AIM: To develop a real - time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac - to - Bac system. METHODS: The recombinant baculovirus containing human IL- 18 gene was produced using Bac -to- Bac system. A 10 -fold serially diluted primary viral stock was used for plaque assay and DNA extraction. Bacmid (baculovirus plasmid) was 10 -fold serially diluted and served as standards. Real - time PCR amplification of the IL - 18 gene was performed in triplicate for each diluted recombinant virus. At the same time, plaque assays were performed using overlay agarose method. RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve. We also find that the"vg/mL"titer value is generally about 10 times than"pfu/mL"titer of the same recombinant virus stock. CONCLUSION: A TaqMan real -time PCR method is established to identify the recombinant baculovirus and determine the"vg/mL"titer of virus. The method is rapid and quantitative over a wide range of virus titers.

Objective To investigate the potential roles of pattern recognition receptor in the pathogenesis of asthma,the concentrations of serum nucleotide -binding oligomerization domain like receptors 1 (NOD1 )and Toll like receptor 1(TLR1)and Dectin and sCD14 protein were determined.Methods Blood samples were obtained from 45 cases of severe asthma(asthma group)during acute attack and convalescent period,the concentrations of serum NOD1,TLR1,Dectin and sCD14 protein were assessed using enzyme linked immunosorbent assay(ELISA),and compared with 30 healthy children(control group).Results The concentrations of NOD1,TLR1,Dectin and sCD14 protein were significantly higher in acute attack period of the asthma group[(65.53 ±19.95)ng/mL,(10.46 ±3.35)ng/mL, (80.38 ±19.51)ng/mL,(4.51 ±1.29)ng/mL]than those in the control group[(25.57 ±9.64)ng/mL,(5.54 ± 1.49)ng/mL,(47.37 ±15.16)ng/mL,(2.21 ±0.68)ng/mL](t =11.57,8.63,7.82,9.95,all P <0.01).Moreo-ver,the concentrations of serum NOD1,Dectin and sCD14 protein were dramatically decreased in convalescent period of the asthma group[(39.67 ±11.25)ng/mL,(60.04 ±16.76)ng/mL,(3.38 ±0.97)ng/mL]compared with acute asthma attack period(t =8.19,6.32,5.42,all P <0.01),while the concentration of serum TLR1 was not different between this two period (9.51 ±3.19)ng/mL(t =1.27,P >0.05).Furthermore,level of NOD1 was positively corre-lated with sCD14(r =0.49,P <0.01).Conclusion Levels of serum NOD1,TLR1,Dectin and sCD14 protein were increased in acute severe asthma,whereas,they were decreased in convalescent period.The elevation of them indicated that pattern recognition reception has synergistic function in the pathogenesis of asthma attack.

Objective To investigate the potential roles of nucleotide -binding oligomerization domain (NOD)-like receptor in the pathogenesis of asthma.Methods Through rat asthma model,24 rats were randomly divided into three groups on average,named asthma group,control group and dexamethasone group.Expression levels of NOD1 and NOD2 mRNA were detected by Real -time PCR in lung tissues.Results The expression levels of NOD1 mRNA in the asthma group,control group and dexamethasone group were (0.62 ±0.34)RQ value,(1 .00 ± 0.00)RQ value,(0.65 ±0.33 )RQ value respectively.The levels of NOD1 mRNA in the asthma group was significantly lower than that in the control group(F =4.75,P <0.05 ),while there was no statistically significant difference of NOD1 mRNA level between the dexamethasone group and asthma group(P >0.05).Moreover,expression levels of NOD2 mRNA in the asthma group,control group and dexamethasone group were (0.92 ±0.32)RQ value, (1 .00 ±0.00)RQ value,(1 .50 ±0.56)RQ value,respectively.There was no statistically significant difference of NOD2 mRNA level between the asthma group and control group (P >0.05 ),but level of NOD2 mRNA in the dexamethasone group was significantly higher than that in the asthma group(F =5.64,P <0.01 ).And there was no significant correlation between level of NOD1 and NOD2 mRNA(r =0.1 5,P >0.05).Conclusion Expression of NOD -like receptor subtype was not at the same level,and their reaction to dexamethasone were different either.

Objective To observe the expressions of grouth-related oncogen (GRO)α, epithelial neutrophil activating protein-78 (ENA-78) and neutrophil-activating peptide-2 (NAP-2) of rat asthma. And to investigate the role of neutrophil in the pathogenesis of asthma exacerbation. Methods In this experi-ment, the rat model of asthma were randomly divided into two groups on average, including asthma group and control group. Levels of ENA-78 at blood neutrophil were detected by flow cytometry method. The ex-pressions of GROα protein at bronchial wall and NAP-2 protein at blood neutrophil were detected by immuno-histochemieal method. Results Levels of GROα, ENA-78 and NAP-2 proteins in asthma group [0.138 ±0.009(A value), 97.65±13.99(MFI), 0.198±0.016(A value), respectively]were significantly higher than those in control group[0.077±0.010(A value), 50.79±8.66(MFI), 0.079±0.015(A value), re-spectively], all P < 0.01. Conclusion Levels of GROα, ENA-78 and NAP-2 were increased at rat asth-ma. They may be participate in inflammation of asthma exacerbation. Neutrophil may promote inflammatory cells influxing into airway wall via increasing synthesis of CXC chemotactic factors.

AIM: To observe the changes of polymorphonuclear leukocytes (PMN), neutrophil elastase (NE) and myeloperoxidase (MPO) expression in rat asthma. METHODS: Eighteen rats were randomly divided into two groups on average, including asthma group and control group. The rat model of asthma was established by the ovalbumin (OVA) challenge methods. Blood PMN were isolated and purified. The protein expression of MPO were detected by immunohistochemistry and chromatometry techniques. NE was detected by ELISA. RESULTS: (1) Immunohistochemistry showed that the levels of MPO expression around bronchus and in purified PMN in asthma group were significantly increased compared with those in control group (P

Objective To investigate possible role of 14 -3 -3 in the pathogenesis of asthma inflammation, the expression of 14 -3 -3 protein was observed in lung tissues of asthmatic rats.Methods Expressions of 14 -3 -3 protein was determined by immunohistochemisty method in lung tissues,and the relationship between 14 -3 -3 and asthma inflammation was analyzed.Results The location of positive expression of 14 -3 -3 protein was mainly at cytoplasm,while little at plasmalemma.The positive expression cell mostly was bronchial epithelial cell,others were lymphocytes,alveolar macrophages and vascular endothelial cells.On the other hand,the bronchial smooth muscle and vascular smooth muscle were negative expressed.Moreover,the expression level of 14 -3 -3 protein in asthma group [(0.353 ±0.023)absorbance]was significantly higher than that in the control group[(0.211 ±0.028 )absorbance] (t =10.969,P <0.01).Conclusion The results showed that the 14 -3 -3 protein was overexpressed in asthmatic lung tissue,it may play an important role in asthma inflammation through bronchial epithelial cells,lymphocytes, alveolar macrophages and vascular endothelial cell.

Objective To investigate the effects of dexamethasone on expressions of epithelial neutrophil activating protein-78 (ENA-78) and transforming growth factor- beta 1 (TGF-β1) in neutrophil of asthma rats.Methods Thirty male SD rats were randomly divided into three groups on average, including asthma group, control group and dexamethasone treated group. In this experiment, the rat model of asthma were established by sentization and challenge with ovalbumin. Blood neutrophil were isolated and purified. The expression of ENA-78 was detected by flow cytometry. The expression of TGF-β1 was detected by immunohistochemical method in blood neutrophil and bronchial wall. Results Expression of ENA-78 in blood neutrophil in dexamethasone treated group(71.82 ±8. 87 mean fluorescence intensity)was lower than that in asthma group, but higher than that in control group(all P ＜0. 01). And expressions of TGF-β1 protein in dexamethasone treated group(0. 173 ± 0. 014,0. 202 ± 0. 019 optical density, respectively) was lower than that in asthma group(all P ＜0. 01) ,but higher than that in control group(all P ＜0. 01). There were significant positive correlation between ENA-78 expression at blood neutrophil and numbers of total inflammation cells in bronchoalveolar lavage fluid (n = 29, γ = 0. 762, P ＜ 0. 01). Conclusion The beneficial effect of glucocorticoid(dexamethasone) on airway inflammation in asthma rats could be at least in part due to their direct inhibitory effect on ENA-78 and TGF-β1 protein generation by neutrophil.