Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.

Objective To investigate the difference between the blood pressure readings between sitting and supine position,and to study the factors that associated with the sitting-supine blood pressure difference in patient with diabetes.Methods We measured the sitting blood pressure first then followed by the supine pressure in 356 diabetic patients,using a standard mercury sphygmomanometer.Patient's body weight,height and blood glucose levels were also measured.Results SBP and DBP were significantly higher in the supine position than in sitting position in diabetic patients(by 3.5?7.6/1.5?4.9 mm Hg,P

The gene encoding the phosphatidylserine synthase in Escherichia coli K12 Sgal-(ExPASy P23830) was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive shuttle vector pBES of Bacillus subtilis, which was constructed in the lab, and the recombinant plasmid pBES-pss was transformed into competent cells of the Bacillus subtilis strain DB104. The positive transformant DB104 (pBES-pss) was grown on Bacillus subtilis common fermentation medium, which contained 30?g/ml kanamycin. After 2 hours cultivation, sucrose was added and increased to the final concentration of 2% for induction and this phosphatidylserine synthase was secreted into the medium. The result of SDS-PAGE showed that the molecular weight of the protein was 52kDa and the result of enzyme coupling colorimetric method showed that the enzyme activity was 1.50U/ml. The recombinant Bacillus subtilis has increased the yield of phosphatidylserine synthase which will be used for industrial biosynthesis of phosphatidylserine.

Objective To explore the application and effect of case-based learning(CBL)in vas-cular surgery clinical teaching. Methods Totally 37 resident doctors were randomly divided into 2 groups respectively: CBL teaching group (n=21)and traditional teaching group (n=16). CBL teaching was con-ducted through the following procedures:selecting typical cases-establishing and applying typical case library-autonomous learning-holding regular seminars. Traditional teaching was conducted through the following procedures: basic theory studying-participating in clinical practice-participating in case discus-sion. Evaluation was conducted based on test socre (written test and clinical operational skill test)and res-idents' feedback of teaching effect. Data were statistically described and independent sample t test was performed. Results Theoretical exam score and clinical skill test score were high in CBL group than in traditional group ((thoretical score:(85.53 ±1.75) vs. (79.94 ±2.29);clinical skill test score:(85.10±1.64)vs.(80.31±1.82)). CBL teaching group had advantages in improving learning efficiency, cultivat-ing clinical thinking,promoting mastery and application of knowledge,broadening knowledge, promoting communication and expression ability and improving study enthusiasm ,et al . Conclusion CBL teaching can effectively improve the teaching quality and obtain higher evaluation. Typical case li-brary should be constantly improved and education of vascular surgical basic theory should be strength-ened to promote CBL.

Objective To investigate the efficacy of intratympanic methylprednisolone perfusion (IMP) for the treatment of sudden sensorineural hearing loss (SSNHL ) which failed to be fully responsive to conventional treatment .Methods The hearing outcomes of 87 patients with unilateral SSNHL were retrospectively analyzed .All of the patients received IMP after not fully responsive to conventional treatment of varying periods of time .They were hospitalized in our department between January 2008 and December 2012 and were followed up for at least one year to exclude recurrent hearing loss .Results The effective rate was 66 .67% and the mean PTA improvement was (18 .53 ± 13 .54)dB of the patients with the time interval between onset of symptoms and IMP within 15 days , 21 .21% and (5 .92 ± 15 .18)dB of the patients with the interval between 16 and 30 days ,4 .76% and (3 .69 ± 7 .00) dB of the patients with the interval more than 30 days respectively .The significant difference in the effective rates were compared among the three groups(χ2 =25 .91 ,P<0 .01) .Regarding to the PTA improvement ,the group with interval within 15 days was better than the other two groups(F=11 .182 ,P<0 .01) .A total of 30 cases acquired more than 15 dB hearing gain after IMP .One of them was hearing loss in low frequencies and the other 29 cases were hearing loss at all frequencies .The mean hearing gains of the 29 cases in 0 .25 kHz ,0 .5 kHz ,1 kHz ,2 kHz , 4 kHz and 8 kHz were 35 .17 ± 18 .15 dB ,35 .38 ± 15 .90 dB ,31 .28 ± 19 .74 dB ,21 .31 ± 17 .34 dB ,14 .97 ± 16 .00 dB and 13 .80 ± 16 .35 dB ,respectively .The mean hearing gains at lower three frequencies (0 .25~1 kHz)were better than those at higher three frequencies (2 k~8 kHz)(F=9 .494 ,P<0 .01) .Conclusion Receiving IMP earlier might help to acquire better hearing gain for the patients with SSNHL after not fully responsive to conventional treat‐ments .The hearing gains at the lower frequencies were better than those at the higher frequencies after IMP .

ObjectiveTo clarify the influence of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 V4 region with mutations at amino acid locuses on its abilities to enter target cells.Methods Based on the facts that ADA strains was a CCR5-tropic strain,only had the ability to infect CCR5 cells; that HXB2 strains was a CXCR4-tropic strain,only had the ability to infect CXCR4 cells,serial glycoprotein 120 mutants with alanine substitution in V4 region of ADA and HXB2 strains,were constructed by overlaping PCR.Eukaryotic expression vectors of mutants and expression vectors of HIV framework gene with luciferase reporter gene were cotransfected into eukaryotic cells to produce pseudoviruse.Concentration of HIV-1 gag P24 in pseudoviruses was detected by enzyme-linked immunosorbent assay(ELISA).U87.CD4.CCR5 and U87.CD4.CXCR4 cells were infected with 20 and 40 ng pseudoviruses,with wild ADA and HXB2 strains as control groups,respectively.The ability to infect cells of pseudovirus of each mutant with HIV-1 V4- region mutated at amine acid locuses 386-417 was measured by detecting the luciferase activity (relative light unit,RLU).ResultsTen mutants with alanine substitution in V4 region of HIV-1 ADA and HXB2 strains were successfully constructed,respectively.Mutants of pseudoviruse with 20 ng and 40 ng at locuses 389-391 and 414-417 with alanine substitution of V4 region in both ADA and HXB2 strains lost completely the abilities to enter CCR5 and CXCR4 expressing cells[ (0 ± 0)％].It was found that introduction of alanine to ADAs 400-403 and ADAs 408-410 increased the ability to infect cells to (124 ± 35)％,(182 ± 29)％ and (127 ± 8)％,( 134 ± 16)％ with pseudoviruse of 20 ng and 40 ng,respectively.Likewise,the ability to infect CXCR4 expressing cells also increased to (144 ± 42 )％ and (121 ± 18 )％ with pseudoviruse of 20 ng and 40 ng,respectively by introduction of alanine to HXB2s 395-397.However,other mutants in V4 region of ADA and HXB2 only maintained partial entry abilities( 15％- 84％).ConclusionsMutants of V4 region of HIV-1 envelope glycoprotein 120 with alanine substitution at locuses 389-391 and 414-417 in both ADA and HXB2 strains have been constructed successfully.They completely lost the ability to enter target cells.

OBJECTIVEIn this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions.

METHODSNeutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot.

RESULTSIn co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05).

CONCLUSIONSCoculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Animals ; Base Sequence ; Bronchi ; cytology ; enzymology ; metabolism ; Cattle ; Cell Adhesion Molecules ; metabolism ; Cell Line ; Coculture Techniques ; DNA Primers ; Down-Regulation ; drug effects ; Epithelial Cells ; enzymology ; metabolism ; Isoflavones ; pharmacology ; NF-kappa B ; metabolism ; Neutrophils ; enzymology ; metabolism ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the cytotoxicity to and inflammation in an immortal human keratinocyte cell line HaCaT during cobalt chloride (CoCl2-induced chemical hypoxia. Methods HaCaT cells were treated with CoCl2 of 2 mmol/L to set up a chemical hypoxia-induced cell model of injury. Then, RNA interference was used to down-regulate the expression of P65 in CoCl2-induced HaCaT cells. After additional culture, cell viability was tested by cell counting kit8 (CCK-8), the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) were detected by ELISA kits, phosphorylated and total P65 protein was measured by Western blot. Results The exposure of HaCaT cells to 2 mmol/L CoCl2 for 0 to 4 hours enhanced the phosphorylation of P65, which began at 0.5 hour, peaked at 1.5 hours, and restored to the normal level at 4 hours, and the level of P65 phosphorylation was about 6.6 times that in the untreated control group. The CoCl2 of 2 mmol/L decreased the cell viability of HaCaT cells in a time dependent manner, and a significant difference was observed in the viability of HaCaT cells between CoCl2-treated and untreated HaCaT cells at 2, 4, and 6 hours (P ＜ 0.05, 0.01, 0.01 ). The release of IL-6 and IL-8 from HaCaT cells was also promoted by CoCl2 treatment. The knockdown of P65 expression with siRNA markedly suppressed the CoCl2-induced cytotoxicity to and increase in the release of IL-6 and IL-8 from HaCaT cells,despite of an increment in cell viability by about 11%. Conclusion The phosphorylated P65 subunit mediates CoCl2-induced cytotoxicity and inflammatory injury to HaCaT cells.

Aged ; Cell Dedifferentiation ; Chondrosarcoma ; pathology ; Humans ; Ribs ; pathology

Objective: To study the dynamic changes of type Th22 cell immunological response during atherosclerosis process in experimental mice in order to provide a new theoretical basis for atherosclerosis therapy. Methods: 8 weeks C57BL/6J mice were divided into 2 groups: Experiment group,n=24 ApoE-/- mice and Control group, n=24 normal mice. All animals received high fat diet and the following indexes were compared between 2 groups at 0, 4, 8, 12 weeks after treatment: aortic atherosclerotic lesions were deifned by Oil red O staining, dynamic changes of Th22 cells in spleen were measured by lfow cytometry, mRNA expressions of interleukin-22 (IL-22), IL-22R1, AhR and T-bet in aorta were examined by RT-PCR, blood levels of IL-22 was detected by ELISA. Results: Compared with Control group, Experiment group had the increased area of aortic atherosclerosis (the ratio of plaque area/lumen area) and Th22 cell (CD4+ IL-22+/CD4+T cell) amount, elevated mRNA expressions of IL-22, IL-22R1, AHR, T-bet in aorta and higher blood levels of IL-22 at all time points, the differences between each time point (except 0 week) had the statistic meaning,P<0.05. In Experiment group, the differences between 2 adjacent time points, for the area of aortic atherosclerosis and mRNA expressions of AHR, T-bet: 4 weeks vs 0 week, 8 weeks vs 4 weeks, 12 weeks vs 8 weeks all had statistic meaning; for Th22 cell amount: 4 weeks vs 0 week, 8 weeks vs 4 weeks had statistic meaning and 12 weeks vs 8 weeks had no real distinction; for mRNA expressions of IL-22, IL-22R1 and blood levels of IL-22: 4 weeks vs 0 week had statistic meaning and 8 weeks vs 4 weeks, 12 weeks vs 8 weeks had no real distinctions. Conclusion: Hyperactive immunological response of Th22 cells might be involved in atherosclerosis process, the relevant mechanism should be further studied.