OBJECTIVE To establish SYBR Green Ⅰ fluorescent quantitative PCR assay for the detection of West Nile virus(WNV),which could be used for early laboratory diagnosis.METHODS A fragment of WNV gene was amplified by PCR,then cloned into pMD-18 T vector.The combinant plasmid was sequenced and analyzed by means of BLAST program,and used as the positive DNA in place of WNV.The SYBR GreenⅠfluorescent quantitative PCR assay was established based on positive plasmid.The sensitivity and specificity of the assay were performed.RESULTS The combinant plasmid was confirmed by sequencing and the fragment belonged to WNV.Ten copies of WNV RNA were detected by SYBR GreenⅠfluorescent quantitative RT-PCR assay.Results of the other members of Flaviviridae were negative,which indicated this assay was specific for WNV.CONCLUSIONS SYBR GreenⅠfluorescent quantitative PCR assay established in this study is highly sensitive and specific,and so it can be used for early diagnosis of WNV infection.

Objective To investigate the effect of rh-resistin on lipid metabolism in HepG2 cells and to elucidate its relation to AMPK pathway. Methods We treated the HepG2 cells with 50 ng/ml rh-resistin and 0.5 mmol/L palmitic acid,used siRNA technique to inhibite α2 subunite expression of AMPK in HepG2 cells and quantitative RT-PCR to detect ACC1,ACC2,and HL mRNA expression levels of related lipid metabolism genes. The P-AMPK-Thr172 of AMPK and P-ACC-Ser79 of ACC were determined by Western blotting. The Lipid accumu-lation in cells was determined by images of Laser Scanning Confocal Microscope after Nile red staining. Results Rh-resistin decreased the AMPKα2,HL mRNA expressions and the phosphorylation level of AMPK and ACC in both basal and insulin-stimulated conditions (P < 0.05),had no influence on ACC2 mRNA expressions (P >0.05),while it increased ACC2 mRNA expressions and cytoplasmic lipid droplets in the same conditions (P <0.05). Conclusion Rh-resistin may affect lipid metabolism via AMPK pathway with increase of fatty acid synthe-sis and inhibition of triglyceride catabolism,which leading to lipid accumulation in HepG2 cells.

Objective To investigate the effect of rh-resistin on glucose metabolism in HepG2 cells and to elucidate whether the underlying mechanisms are related to AMPK pathway. Methods Cells transfected with control siRNA or AMPKα2 siRNA were cultured in 6-well plates and then treated with 50 ng/mL rh-resistin for 24 hours , while untransfected cells were treated with or without 50 ng/mL rh-resistin on the same conditions , followed by serum-starving in glucose-free DMEM for 3 ~ 5 hours in the continued absence or presence of rh-re-sistin. Then the cells were treated with or without insulin for 2 hours. AMPKα2, G6Pase, PEPCK and Glut2 mRNA expression levels were determined by quantitative RT-PCR. The phosphorylation state of AMPK was deter-mined by Western blotting. Glycogen synthesis was measured by the incorporation of D-[U-14C] glucose to glycogen. Results Rh-resistin suppressed the AMPKα2, Glut2 mRNA expressions, and reduced the phosphory-lation level of AMPK and glycogen synthesis on both basal and insulin-stimulated conditions (P < 0.05), while it accelerated G6Pase and PEPCK mRNA expressions on the same conditions (P < 0.05). The mRNA expression levels of G6Pase , PEPCK , Glut2 and the phosphorylation level of AMPK and glycogen synthesis were signifi-cantly different between the rh-resistin group and the rh-resistin in conjunction with AMPKα2 siRNA-treated group. Conclusion Rh-resistin may affect glucose metabolism in HepG2 cell via AMPK pathway.

BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation.TIME AND SETTING: The experiment was performed in the Clinical Molecular Biology Laboratory, the Second Affiliated Hospital of Sun Yet-sen University from April 2007 to January 2008.MATERIALS: QuikChange II Site-Directed Mutagenesis Kit was produced by Stratagene. Eukaryotic expression vector pcDNA3.1(+) and E. coll DH5a were provided by the laboratory. Human skeletal muscle tissue was from patients who received amputation surgery in the Second Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from the patients, and fresh samples were collected and frozen in liquid nitrogen.METHODS: The human AMPKo2 subunit gone was amplified from human skeletal muscle by RT-PCR, cloned into T vector, and the recombinant plasmid was confirmed by sequencing. In vitro site-directed mutagenesis was carded out with Quickchange site-directed mutagenesis kit. The wild-type and mutant coding genes were subcloned into eukaryotic expression vector pcDNA3.1, and the recombinant plasmids were validated by enzyme digestion and sequencing.MAIN OUTCOME MEASURES: ①The cloning of aim gone; ②site-directed mutagenesis; ③ eukaryotic expression plasmid. RESULTS: The human AMPKα2 subunit gene (about 1700 bp) was successfully cloned, with 99％ homology to the reported AMPK α2 gene. A GenBank accession number was EF056019, The achieved mutation of the 45<'th> Lysine (AAA) was found to Arginine(AGA). The wild-type and mutant pcDNA-AMPKα2 recombinant plasmids were constructed successfully. CONCLUSIONS: The human AMPKα2 subunit gene was cloned successfully from human skeletal muscle and its wild-type and mutant eukeryotic expression vectors were constructed successfully in the experiment.

OBJECTIVE To detect serotype of dengue virus in sera from patients with fever.METHODS A pair of degenerated primers and one MGB probe were designed targeting the conserved region at the E gene of dengue type 1 virus.TaqMan MGB real-time PCR assay was developed with plasmid including E gene of dengue type 1 virus as standard sample.The sera of 10 patients with fever were used to extract RNA,and convert into cDNA.Then cDNA were detected by TaqMan MGB real-time PCR assay and the amplified products were analyzed at the same time.RESULTS The sera of 9 patients from 10 samples were observed to generate a fluorescent signal,and about 100 bp fragment was obtained simultaneously.CONCLUSIONS Dengue fever on 2006 in Guangzhou is caused by the dengue type 1 virus.TaqMan MGB real-time PCR assay is rapid and sensitive to detect dengue virus infections.

Objective To observe the clinical efficacy of infrared thermal therapy against pseudomonas ae-ruginosa infection on deep partial-thickness burn wound. Methods Forty-three patients in our hospital with main-ly deep partial-thickness burn wound from January 2015 to October 2016 were randomly enrolled to the treatment group(TG,treated with sulfadiazine silver + infrared thermal therapy)and the control group(CG,treated with sulfadiazine silver only). Scores of wound exudation,positive rate of pseudomonas aeruginosa on wound,wound healing rate,wound healing time and overall evaluation of wound healing on the day of 0,3,7,14,21,28 after treatment were conventionally recorded. Adverse effects in TG and CG were also observed. Results (1)Age,sex and burn surface area of patients were found no statistically significant difference between the two groups(P>0.05). (2)On the day of 3,7 and 14,wound exudation score of TG was significantly lower than that of CG(P<0.05);On the day of0,21 and 28,wound exudation score of the two groups were almost same.(3)Positive rate of pseudomonas aeruginosa between the two groups on the day of 7,14 and 21,TG was significantly lower than CG(P < 0.05). (4)Wound healing rate of TG on the day of 7,14 and 21 was higher than CG,which was statistically significant difference in the 2 groups(P<0.05);Wound healing time of TG patients[(21.1 ± 6.5)day]was significantly shorter than that of CG patients[(26.2 ± 6.5)day](P<0.05).(5)Overall evaluation of wound healing of TG was better than that of CG on the day 14 and 21(P<0.05). Conclusions Infrared thermal therapy could reduce the secretion of deep partial-thickness burn wound and effectively control pseudomonas aeruginosa infection. Furthermore,infrared thermal therapy finally improved wound healing rate and shortenedwound healing time of burn wound.

AIM: To investigate the expression of survivin in pancreas in the streptozotocin-induced diabetic mice. METHODS: Low dose of streptozotocin was used to induce diabetes mellitus in BALB/c mice. Body weight and blood glucose concentrations were examined at 1, 2, 3 and 4 weeks after the streptozotocin injection. Expression of survivin mRNA was detected by real-time FQ-PCR. RESULTS: Survivin was expressed in the pancreas of normal BALB/c mice. Low dose of streptozotocin provoked hyperglycaemia with increased survivin expression in the pancreas, but blood glucose concentration and expression of survivin was not significantly changed in control group. CONCLUSION: Survivin is expressed in the pancreas of normal BALB/c mice. Streptozotocin increases survivin expression in the pancreas, which may be related with islets regeneration.

AIM: To study the effects of insulin on the proliferation and function of osteoblasts and the relationship between insulin post-receptor change in osteoblasts and osteoblastic cell growth.METHODS: The effects of different levels of insulin on osteoblasts were assessed by MTT colorimetry.Osteocalcin in medium was measured by RIM.IGF-1 mRNA expression levels were determined by RT-PCR.The concentrations of free IGF-1 protein in serum-free medium were measured by ELISA.In addition,the protein level and phosphorylated protein of P~(44/42)MAPK were determined by Western blotting analysis.RESULTS: Insulin enhanced the proliferation of osteoblasts,depending on its dose and exposure time.Insulin at concentration of 10~(-7) mol/L showed the strongest effect,and the action attained the plateau phase beyond 96 h.The best concentration that stimulated synthesis of osteocalcin by insulin was 10~(-7) mol/L.When the insulin concentration beyond 10~(-7) mol/L,the osteocalcin concentration was decreased.Exposure time had no effect on insulin-stimulated synthesis of osteocalcin of osteoblastic cells.When the concentration of insulin reaches 10~(-6) mol/L,the IGF-1 mRNA expression stimulated by insulin was also decreased.The concentrations of free IGF-1 protein in insulin-stimulated groups were all higher than that in control group(P0.05).Insulin acute stimulation rapidly induced the activity of tyrosine phosphorylation of P~(44/42)MAPK.The degree of tyrosine phosphorylation of P~(44/42)MAPK was increased step by step along with the increasing doses of insulin from 0 to 10~(-7) mol/L(P

Objective: To establish 3-{4-[2-hydroxyl-(1-methylethylamino) propoxy] phenyl} propionic acid cetylesters (PAC) modified nanoparticles, and preliminarily explore its cardiomyocyte-targeting function and protection effects on myocardium. Methods: (1) HL-1 myocardial cells were divided into cyanidin-3 (Cy3) marked non-targeted small interference RNA (Cy3-siNC) group and Cy3 marked small interference RNA designed for the nuclear factor kappa B (NF-κB)-p65 gene (Cy3-si435) group according to the random number table, with 3 wells in each group. Cells in Cy3-siNC group were transfected with Cy3-siNC, while cells in Cy3-si435 group were transfected with Cy3-si435. At transfection hour 24, the mRNA expression of NF-κB-p65 of cells was determined by real-time fluorescent quantitative polymerase chain reaction. (2) Multiple emulsificating solvent evaporating method was adopted to prepare PAC modified nanoparticles carried with Cy3-siNC (Cy3-siNC-PAC) and PAC modified nanoparticles carried with Cy3-si435 (Cy3-si435-PAC). The morphology of Cy3-si435-PAC nanoparticles was observed with scanning electron microscope, and the size and potential of Cy3-si435-PAC nanoparticles were detected by nanometer particle size and zeta potential analyzer. The entrapment efficiency and drug loadings of Cy3-si435-PAC nanoparticle were determined with ultraviolet spectrophotometer. The release of Cy3-si435 of Cy3-si435-PAC nanoparticles was determined by dialysis method. (3) Another batch of HL-1 cells were divided into 4 groups according to the random number table, with 9 wells in each group. Cells in negative control group were added with 5 μL phosphate buffer. Cells in 25, 50, and 100 mg/mL Cy3-si435-PAC nanoparticles groups were added with 5 μL 25, 50, and 100 mg/mL Cy3-si435-PAC nanoparticles, respectively. At transfection hour 6, 12, and 24, proliferation activity of cells in 3 wells of each group was detected by methyl thiazolyl tetrazolium method, respectively. (4) Another batch of HL-1 cells were cultured for 24 h, and then treated with 100 μL Cy3-si435-PAC nanoparticles. At transfection hour 0, 4, 8, 12, and 24, the percentage of cells uptaking Cy3-si435-PAC nanoparticles in 3 wells were detected by flow cytometry, respectively. (5) Another batch of HL-1 cells were divided into 2 groups according to the random number table, with 3 wells in each group. Cells in Cy3-siNC-PAC group were added with 100 μL Cy3-siNC-PAC nanoparticles, while cells in Cy3-si435-PAC group were added with 100 μL Cy3-si435-PAC nanoparticles. At transfection hour 24, the mRNA expression of NF-κB-p65 of cells was determined by real-time fluorescent quantitative polymerase chain reaction. (6) Six male C57BL/6J mice were divided into 2 groups according to the random number table, with 3 mice in each group. Mice in Cy3-siNC-lipopolysaccharide (LPS) group and Cy3-si435-LPS group were respectively injected with 500 μL Cy3-siNC-PAC nanoparticles and Cy3-si435-PAC nanoparticles (50 mg/mL) in the tail vein. At injection hour 24, mice in the two groups were intraperitoneally injected with 10 mg/kg LPS to induce myocardial injury. At post injury hour 24, the distribution of nanoparticles in mice was detected with small animal imager. (7) Another 9 male C57BL/6J mice were divided into 3 groups according to the random number table, with 3 mice in each group. Mice in Cy3-siNC-normal saline (NS) group and Cy3-siNC-LPS group were injected with 500 μL 50 mg/mL Cy3-siNC-PAC nanoparticles in the tail vein, while mice in Cy3-si435-LPS group were injected with 500 μL 50 mg/mL Cy3-si435-PAC nanoparticles. At injection hour 24, mice in Cy3-siNC-NS group were intraperitoneally injected with NS, while mice in Cy3-siNC-LPS group and Cy3-si435-LPS group were injected with 10 mg/kg LPS to induce myocardial injury. At post injury hour 24, pathological changes of myocardium of mice in each group were observed with HE staining. Data were processed with t test and one-way analysis of variance. Results: (1) The mRNA expression of NF-κB-p65 of cells in Cy3-si435 group was 0.183±0.004, significantly lower than 1.003±0.092 in Cy3-siNC group (t=15.46, P<0.01). (2) The form of prepared Cy3-si435-PAC nanoparticles was good, with particle size of 146.0 nm, potential of -29.2 mV, entrapment efficiency of (86.9±1.1) %, drug loadings of (25.4±0.9) %, and stable Cy3-si435 release. (3) At transfection hour 6, 12, and 24, there were no statistically significant differences in proliferation activity of cells in the 4 groups (with F values from 0.129 to 2.512, P values above 0.05). (4) At transfection hour 0, 4, 8, 12, and 24, the percentages of cells uptaking Cy3-si435-PAC nanoparticles were (0.79±0.06)%, (31.04±1.59)%, (51.64±2.67)%, (68.15±2.60)%, and (83.68±4.67)%, respectively. (5) The mRNA expression of NF-κB-p65 of cells in Cy3-si435-PAC group was 0.286±0.015, significantly lower than 1.002±0.073 in Cy3-siNC-PAC group (t=16.62, P<0.01). (6) At post injury hour 24, uniform distribution of nanoparticles could be observed in cardiomyocytes of mice in Cy3-siNC-LPS group and Cy3-si435-LPS group. (7) The structure of myocardial fibers of mice in Cy3-siNC-NS group was dense, with no inflammatory cells infiltration and uniform distribution of cytoplasm. The structure of myocardial fibers of mice in Cy3-siNC-LPS group were loose, with inflammatory cells infiltration and scattered distribution of cytoplasm. The structure of myocardial fibers of mice in Cy3-si435-LPS group was denser, with no obvious inflammatory cells infiltration and uniform distribution of cytoplasm. Conclusions Cy3-si435-PAC nanoparticles have good morphology, uniform particle size, normal potential distribution, and no cell cytotoxicity. Cy3-si435-PAC nanoparticles can be effectively uptaked by HL-1 cells and suppress NF-κB-p65 mRNA expression. They also can effectively target to mice cardiomyocytes to reduce inflammatory cells infiltration and alleviate the myocardial injury of mice induced by LPS.

As a common complication in patients with sepsis, cardiac dysfunction may significantly increase mortality of these patients, but its mechanism is still unclear. Nuclear factor-κB (NF-κB) is a pleiotropic transcription inducing factor, which involves in the regulation of multiple biological phenomena and disease status. NF-κB activation participates in cardiomyocyte apoptosis, cardiomyocyte autophagy, and release of inflammatory cytokines in patients with sepsis, indicating its important role in sepsis-induced myocardial dysfunction.