Abdominal aortic aneurysms (AAA) is one kind of common vascular disease. Inflammation and matrix degradation in the vasculature are crucial for AAA formation, key mechanism of which include vascular smooth muscle cell(VSMC) senescence, oxidative stress, increased local production of proinflammatory cytokines, and increased activities of matrix metalloproteinase (MMPs). Cyp A, the member of cyclophilins,has a variety of physiological functions. It is highly expressed in VSMC, and plays an important role in the development of AAA, making it a new therapeutic target for such disease.

Objective To investigate the relationship between HBV polymerase gene mutations and HBV genotypes in chronic hepatitis B (CHB) patients resistant to adefovir dipivoxil (ADV).Methods Blood samples were collected from 114 ADV-resistant CHB patients during February 2010 and May 2012.The HBV polymerase regions from serum samples were amplified with real-time PCR,and the PCR products were sequenced.Normal distribution data were presented as x ± s,and non-normal distribution data were presented as M (P25-P75) ; for homogeneous data analysis of variance and LSD-t test were performed.Results There were 8 types of HBV polymerase gene mutations in 114 CHB patients; single point mutation was detected in 102 patients (89.47％) and double or triple points mutations were detected in 12 patients (10.53％).rtA181V/T/S (57.89％),rtN236T (14.91％) and rtA181V/T/S + N236T (9.65％) were the predominant mutations.For 21 patients (18.42％) with HBV genotype B,rtN236T mutation was prevalent (47.62％,10/21) ; while for those with HBV genotype C (93,81.58％),rtA181V/T/S mutation was the predominant (65.59％,61/93).The differences of rtA181V/T/S (x2 =12.269,P ＜0.01) and rtN236T (x2 =18.658,P ＜0.01) mutation rates between B and C genotypes were statistically significant.Conclusion rtA181V/T/S,rtN236T and rtA181V/T/S + rtN236T are the major HBV polymerase gene mutation types in ADV resistant CHB patients,and mutation types are related with HBV genotypes.

OBJECTIVETo explore the effect of silencing the Notch2 gene by small interfering (si)RNA on the proliferation of the HepG2 human hepatocellular carcinoma (HCC) cells.

METHODSNotch2-siRNA was transfected as a liposomal formulation into HepG2 cells. The non-HCC cell lines SG07901 (gastric cancer) and SW620 (colon cancer) were used as controls. The mRNA expression of Notch2 and Hesl were detected by RTPCR, and the protein expression of Notch2 was detected by western blotting. The proliferation of transfected HepG2 cells was assessed by the cell counting kit-8 (CCK8) colorimetric assay.

RESULTSThe untransfected HepG2 cells showed significantly upregulated transcript expression of Notch2, and not of Notch1, Notch3 or Notch4, compared to the other non-HCC cell lines. Following transfection of Noteh2-siRNA into HepG2 cells, the mRNA expression of Notch2 and Hes1 and the protein expression of Notch2 were significantly decreased. The rales of proliferation inhibition in HepG2 following transfection of Notch2-siRNA showed an increasing time-related trend, with 2.64% ± 1620% at 12 h, 38.34% ± 8.80% at 24 h, 70.05% ± 7.80% at 48 h, 70.78% ± 10.00% at 72 h, and 74.22% ± 4.80% at 96 h.The inhibition rate at 24 h of transfection was significantly different from that of the groups of control cells.

CONCLUSIONNotch2 is upregulated in the common HCC cultured cell line HepG2. siRNA-mediated silencing of Notch2 exerts inhibition effects on HepG2 proliferation, suggesting the potential for this approach as targeted therapy for treating HCC.

Carcinoma, Hepatocellular ; pathology ; Cell Proliferation ; Down-Regulation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; RNA Interference ; RNA, Small Interfering ; Receptor, Notch2 ; metabolism

Objective To comparatively analyze the immunological characteristics of patients with mild and severe influenza A (H1N1), and to provide the evidence for condition monitoring and treatment . Methods 52 cases with mild influenza A ( H1N1), 152 cases with severe influenza A ( H1N1) and 26 healthy subjects from July 1, 2009 to December 31, 2009 were enrolled in the study.Lymphocyte subsets in peripheral blood were analyzed by flow cytometry and the serum concentrations of interferon -γ( IFN-γ) and interleukin-4 (IL-4) were detected by enzyme-linked immune-sorbent assay (ELISA).Results The total lymphocyte counts were decreased obviously in patients with severe influenza A ( H1N1) than in mild pa-tients and in healthy subjects (P<0.01).The T lymphocyte, NK cells, CD4+T lymphocyte and CD8+T lym-phocyte were also decreased obviously in severe patients than in mild patients (P<0.01).The B lymphocyte and CD4+/CD8+were also decreased in severe patients than in mild patients but had no significant statistical difference (P=0.11, 0.175).The serum IFN-γlevels in patients with mild and severe influenza A (H1N1) were lower than those in control group, especially in patients with severe influenza A (H1N1) (compared with control group and mild group , P<0.01).And the changes of serum IL-4 levels were the same with the former, but there were no statistically significant differences in three groups (P>0.05).Con-clusion Immune dysfunction in patients with influenza A (H1N1) infection is associated with the severity of disease, especially cellular immunity .Therefore, monitoring of the immune system is valuable for the diag-nosis of influenza A(H1N1) infection.

Objective To study the feasibility, safety and clinical effects of spleen and splenic vessel-preserving distal pancreatectomy. Methods A retrospective study was performed in 26 patients undergoing distal pancreatectomy for benign or low grade malignant disease with splenectomy (n = 13) or splenic preservation (n = 13 ) at the First Hospital of Sun Yat-sen University and Guangdong General Hospital from May 2002 to April 2009. Results All 26 pancreatectomy with splenectomy or splenic preservation were performed successfully. There was no statistically significant difference between two groups in average operative time[(172±47) min vs. (157±52) min, P ＞ 0.05 ], intraoperative estimated blood loss [( 183 ± 68 ) ml vs. ( 160 ± 51 ) ml, P ＞ 0.05 ], incidence of noninfectious and infection complication and postoperative hospital stay [(10.1±2.2) d vs. ( 12. 1 ± 4. 6 ) d, P ＞ 0.05 ]. The platelet counts examined one week after operation were significantly higher in the distal pancreatectomy with splenectomy group than that in spleen-preserving group [(37.3 ± 12.8)×109/L vs. (54.7 ± 13.2) × 109/L, P＜0.05 ]. Conclusions Spleen-preserving distal pancreatectomy appears to be a feasible and safe procedure in selected cases of benign or low-grade pancreatic malignant disease necessitating a distal pancreatectomy.

Objective To analyze the prevalence status and the genetic characterizations of influenza B viruses isolated in Hunan Province after pandemic influenza A (H1N1) 2009,and to explore possible reasons for the prevalence.MethodsThroat swabs were collected from outpatients with influenza-like illness in 23 sentinel hospitals of Hunan Province in 2010.Influenza viruses were isolated with Madin-Darby canine kidney (MDCK) cells and identified by haemagglutination inhibition test.The genomes of 10 selected influenza B viruses were sequenced and analyzed for phylogenetic and molecular characterization.ResultsWith the reduction of isolation of pandemic influenza A (H1N1)2009 viruses,influenza B virus became the predominant isolated strain in the first half of 2010.Epidemic viruses mainly belonged to the B/Victoria lineage,and both two lineages co-circulated.Seven out of 11 influenza outbreaks caused by type B.Ten strains were filled into 2 branches of BV and BY which were classified by their lineage types in polymerase (PB2,PB1,PA),hemagglutinin (HA),neuraminidase (NA),NB,membrane protein (M1),influenza B virus membrane protein M2 (BM2),and non-structural protein (NS1,NS2) phylogenetic trees except the NP phylogenetic tree in which 10 strains were all in the BY branch.Compared with World Health Organization (WHO) vaccine strains,the amino acid identity of 11 proteins of the 10 strains was high (97.2％-100.0％).However,some amino acid point mutations were found.No mutation was found in drug resistance mutation sites.Some mutations in NA,NB,PB1,PB2 and NS2 molecules were found in 2 strains isolated from outbreaks compared with strains from sentinel surveillance.Conclusions The point mutations,insertions and genetic reassortment indicate viruses sustaining evolution,which is probably the reason for predominant influenza B viruses after pandemic influenza A (H1N1) 2009 in Hunan Province.

OBJECTIVETo observe the effect of normobaric hyperoxia exposure on the functions of N9 microglia and explore the underlying mechanism of hyperoxia-induced immature brain injury.

METHODSN9 microglial cells were exposed to 900 ml/L O(2) for 2, 6, 12, 24 or 48 h, and the cell apoptotic rate was assessed using flow cytometry. The intracellular oxidative stress was measured using a fluorescent DCFH-DA probe, and the expression of Toll-like receptor 4 (TLR4) mRNA was detected using RT-PCR. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations in the supernatant of the cell cultures were tested with ELISA following the exposures. TLR4 protein expression was observed using immunofluorescence staining.

RESULTSSignificant cell apoptosis was detected after oxygen exposures for 12-24 h. Accumulation of reactive oxygen species (ROS) were detected after a 2-h exposure. After prolonged hyperoxia exposure, TLR4 expression and IL-1β and TNF-α levels significantly increased in the cells.

CONCLUSIONHyperoxia exposure activates TLR4 signaling pathway in N9 microglial cells in vitro, leading to massive production of ROS, IL-1β, and TNF-α and thus triggering cell apoptosis.

Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cells, Cultured ; Interleukin-1beta ; metabolism ; Mice ; Mice, Inbred ICR ; Microglia ; cytology ; drug effects ; physiology ; Oxygen ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism

Objective To analyze the conding region of hantanvirus S gene and predict the structure of nucleoprotein for diagnostic antigen study.Methods RT-PCR was used to amplify the S gene of hantanvirus Hunan03 strain after designing specific primers.The amplification product was cloned into pGM-T vector and then the recombinant vector was transformed into E.coli TOP10,gene sequencing was carried out after blue-white selection and PCR screening for positive clones.The database of NCBI and Swiss-Prot/TrEMBL were used to predict and analyze the structure,biological characteristics and protein structures of S gene.Results The amplification product was about 1290 bp,the pGM-T/S vector was constructed and successfully sequenced,the whole length of the open reading frame (ORF) was composed of 1290 nucleotide residues,among them the GC content was 44.11％ and the AT content was 55.89％,it was composed of 429 amino acids (20 kinds),the accession number of the sequence submitted to GenBank was JN712306,its homology of nucleotides to the 76-118 strain was 83％ and the homology of amino acids was 98％,ten nonspecific variation sites were found.The grand average of hydropathicity was-0.405.There were three transmembrane domains and four non transmembrane domains in the secondary structure of nucleoprotein including 55％ of helix structure,6.1％ of sheet structure and 38.9％ of loop structure.Conclusion The bioinformatics analysis of Hunan03 strain S gene might be important for provide the substructure data to reveal the significance of S gene characteristics on hemorrhagic fever renal syndrome (HFRS) prevention and control.

Objective To improve the diagnostic and therapeutic ability of Brucellosis by analyzing the epidemic and clinical characteristics.Methods A retrospective analysis was done on the data of Brucellosis patients treated in our hospital from 2007 to 2016.Results Since the first case was diagnosed in 2012,27 patients [19 male and 8 female,mean age (44.4 ± 16.9) years] were confirmed by clinical manifestations and positive bacterial cultures results.The annual number of cases from 2012 to 2016 was 1,1,6,4 and 15.Among them,10 cases (37.0％) had a history of close contact with goat,7 cases (25.9％)with raw mutton,1 case (3.7％) with raw beef and 1 case (3.7％) with suspicious laboratory contamination while 8 cases (29.6％) had no evident risk factors for Brucellosis.The common clinical manifestation included fever (81.5％),lumbago/joint pain (55.6％),fatigue (33.3％) and hyperhidrosis (22.2％).The white blood cell count was normal among 20 cases (74.1％) while 6 cases (22.2％) with leukopenia.Mild to moderate anemia in 20 cases (74.1％) and decreased platelet number in 4 case (14.8％).The percentage of elevated alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase was 32.0％,48.0％ and 100％,respectively.Decreased albumin level was found in 23 cases (92.0％).The percentage of elevated erythrocyte sedimentation rate (ESR),C-reactive protein and serum ferritin 75.0％,82.3％ and 77.8％,respectively,while 12 cases (85.7％) with procalcitonin level below 0.5 ng/L.According to follow-up for at least half year,all the cases were cured by active medical management.Conclusions The number of Brucellosis cases is rapidly increasing in our hospital.It's of great significance to know the epidemic and clinical characteristics of Brucellosis.

OBJECTIVETo investigate the effect of co-expression of bone morphogenetic protein 2 (BMP2) and Sox9 on chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro and provide experimental evidence for tissue engineering of cartilage.

METHODSMouse embryonic bone marrow MSC C3H10T1/2 cells were infected with recombinant adenovirus expressing BMP2, Sox9 and green fluorescent protein (GFP) for 3-14 days, with cells infected with the adenovirus carrying GFP gene as the control. The mRNA expression of the markers of chondrogenic differentiation, including collagen type II (Col2a1), aggrecan (ACAN), and collagen type X (Col10a1), were determined by real-time PCR. Alcian blue staining was used for quantitative analysis of sulfated glycosaminoglycan in the cellular matrix. The expression of Col2a1 protein was assayed by immunohistochemical staining and Western blot analysis.

RESULTSAdenovirus-mediated BMP2 expression induced chondrogenic differentiation of C3H10T1/2 cells. Overexpression of Sox9 effectively enhanced BMP2-induced expression of the chondrogenic markers Col2a1, aggrecan and Col10a1 mRNAs, and promoted the synthesis of sulfated glycosaminoglycan and Col2a1 protein in C3H10T1/2 cells.

CONCLUSIONCo-expression of BMP2 and Sox9 can promote chondrogenic differentiation of MSCs in vitro, which provides a new strategy for tissue engineering of cartilage.

Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Cartilage ; cytology ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; SOX9 Transcription Factor ; genetics ; metabolism ; Tissue Engineering