA sequencing.Results Each sample was determined by double tubes, and two melting curves were analysis. As compared the Tm value of samples with the Tm of standard substance, the apoE genotype of samples was determined. The apoE genotype of 30 samples were E3/3 (27/30) and E3/4 (3/30) respectively, which was accordant with the results of PCR-RFLP and DNA sequencing.Conclusion The presented allelic assay was specific, easy to operate and applicable for discrimination of apolipoprotein E genotyping of human blood.

Objective To detect differentially expressed genes between human normal skin and photoaging skin, and to investigate the molecular and biological mechanisms of human skin photoaging at transcriptional level. Methods Full-thickness skin specimens were obtained during full-face rhytidectomy from sun-exposed (anterior ear skin) and sun-protected (retroauricular skin) sites of 6 patients with facial photoaging from 2007 to 2008. Genomic microarray analysis was conducted to identify differentially expressed genes between the two groups of specimens followed by gene-cluster analysis. Results The normalization of microarray data showed that the number of differentially expressed genes was 2163 between skin samples from sun-exposed and sun-protected sites in one patient, significantly higher than that in the other 5 patients (less than 200);therefore, the data from the patient with 2163 differentially expressed genes were excluded from further analysis.Totally, 172 differentially expressed genes were identified with Beadarray chip, including 99 up-regulated genes and 73 down-regulated genes. Based on Genebank research, 118 functionally classified genes werefound, which were associated with a series of biological processes, including cell adhesion, receptor regulation,signal transduction, metabolism, and so on. Conclusions There are a lot of differentially expressed genes between human photoaging skin and normal skin. Rhytidectomy may be associated with the differential expression of skin photoaging-related genes.

Chronic liver diseases can further develop to liver fibrosis and cirrhosis. Currently, there is no effective treatment except liver orthotopic transplantation at this point. The extreme shortage of liver organ source forced people to find alternative treatment strategies. Mesenchymal stem cells ( MSCs) have the abilities of immunomodulatory, hepatocyte differentiation, promotion of liver cells regeneration in situ and inhibiting the activation of hepatic stellate cells. Therefore, MSCs transplantation provides a very broad prospect for cell therapy. It is important to provide preclinical evaluation of the efficacy and safety before the application of cell therapy in clinical trials. The progress of various animal models of human liver diseasees and significance of using MSCs to treat liver diseases in preclincal studies based on these animal models were reviewed in this paper.

Objective To investigate the diagnosis and treatment of renal neoplasm with calcification .Methods Retrospectively summarized the clinical data of the 2 patients with calcific renal neoplasm admitted in our hospital from the May to July in 2014, then analyzed and discussed the clinical manifestations , diagnosis and treatment com-bined with the literatures .Results The two cases were both suspected of renal malignant tumor preoperatively .The case 1 was a 32-year-old male , laparoscopic partial resection of the left kidney was performed , and the postoperative pathology was clear cell carcinoma (Fuhrman levelⅠ).The case 2 was a 18-year-old male, partial resection of the right kidney was performed because of the tumor size , and the postoperative pathology was adult nephroblastoma . Conclusions The calcific renal neoplasm is a rare disease , the property determination depends on postoperative pa-thology, and as to the choice of surgical method , the patients'age, the tumor size and the tumor location should be taken into consideration , and intraoperative frozen should be performed when necessary .

Objective To observe the effect of sinomenine (SIN) on the expression of cyclooxygenase (COX2),alpha-7 nicotinic acetylcholine receptor(α7nAChR) and adenosine receptor(A2A) in A549 cells,and to explore the relative mechanism for cell proliferation.Methods The effect of SIN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the proliferation of A549 cells was determined by methyl thiazolyl tetrazolium (MTT) assay.The effect of SIN and NNK on the migration of A549 cells was detected by cell wound scratch assay.The effect of SIN and NNK on COX2 expression in A549 cells was determined by Western blotting method.The effect of SIN and NNK on the expression of α7nAChR and A2A mRNA and protein was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting method.Results NNK increased the proliferation and migration of A549 cells,while SIN inhibited the proliferation and migration of A549 cells.COX2 expression level was increased in NNK group but was decreased in SIN group.The expression levels of α7nAChR and A2A were up-regulated in NNK group but were down-regulated in SIN group.Conclusion SIN plays a role in inhibiting the proliferation and migration of A549 cells by suppressing COX2 expression.SIN has an inhibitory effect on the expression of α7nAChR and A2A.