Objective To construct RNAi combinant adenoviral expressive vectors specific to p65 subunit of NF-?B and to observe their gene silencing effect on p65 subunit.Methods Three pairs of complementary. single-strand DNA oligos targeting three various sites of p65 mRNA were designed and synthesized.Annealling was used to generate double-strand oligos(ds-oligos),and then the ds-oligos were cloned into pENTR~TM/u6 to generate the entry clone named pENTR.Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT~TM- DEST was used to creat the adenovirus plasmid which contains the RNAi cassette.Then,the adenovirus plasmids digested with PacI were transfected into HEK293A cells to product adenovirus,and latter infected the HEK293A cells to amplify the adenoviral stock.Plaque forming assay was used to titer the adenoviral stock.The p65 gene silencing effect induced by the RNAi adenovirus was detected by Western blot and immunocytochemistry assay in ECV304 cells.Results The RNAi adenovirus specific to p65 subunit of NF-?B were produced with titer of 3.0 x 10~9pfu/ml to 2.5?10~10pfu/ml.The expression of p65 protein in ECV304 cells could be down-regulated efficiently by the RNAi adenovirus 48-72 h after infection,which would last for more than 6 days after infection.Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently.

OBJECTIVETo explore the clinical significance of serum matrix metalloproteinase-3 (MMP-3), tissue inhibitor of metalloproteinase-1 (TIMP-1), and monocyte CD147 in rheumatoid arthritis (RA) patients of damp-heat Bi-syndrome (DHBS) and of cold-damp Bi-syndrome (CDBS).

METHODSThe clinical data of 22 patients from inpatients and outpatients with RA were collected, and their peripheral blood was withdrawal. The disease activity scores [DAS28(4)] were assessed. The serum levels of MMP-3 and TIMP-1 were detected by double antibody sandwich enzyme linked immunosorbent assay (ELISA). The mean fluorescence intensity (MFI) and the expression percentage of CD147 on CD14+ monocytes were detected by flow cytometry. The difference of each index between RA patients of DHBS and RA patients of CDBS was analyzed.

RESULTSThe level of serum MMP-3 and the MFI of CD147 on the monocyte surface were obviously higher in RA patients of DHBS than in those of CDBS and the normal control group (P < 0.05). The concentration of serum TIMP-1 was obviously higher in RA patients of DHBS than in those of the normal control group (P < 0.05), while there was no statistical difference between the two syndrome types. The percentage of CD147 expression was obviously lower in DHBS than in those of CDBS and the normal control group (P < 0.05).

CONCLUSIONSIncreased serum MMP-3 level of RA patients of DHBS might result in destroy of joint cartilages and sclerotin. The significant increase of MFI and decreased expression percentage of monocyte CD147 might be the results of increased disease activity of RA and monocyte migration to the synovial membrane tissue.

Adult ; Aged ; Arthritis, Rheumatoid ; blood ; diagnosis ; Basigin ; metabolism ; Case-Control Studies ; Female ; Humans ; Male ; Matrix Metalloproteinase 3 ; blood ; Medicine, Chinese Traditional ; Middle Aged ; Monocytes ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; blood

Objective To explore the effect of edaravone (ED) on the neurological functionaldeficits, apoptosis and expression of caspase-3 protein following focal cerebral ischemia/reperfusion(I/R)injury in rats. Methods A total of 24 male Sprague-Dawley (SD) rats were randomly allocated intothe sham-operation group, cerebral I/R group, normal saline treatment group and ED treatment group, 6rats in each group. Rat models with focal cerebral I/R injury induced by middle cerebral artery occlusion(MCAO) were established using a modified suture method. ED (3mg/kg) or equal volume of normalsaline was injected intraperitoneally immediately after cerebral ischemia and 12 h after reperfusion in thetreatment groups;the rats in sham-operation group underwent the same modeling procedure withoutischemia by nylon suture. The neurological behavioral deficits were evaluated 24 h after I/R injury;,immunohistochemical staining and Western blot assay were applied to detect the change in the expressionof caspase-3 protein; in situ TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay was used tostudy the change in neuronal apoptosis. Results The scores of neurological behavioral deficit scale,the positive cells and expression of caspase-3 protein, and the apoptotic cells in the ED treatment groupwere significantly decreased, compared with that of the I/R group and normal saline treatment group(P<0.05 for each comparison). Conclusion ED may effectively reduce neuronal apuptosis andneurological functional deficits after cerebral I/R injury, which might be related with the inhibition of thecaspase-3 protein expression.

Objective To investigate the relationship between the levels of plasma adrenaline and norepinephrine and gene polymorphism of β1 adrenergic receptor G1165C in children with enterovirus 71 (EV71) infection in hand foot and mouth disease (HFMD). Methods The polymerase chain reaction (PCR) was used to detect the expression of gene polymorphism of β1 adrenergic receptor G1165C in vitro. The levels of plasma adrenaline and norepinephrine were measured by enzyme-linked immunosorbent assay (ELISA). Results The plasma norepinephrine level of severe group was significantly higher than the mild group in children with EV71 infection in HFMD (P < 0.05); however, the levels of plasma adrenaline in two groups had no statistical differences (P > 0.05); There was no significant difference in the distribution of β1 adrenergic receptor G1165C genotype and allele between EV71 infection group and healthy control group (P > 0.05). Further analysis of EV71 infection group by dividing it into mild and severe groups showed that there was no significant difference in the distribution of genotype and allele between these two groups as well (P > 0.05). There was no significant difference in the levels of epinephrine and norepinephrine in different genotypes of EV71 infection group (P > 0.05), and in the levels of plasma epinephrine and norepinephrine in the mild and severe groups (P > 0.05). Conclusions As the disease gets worse, the plasma norepinephrine level has a rising trend in children with EV71 infection in HFMD, which is an important indicator to evaluate the progress of the disease. However, the gene polymorphism of β1 adrenergic receptor G1165C have no significant correlation, not only with the susceptibility and severity of EV71 infection in hand, foot and mouth disease, but also with the levels of catecholamine.