Objective To investigate the effects of application of pseudoreplica technique in electron microscope observation of A?.Methods With the modification of classical pseudoreplica technique,ADDLs were spotted and concentrated on the gel before negative staining.Results The structures of 5nm to10nm spherical granules and 20 to 30nm coils were visualized by transmission electron microscopy(TEM).Conclusion Pseudoreplica is rapid and effective in ADDLs condensation,negative staining especially in further ultra miero structure observation and research.

Objective To express and characterize the virus-like particles( VLPs) of H5 subtype containing of hemagglutinin ( HA ) and matrix 1 ( M1 ) protein by using Baculovirus-insect cells .Methods Full length genes encoding HA protein from the A/Indonesia/05/2005(H5N1) strain and the M1 protein from the A/Anhui/01/2005 ( H5N1 ) strain were cloned into a baculovirus expression vector to construct pFBD-M1-HA.The expression of HA and M1 proteins were detected by Western blot and indirect immunoflu-orescence after the transfection of Spodoptra frugiperda (Sf9) insect cells with recombinant baculovirus.Pu-rified VLPs were analyzed by SDS-PAGE and visualized with transmission electron microscope.The biologi-cal activity of purified VLPs was detected by hemagglutination test.Results The HA and M1 proteins of H5 subtype expressed by baculovirus-insect cells could be self-assembled into the functional mature VLPs.The hemagglutination titer of VLPs was as high as 1024 HAU/50μl.Conclusion The H5 subtype VLPs as pre-pared in this study would pave a way for the development of a candidate recombinant A ( H5) vaccine.

Objective To investigate the relationship between the sensitivity of gastric cancer cells to arsenic trioxide（As2O3）and the level of reactive oxygen species（ROS）production.Methods The viability of gastric cancer cell lines MGC803,BGC823 and SGC7901 treated with As2O3 was determined by MTT assay.ROS levels of the gastric cancer cells before and after the treatment of As2O3 were detected by flow cytometry.Results Cell growth was significantly inhibited by As2O3 in time-and dose-dependent manner in three gastric cancer cell lines.The IC50（72 h）of As2O3 for MGC803,BGC823 and SGC7901 was about 2.8,3.1 and 10.2 μmol/L,respectively.IC50（72 h）of MGC803 and BGC823 was lower than that of SGC7901（P 0.01）,gastric cancer cell line SGC7901 was less sensitive than the others.The inherent ROS level of MGC803,BGC823 and SGC7901 was 20.3±2.0,64.2±3.3 and 57.7±2.0.After treatment with As2O3 5 μmol/L for 24 h,the peak level of ROS in MGC803 and BGC823 cells increased to 100.8±3.8 and 103.5±2.3,compared with inherent ROS level,the difference had statistical significance（P 0.001,P 0.01）,but the inherent ROS level in SGC7901 cells was 56.5±2.4（P 0.05）.Conclusion The sensitivity of gastric cancer cells to arsenic trioxide is associated with the increase of reactive oxygen species level.

Objective To explore the effect of airway humidification on lung injury as a result of mechanical ventilation with different tidal volume(VT). Methods Twenty-four male Japanese white rabbits were randomly divided into four groups:low VT with airway humidification group,high VT with airway humidification group,low VT and high VT group without humidification,with 6 rabbits in each group. Mechanical ventilation was started after intubation and lasted for 6 hours. Low VT denoted 8 mL/kg,while high VT was 16 mL/kg,fraction of inspired oxygen (FiO2)denoted 0.40,positive end-expiratory pressure(PEEP)was 0. Temperature at Y piece of circuit in airway humidification groups was monitored and controlled at 40℃. Arterial blood gas analysis,including pH value,arterial partial pressure of oxygen(PaO2),arterial partial pressure of carbon dioxide(PaCO2),lung mechanics indexes, including peak airway pressure(Ppeak)and airway resistance(Raw),and lung compliance was measured at 0,2,4, 6 hours of mechanical ventilation. The levels of tumor necrosis factor-α(TNF-α)and interleukin-8(IL-8)in plasma and bronchoalveolar lavage fluid(BALF)were determined by enzyme linked immunosorbent assay(ELISA). The animals were sacrificed at the end of mechanical ventilation. The wet to dry(W/D)ratio of lung tissues was calculated. Histopathologic changes in the lung tissueies were observed with microscope,and lung injury score was calculated. Scanning and transmission electron microscopies were used to examine the integrity of the airway cilia and the tracheal epithelium. Results Compared with low VT group,pH value in high VT group was significantly increased,PaCO2 was significantly lowered,and no difference in PaO2 was found. Ppeak,Raw,and lung compliance were significantly increased during mechanical ventilation. There were no significant differences in blood gas analysis and lung mechanics indexes between low VT with airway humidification group and low VT group. Compared with high VT group,PaCO2 in high VT with airway humidification group was significantly decreased,Ppeak raised obviously,and no difference in pH value,PaO2,Raw and pulmonary compliance was found. Compared with low VT with airway humidification group,no difference in blood gas analysis(PaCO2,mmHg,1 mmHg=0.133 kPa)was found,but Ppeak(cmH2O,1 cmH2O=0.098 kPa),Raw(cmH2O),and lung compliance(mL/cmH2O)were increased significantly in high VT with airway humidification group(PaCO2 at 2 hours:27.96±4.64 vs. 36.08±2.11,4 hours:28.62±2.93 vs. 34.55±5.50, 6 hours:29.33±2.14 vs. 35.01±5.53;Ppeak at 0 hour:14.34±1.97 vs. 8.84±1.32,2 hours:17.33±0.52 vs. 11.17±2.14,4 hours:17.83±0.98 vs. 12.67±2.06,6 hours:18.67±1.22 vs. 13.50±2.16;Raw at 0 hour:37.36±5.14 vs. 27.05±2.93,2 hours:43.94±6.58 vs. 31.95±3.56,4 hours:48.04±6.07 vs. 35.24±3.50, 6 hours:50.33±6.34 vs. 36.66±3.64;pulmonary compliance at 6 hours:2.28±0.18 vs. 1.86±0.37,all P<0.05). The lung W/D ratio in high VT group was significantly higher than that of the low VT group(6.17±2.14 vs. 3.50±1.52, P<0.05). W/D in high VT with airway humidification group was higher than that of low VT with airway humidification group but without statistically significant difference(5.17±2.14 vs. 3.00±1.10,P>0.05). Microscopic observation showed that cilia were partially detached,adhered and sparse in low VT group,while cilia in high VT group showed serious detachment and lodging. Remaining cilia were sparse,with lodging,and cellular structure was damaged. Lung tissue pathological injury score in the high VT group was significantly higher than that of low VT group(6.17±2.14 vs. 3.50±1.52,P<0.05). Cilia density and cellularity were normal in low VT with airway humidification group,and no difference in lung tissue pathological injury score was found compared with low VT group(3.00±1.10 vs. 3.50±1.52, P>0.05). Cilia were severely detached,adhered and lodging,and cellularity were not obvious in high VT with airway humidification group,and lung tissue pathological injury score was elevated significantly than that of the low VT with airway humidification group but without statistically significant difference(5.17±2.14 vs. 3.00±1.10,P>0.05). TNF-α and IL-8 concentrations showed no change in plasma and BALF in all groups during ventilation,and no significant difference was found among the groups. Conclusions Airway humidification can alleviate pathological lung injury,damage of cilia and cellular structure in trachea caused by mechanical ventilation with low and high VT. High VT with humidification can result in serious pulmonary edema.

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.
Animals ; Antibodies, Viral ; blood ; Baculoviridae ; genetics ; metabolism ; Capsid Proteins ; biosynthesis ; genetics ; Cell Line ; Cloning, Molecular ; Genetic Vectors ; genetics ; Parvovirus B19, Human ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Virion ; genetics ; metabolism