Objective To examine the expression and analyze the significance of autophagy-related gene microtubule-associated protein 1 light chain 3 (MAP1-LC3,LC3),p62 and lysosorne-associated membrane protein 2 (LAMP-2) in pancreatic tissues of mice with acute pancreatitis (AP).Methods Twenty mice were randomized into AP group and control group,and the number of mice was equal between two groups.AP group was intra-peritoneally injected by 20％ L-arginine solution (two injections of 4 g/kg body weight,every 1 h) in the dosage of 4 g/l kg twice every 1 hour to establish AP model,while control group was administered with equal volume of normal saline by intra-peritoneal injection.All the mice were euthanized at 24 hour after the last injection.Pancreatic histopathological changes were measured.In addition,the protein expressions of LC3,p62 and LAMP-2 were detected by Western blot.Results No obvious pathological changes were observed in control group.Pancreatic acinar edema,structure destruction,missing,the obvious widening of interlobular septum,small interlobular septum and acinar septum,and the necrosis of acinar cells at different degrees were observed in AP group.The pathological score for tissue edema,hemorrhage,necrosis and inflammation in AP group was 3.13 ± 0.50,2.83 ± 0.32,3.25 ± 0.46 and 3.16 ± 0.47,respectively,which was all 0 in control group.The differences were statistically significant between AP group and control group (P ＜ 0.01).In AP group,the ratio of LC3-Ⅱ/LC3-Ⅰ,p62 and LAMP-2 protein in pancreatic tissue were 1.16 ± 0.08,0.94 ± 0.04 and 0.35 ± 0.04,respectively,which were 0.24 ± 0.02,0.34 ± 0.03 and 0.95 ± 0.03 in control group.The ratio of LC3-Ⅱ/LC3-Ⅰ and p62 protein in pancreatic tissue in AP group were much higher than those in control group,while LAMP-2 in AP group was lower than that in control group,and there was statistically significant difference between two groups (all P ＜0.01).Conclusions Intraperitoneal injection of L-arginine could induce acute pancreatitis,and autophagy is impaired,which was associated with decreased LAMP-2 protein expression.

Objective To explore the alteration and effect of autophagy in pancreas tissue of rat injected by exenatide.Methods Diabetes model rats were induced by two-month high-sugar and high-fat diet and streptozotocin injection (35 mg/kg) in normal rats.50 SD male rats were divided into four groups according to the principle of complete random design,namely normal control group (n=10),normal exenatide-injected group (n=10),diabetes-model control group (n=l5) and diabetes-model exenatide-injected group (n=15).Rats in exenatide-injected groups were subcutaneously injected with exenatide respectively in 5 μg/kg dose each time,twice a day,at 8 a.m.and 6 p.m.Animal weights were weighted weekly and the dose of exenatide was adjusted according to current weight.Rats in the two control groups were injected with the corresponding amount of saline.Mter 10 weeks of treatment,all rats were killed and pancreatic tissues were disposed.Immunohistochemistry was used to measure the expression of GLP-1R in pancreatic tissues.Western blot was used to test the expressions of LC3-Ⅰ,LC3-Ⅱ and p62 in pancreatic tissues,and LC3-Ⅱ/Ⅰ ratio and p62 were compared between any two groups.All specimens were stained with hematoxylin-eosin (HE).The data were expressed as means ± standard deviation and were analyzed by unpaired Student t test using SPSS 18.0 statistics software.P value ＜0.05 was considered to be statistically significant for all tests.Results The pancreatic tissues from 13 rats (6 from the normal exenatide-injected group and 7 from the diabetes-model exenatide-injected group) appeared pathological changes such as gland structure damage,pancreatic cells atrophy and cells compartment broadening.The expressions of GLP-1R,LC3-Ⅱ and p62,and LC3B-Ⅱ/Ⅰ ratio in the two exenatide-injected groups were higher than those in the respective control group,and the differences had statistical significance (P＜0.05).Conclusions Long-term subcutaneous injection of exenatide can upregulate the expression of GLP-1R in rat pancreatic acinar cells and may induce the impaiment of autophagy flux in rat pancreatic cell.

Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; virology ; Genes, p53 ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; virology ; Loss of Heterozygosity ; Nuclear Proteins ; metabolism ; Point Mutation ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo explore the effects of hepatitis B virus X gene and p53 on hepatocellular growth.

METHODSTwo kinds of plasmids containing sense and antisense human wild p53 gene respectively were constructed. SMMU-7721 cells were transfected with HBx, sense-wtp53 antisense-wtp53 separately or cotransfected with either HBx and sense-wtp53 or HBx and antisense-wtp53. Flow cytometry was adopted to measure the apoptosis rates and the effects of HBx on cell cycle progression. The activity of p21(Waf1) promoter-luciferase construct was detected. Growth curves for SMMU-7721 stably transfected with pcDNA3 and pcDNA3HBx were analyzed.

RESULTSAfter doxorubicin administration, HBx was noticed able to initiate apoptosis of the liver cells. The apoptosis rate was 5.32% in the pcDNA3 transfected and 12.66% in the pcDNA3HBx transfected groups respectively. HBx could also abrogate p53-mediated apoptosis. The apoptosis rate in groups transfected with pcDNA3, pcDNA3wtp53 and pcDNA3HBx + pcDNA3wtp53 was 5.32%, 11.72% and 4.67% respectively. In compared with the normal group, the number of cells in transiently HBx-expressed group and HBx-transfected group decreased 4.79% and 10.25% respectively. HBx inhibited the activity of p21(Waf1) promoter-luciferase constructed (P < 0.05) and promoted cell growth. The growth rate of HBx expression cells was faster.

CONCLUSIONUnder DNA damage, HBx reduced expression of p21(Waf1) by repressing the activity of p53 protein, followed by disturbing the regulation of G(0)-G(1) cell cycle checkpoint, and promoted the growth rate of hepatoma cells.

Apoptosis ; Carcinoma, Hepatocellular ; pathology ; virology ; Cell Division ; Cell Line, Tumor ; Genes, p53 ; Hepatitis B Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; pathology ; virology ; Trans-Activators ; biosynthesis ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo evaluate the capillary isoelectric focusing (CIEF) method for the estimation of blood hemoglobin A2 (Hb A2) concentrations in routine thalassemia screening.

METHODSA total of 105 samples from healthy adults and 93 samples with positive phenotypes were collected by routine thalassemia screening. CIEF was compared with Helena spife combo electrophoresis system for Hb A2 measurement and its precision and reproducibility were tested by analyzing intra-assay or inter-assay coefficient of variations(CVs). The reliability and veracity of Hb A2 measurement by CIEF for the detection of alpha- and beta- thalassemia including Hb E were evaluated by genotyping of 93 consecutive samples for routine thalassemia screening.

RESULTSBy us e of CIEF for measurement of Hb A2 in a local healthy adult population, the range of reference value(3.59%-5.23%) was obtained. The results of CIEF showed good linearity relation to that of conventional Hb electrophoresis assay. All thalassemia carriers (43 cases of alpha-thals and 44 of beta-thals) or Hb E carriers (6 cases) presumptively identified by the present CIEF for the quantification of Hb A2, combined with routine RBC parameters for indicating microcytosis and hypochromia were confirmed to be the heterozygous or compound heterozygous defects of alpha- or beta- globin gene by molecular diagnosis, without any false positive or false negative results.

CONCLUSIONThe measurement of Hb A2 by CIEF method is rapid, precise and reproducible; it could be used in routine screening for alpha- or beta- thalassemia.

Adult ; Electrophoresis, Capillary ; methods ; Female ; Genotype ; Hemoglobin A2 ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; alpha-Thalassemia ; blood ; genetics ; beta-Thalassemia ; blood ; genetics

Objective To explore the effects of salvia miltiorrhizae (SM) injection on the apoptosis of cultured rat neuronal stem cells induced by hypoxia and the activity of Caspase-3, in order to provide the further evidence for the molecular mechanism of neuroprotection of SM injection. Methods The neuronal stem cells from neonatal rat hippocampus were cultured and divided randomly into normal control group, hypoxia group and SM treatment group. After Hoechst staining, the apoptotic morphological change and apoptosis percentage were observed under fluorescence microscope. The activities of Caspase-3 in the 3 groups were evaluated by the colorimetric assay. Results Compared with normal control group [(2.75±0.28)%, 1.16±0.07], the percentage of apoptosis and the activity of Caspase-3 were increased significantly in neuronal stem cells cultured in hypoxia [(30.12%±2.09)%,3.85±0.41, P<0.05). Application of SM injection reduced markedly the percentage of apoptosis and the activity of Caspase-3 of the neuronal stem cells cultured in hypoxia [(9.16±1.34)%, 1.50±0.09, P<0.05].Conclusion SM injection can depress the apoptosis of the rat neuronal stem cells induced by hypoxia,so as to exert the neuroprotection.

OBJECTIVETo explore the effects of hepatitis B virus X protein (HBx) on hepatoma cell growth through p14(ARF)-dependent and p14(ARF)-independent pathways.

METHODSHBx and p14(ARF) were transfected either separately or in combination into HepG2 cells containing wt-p53 but not expressing p14(ARF). The cells were divided into 4 groups, namely pcDNA3 (control), pcDNA3HBx, pcDNA3p14(ARF), and pcDNA3HBx + pcDNA3p14(ARF) groups. Flow cytometry was used to examine the apoptosis rates and cell cycle progression of HepG2 cells in different groups. The expression of p14(ARF), MDM2, p53, and p21(WAF1) proteins were investigated by detecting the activity of p21(WAF1) promoter-luciferase and using Western blotting.

RESULTSThe apoptosis rates of HepG2 cells in pcDNA3HBx and pcDNA3p14(ARF) groups were significantly higher than that in the control group (14.11%, 13.72% vs 10.66%). Compared with the control group, pcDNA3HBx and pcDNA3p14(ARF) groups also showed significantly higher cell percentages arrested at G(0)/G(1) phase (63.62%, 61.75% vs 57.42%), luciferase activity of p21 promoter (1.25-/+0.05, 1.09-/+0.06 vs 0.77-/+0.03) and expressions of p53 and p21(WAF1). The cell apoptosis rate, percentage of cells in G(0)/G(1) phase and expression level of p14(ARF) were even higher in pcDNA3HBx+pcDNA3p14(ARF) group (18.61%, 66.74%, and 3.53-/+0.43, respectively) than in either p14(ARF) or HBx group.

CONCLUSIONHBx induces p53 expression through p14(ARF)-dependent and independent pathways to activate p21(WAF1) promoter, leading to G(0)/G(1) arrest and apoptosis of HepG2 cells.

Carcinoma, Hepatocellular ; genetics ; pathology ; virology ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Humans ; Liver Neoplasms ; genetics ; pathology ; virology ; Promoter Regions, Genetic ; Trans-Activators ; genetics ; Transfection ; Tumor Suppressor Protein p14ARF ; genetics ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVESTo study the interaction of hepatitis virus B (HBV) and tumor suppressor p53.

METHODSPlasmid pCMVp53 was transfected or cotransfected with pCMVHBVa (wild type HBV) or PCMVHBVb (mutant type HBV) into the hepatoma cell line SMMC-7721 by lipofectamine. Apoptosis cells were labeled by annexin V-FITC and confirmed by flow cytometry. Reporter plasmids PG13-CAT or p21-luc were cotransfected respectively in each group to indicate transactivation activity of p53 and it's effect on p21 promoter. Western blot was performed to observe p53 expression in each group.

RESULTSThe group transfected by pCMVp53 alone exhibit higher luciferase activity and higher apoptosis rate, otherwise, p53 expression, enzyme activity of PG13-CAT or p21- luc and cell apoptosis rate were much higher in the group cotransfected by pCMVp53 and pCMVHBVa, but not in the other cotransfected group; HBV replication was enhanced in p53 cotransfected group.

CONCLUSIONp53 expression and effects could be enhanced by HBV and p53 had positive regulation effect on HBV replication.

Apoptosis ; Carcinoma, Hepatocellular ; genetics ; pathology ; virology ; Cell Line, Tumor ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; genetics ; pathology ; virology ; Luciferases ; metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins p21(ras) ; genetics ; Trans-Activators ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Virus Replication

Objective To explore whether exendin-4 inhibits AR42J cells and its mechanism.Methods AR42J cells were treated with exendin-4 under multiple concentrations(1,5,10 pmol/L) at 24,48,72,96,120 h to assess its cell viability by MTT assay and got the IC-50 and time points.Then checking whether exendin-4 could induce the AR42Jcells apoptosis by setting normal control (NC) group,exendin-4 (Ex-4) group and Ex-4+ z-VAD-fmk (apoptosis inhibitor) group,and exploring whether 3-MA which is autophagy inhibitor could inhibit the AR42J cells apoptosis induced by exendin-4 by setting NC group,Ex-4 group and Ex-4+3-MA group.Cell viability was analyzed by MTT and the cells apoptosis was detected by flow cytometry and the protein levels of caspase-3,LC3 and p62 were studied by Western blot.Results Concentration of 10 pmol/L exendin-4 and and time point 72 h were selected for the further study.z-VAD-fmk pretreatment can significantly inhibit the cell viability of exendin-4 by (81.2±3.3)％ vs.(49.4±3.0)％ (P＜0.05).Flow cytometry showed that exendin-4 could induce the AR42J cells apoptosis by (28.2± 1.4)％ vs.(3.6±0.8)％,and increased the caspase-3 level by Western blot,which both can be reversed by (79.1 ±2.3) ％ vs.(49.8±2.5)％ (P＜0.05) when the cells were treated for 72 h,as was apoptosis ratio by (14.5±2.1)％ vs.(29.2±3.2)％.Western blot showed that exendin-4 can upregulate protein levels of LC3B-Ⅱ,p62 和 caspase-3 and 3-MA,and pretreatment can inhibit the upregulation of LC3B-Ⅱ and caspase-3 but further increased the upregulation of p62 induced by exendin-4.Conclusions Exendin-4 can induce AR42J cells apoptosis and 3-MA pretreating can inhibit exendin-4 cytotoxicity through downregulating autophagy.So auto phagy inhibitor 3-MA could potentially extenuate the cytotoxicity of exendin-4 in pancreatic acinar cells.

OBJECTIVEThe purpose of this study was to investigate the clinical characteristics of lymph node metastasis in the contralateral mediastinum and scalene in patients with potentially operable nonsmall cell lung cancer (NSCLC).

METHODSCervical mediastinoscopy was performed for 89 patients with clinical stage I-III A non-small cell lung cancer prior to thoracotomy. Of these, 12 underwent cervical medistinoscopy combined with right scalene lymph node biopsy and 10 with anterior para-mediastinal small incision.

RESULTSA total of 9 patients were found have N3 disease on mediastinosopy, with cancer-cell-positive lymph nodes in the contralateral mediastinum in 6 and 3 in the right scalene. Statistical analysis revealed that the incidence of N3 disease in adenocarcinoma group was higher than that in patients with nonadenocarcinoma (P < 0.05), which was also higher in the patients with serum CEA >5 ng/ml than that in the patients with CEA <5 ng/ml (P < 0.05), and it was higher in the patients with ipsilateral mediastinal multi-station lymph node metastasis than that in the patients with uni-station lymph node metastasis (P < 0.05).

CONCLUSIONBiopsy of contralateral mediastinal lymph nodes or scalene lymph node should be performed in order to exclude N3 disease for potentially operable NSCLC patients with adenocarcinoma, serum CEA >5 ng/ml or ipsilateral multi-station mediastinal lymph node metastasis.

Adenocarcinoma ; blood ; pathology ; therapy ; Adult ; Aged ; Biopsy ; Carcinoembryonic Antigen ; blood ; Carcinoma, Non-Small-Cell Lung ; blood ; pathology ; therapy ; Carcinoma, Squamous Cell ; blood ; pathology ; therapy ; Chemotherapy, Adjuvant ; Female ; Humans ; Lung Neoplasms ; blood ; pathology ; therapy ; Lymph Node Excision ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Mediastinoscopy ; Mediastinum ; Middle Aged ; Neck Muscles ; Neoplasm Staging ; Pneumonectomy