Exchange proteins activated directly by cyclic adenosinemonophosphate (Epac) is one of cyclic adenosinemonophosphate (cAMP)-regulated guanine nucleotide exchange factors.When Epac closely binds with cAMP,it can activate many downstream signal molecules,such as Rap and Ras,etc.By activating several effectors,Epac regulates a multitude of important physiological and pathological processes.Ras and Rap play important effects on the differentiation and proliferation of photoreceptors.Ras may participate in the pathogenesis and development of age-related macular degeneration (AMD) and prolifereative vitreo-retinopathy (PVR) through Ras/Raf-1/MEK/ERK cascade.Therefore,further studies about the role of Epac and its downstream signals in the retina may provide numerous directions both in research and treatment of retinal diseases.The researching progress in Epac and the functions of the downstream signaling molecules of Epac in the retina were reviewed.

Objective To evaluate the correlation between transient elastrography (TE) and liver function Child-Pugh grade in patients with liver cirrhosis. Methods The clinical data of 120 patients with liver cirrhosis were retrospectively analyzed. Among the patients, hepatitis-B virus-related cirrhosis was in 103 cases, and 57 patients had ascites. The liver stiffness measurement (LSM) was measured by FibroScan. The liver function Child-Pugh grade was evaluated by liver function Child-Pugh system score. The LSM was compared in patients with different liver function Child-Pugh grade. Results Among the 120 patients with liver cirrhosis, Child-Pugh A grade was in 39 cases, Child-Pugh B grade in 28 cases, and Child-Pugh C grade in 53 cases. The LSM in Child-Pugh B grade patients and C grade patients were significantly higher than that in Child-Pugh A grade patients: (20.2 ± 1.1) and (30.8 ± 1.2) kPa vs. (15.7 ± 1.4) kPa, the LSM in Child-Pugh C grade patients was significantly higher than that in Child-Pugh B grade patients, and there were statistical differences (P<0.05). Among the 103 patients with hepatitis-B virus-related cirrhosis, Child-Pugh A grade was in 33 cases, Child-Pugh B grade in 24 cases, and Child-Pugh C grade in 46 cases. The LSM in Child-Pugh B grade patients and C grade patients were significantly higher than that in Child-Pugh A grade patients: (18.7 ± 0.9) and (26.9 ± 0.6) kPa vs. (12.6 ± 1.7) kPa, the LSM in Child-Pugh C grade patients was significantly higher than that in Child-Pugh B grade patients, and there were statistical difference (P<0.05). Among the 57 patients associated ascites, Child-Pugh B grade was in 11 cases, and Child-Pugh C grade in 46 cases. The LSM in Child-Pugh C grade patients was significantly higher than that in Child-Pugh B grade patients:(42.3 ± 1.4) kPa vs. (35.1 ± 1.0) kPa, and there was statistical difference (P<0.05). Among the 103 patients with hepatitis-B virus-related cirrhosis, associated ascites was in 49 cases, Child-Pugh B grade was in 10 cases, and Child-Pugh C grade in 39 cases. The LSM in Child-Pugh C grade patients was significantly higher than that in Child-Pugh B grade patients: (40.6 ± 0.9) kPa vs. (33.2 ± 1.5) kPa, and there was statistical difference (P<0.05). Conclusions The LSM values of patients with liver cirrhosis are higher with the elevation of liver function Child-Pugh grade. There is a correlation between LSM values and Child-Pugh scores. The LSM can partly evaluate the severity of liver disease in patients with liver fibrosis.

Objective To investigate the effect of MicroRNA-9 (miR-9) on cell proliferation and collagen synthesis in hepatic stellate cells (HSCs),and to explore the potential mechanism.Methods HSC-T6 cells were cultured and transfected with miR-9 mimics with lipofectamine 2000.After incubation 48 h,the cells were collected and total proteins and RNAs were extracted.The expression of miR-9 was detected by quantitative real time polymerase chain reaction (qRT-PCR).The protein expression of type Ⅰ collagen and type Ⅲ collagen were measured by Western blot.The methyl thiazolyl tetrazolium (MTT) method was used to asses the proliferation of HSC-T6 cells.The expression of transforming growth factor-β1 receptor 2 (TGFBR2) was detected by qRT-PCR and Western blot.Results Compared to the control group,miR-9 expression in HSCs was increased in the miR-9 mimics group (P ＜ 0.05),type Ⅰ and type Ⅲ collagen protein expression was reduced by (44 ± 2) ％ and (50 ± 3) ％ (P ＜ 0.01),respectively.The proliferation activity of HSCs was decreased by (48 ± 4)％ (P ＜ 0.05).The expression of TGFBR2 was inhibited in the miR-9 mimics group.Conclusions Upregulation of miR-9 plays a role on suppressing cell proliferation and collagen synthesis in HSCs.This process might be mediated by downregulation of TGFBR2.

Objective To investigate the effect of miRNA-200b-specific inhibitor on hepatic stellate cells(HSCs) activation,proliferation,and extracellular matrix production.Methods The miRNA-200b-specific inhibitors were designed,synthesized,and transfected into HSCs with lipofectamine 2000.The supernatant and HSCs were collected after incubation for 48 h.The expression of miR-200b was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).The expression ofα-smooth muscle actin (oα-SMA) protein in HSCs was detected by Western blotting.The cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) method.Contents of type Ⅲ procollagen and hyaluronic acid in supernatant were determined by radioimmunoassay.Results Compared to the control group,miRNA-200b expression was decreased in the miRNA-200b inhibitor group by 82％ (P ＜ 0.01),α-SMA protein expression was reduced in the miRNA-200b inhibitor group by (19 ± 3) ％ (P ＜ 0.05),and the activity of HSCs proliferation was reduced by(33 ± 5)％ (P ＜0.01),and the contents of type Ⅲ procollagen and hyaluronic acid in supernatant were reduced in miRNA-200b inhibitor group by (35 ± 4)％ and (31 ± 2)％,respectively(P ＜0.01).Conclusions The miRNA-200b-specific inhibitor could significantly reduce the expression of miRNA-200b,and inhibit HSC proliferation,activation,and extracellular matrix production.

BACKGROUND: Vascular endothelial growth factor (VEGF) can promote angiogenesis, and has been extensively used in treatment of bone defect. However, few studies have addressed its isomer VEGF121. OBJECTIVE: To construct adenovirus vector carrying VEGF121-FLAG and humanized Renilla reniformis green fluorescent protein 1(hrGFP-1) and observe its expression in bone marrow stromal stem cells (BMSCs). METHODS: Using polymerase chain reaction technique, VEGF121 gene contained in the plasmid of pTG19T-VEGF121 was used to remove termination codon. NotI and Xho I restriction sites were added before and after gene sequence. Obtained gene subclone was moved onto pMD19-T plasmid. The pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids underwent double enzymatic digestion. Small fragment and big fragment were retrieved utilizing gel. Subsequently, coupled reaction was conducted to complete the construction of shuttle plasmid. After measuring virus titer, BMSCs were transfected and the fluorescence intensity was observed under fluorescence microscope.RESULTS AND CONCLUSION: Recombinant adenovirus plasmid was successfully constructed by enzymatic digestion determination and gene sequence. Fluorescence microscope has shown that BMSCs transfected with recombinant adenovirus presented significantly green fluorescence expression. Thus, adenovirus vector carrying VEGF121-FLAG and hrGFP-1 gene can express in eukaryotic cells, which can be used for gene therapy for ischemic disease.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) can regulate the co-expression of various genes, and can induce angiogenesis with integrated physiological function.OBJECTIVE: To construct a novel adenoviral eukaryotic expression vector that can co-express mutant hypoxia-inducible factor-1 alpha (HIF-1a) target protein and humanized Renilla reniformis green fluorescent protein (hrGFP) reporter molecule under normoxic conditions.METHODS: The human HIF-1α gene carried by target gene donor plasmid pCMV6-XL5-HIF1α was sequenced and the site of restriction enzyme in above gene was analyzed. Site-directed mutagenesis of three amino acids including the 402 location, the 564 location, and the 803 location in gene coding region in HIF-1α were performed by polymerase chain reaction and sequencing was also done for monitoring mutation. The HIF-1α gene mutated correctly (HIF-1αmu) was coupled to adenoviral shuttle vector pShuttle-CMV-IRES- hrGFP-1. The recombinant adenovirus shuttle vector carrying HIF-1αmu gene was transferred to BJ5183-AD-1 electroporation competent cells after sequencing identification and Pme I restriction enzyme linearization.HIF-1αmu and hrGFP gene as well as hemeo-expression elements of hrGFP gene were reconstructed into adenoviral genome plasmids using homologous recombination mechanism in bacterium. Recombinants were obtained by Pac I restriction enzyme digestion and sequencing identification.RESULTS AND CONCLUSION: Amino acids including the 402 location, the 564 location and the 803 location in gene coding region in HIF-1α had become alanine after site-directed mutagenesis. Recombinant adenoviral expressing vector was successful as confirmed by restriction enzyme digestion and sequencing. These findings demonstrate that a novel recombinant adenoviral mutant eukaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 was successfully constructed.

Proteomic analysis is an effective way to identify protein constituent in Lewy bedy-like inclusions (or aggresome) in vitro. Exposure to synthetic proteasome inhibitor (PSI, 10 μmol/L) for 48 hours was used to induce the formation of cytoplasmic proteineous inclusions (termed as PSi-induced inclusions) in PC12 cells.The proteomic approaches of biochemical fractionation, two-dimensional electrophoresis (2-D) and identification via peptide mass fingerprints (PMF) were deployed, and 20 protein components of LBs were identified,i ncluding 2 proteins involved in the production of synaptic neurotransmitter, 6 subunits of the 26 S proteasome,2 cytoskeleton proteins, 2 subunits of mitochondrial complexes, 1 anti-oxidant protein, and 7 chaperone proteins and (or) chaperone-like proteins. The results suggested that these LB protein components might had been recruited in PSI-induced inclusions formed in PC12 cells under the condition of proteasome inhibition.

Objective To estimate the clinical effect of fuzhenghuayu capsule in treatment of early hepatocirrhosis by liver instantaneous elastic imaging. Methods Eighty patients with early hepatocirrhosis were divided into experiment group and control group according to the treatment method with 40 cases each. All the patients in 2 groups were given the same antiviral treatment with entecavir dispersible tablets, and the patients in experiment group combined with fuzhenghuayu capsule. All treatment lasted for 6 months. The liver stiffness measurement (LSM) was measured before treatment and 1, 3 and 6 months after treatment by liver instantaneous elasticity imaging. The serum hyaluronic acid was measured, and the Child-Pugh score was evaluated at the same time. Results The LSM, hyaluronic acid and Child-Pugh scores 6 months after treatment in experiment group were significantly lower than those in control group:(19.3 ± 0.9) kPa vs. (29.6 ± 1.3) kPa, (215.6 ± 59.3)μg/L vs. (344.4 ± 39.6)μg/L and (2.1 ± 1.3) scores vs. (3.9 ± 0.9) scores, and there were statistical differences (P<0.05). No obvious adverse reactions related to the use of fuzhenghuayu capsule were found during the course of treatment. Conclusions Liver instantaneous elasticity imaging can be used to evaluate and monitor early hepatocirrhosis. Fuzhenghuayu capsule on patients with early hepatocirrhosis has a certain degree of curative effect.

BACKGROUND:The structure of tissue engineering carrier affects the bio-action of cells greatly.OBJECTIVE: To investigate the biological characteristics of bone marrow stem cells (MSCs) in different concentrations of alginate combined with de-antigen bone xenograft (DBX).DESIGN: Observational trial.SETTING: PLA Institute of Trauma and Orthopedics, the Fourth Military Medical University of Chinese PLA.MATERIALS: Alginate, calcium chloride, MSCs, bone xenograft.METHODS: Bovine cancellous bone was out into cubes, which were degreased, deproteinized and then lyophilized.Cubes in pore size within 300-500 μm were selected for use after ethylene oxide sterilization. The purified sodium alginate was dissolved in DMEM cell culture medium of concentrations as different as 0.5%, 2%, 8% and 16%; 1×1012 L-1 induced MSCs were blended with isopyknic alginate-DMEM and compounded with DBX at a status of 0.5 Mpa negative pressure for 5 minutes in order to make a cell suspension fully fill into the pores of the cancellous bone. Then alginate was crosslinked with 50 g/L calcium gluconic acid for 30 seconds. The complex was put into a CO2 incubator and cultured for 4 days. The gel compound and cell growth in the pores of the complex were grossly observed with an inverted microscope. Status of cell growth in the complex with different concentrations of alginate was observed with scanning electron microscopeMAIN OUTCOME MEASURES: Compound status of alginate and bone xenograft, cell growth status and matrix secretion in compound carries.RESULTS: When the concentration of alginate was 0.25% or 1%, alginate was equally combined in DBX, while that of 4% and 8% only combined on the surface of cancellous bone. After in vitro cultured for 4 days, alginate of 0.25% were broken off from DBX surface. But alginate of 1% was equally combined with DBX pores with cells secreting well in alginate. Development of cells in alginate of 4% was restricted and no cells were seen in alginate of 8%.CONCLUSION: Alginate of 1% is suitable for constructing the carrier of bone tissue engineering with bone xenograft.

To survey the serum uric acid (SUA) levels and associated risk factors of hyperuricemia among youth and middle-aged residents in Guangzhou.A total of 1420 subjects,aged from 20 to 60 years,receiving health check-up at our hospital in 2010 were enrolled.The total prevalence of hyperuricemia was 22.04％,32.01％ in males and 14.07％ in females.The average SUA was (388 ±78) μmol/L in males and (288 ± 63) μ mol/L in females.The prevalence of hyperuricemia in males was 30.11％ before 40 years of age and 33.81％ between 40 and 60 years of age.The average level of SUA in males was significantly higher than that of females.logistic regression analysis showed that BUN,body mass index (BMI) and hypertriglyceridemia were the independent risk factors of disease while HDL-C and gender (females) the protective factors.