5um)were more.The material middle diameters(MMD)of samples all surpassed 5?m.

Objective To investigate the clinical value of urinary retinol binding protein(RBP) and N-acetyl-β-glucosaminidase(NAG) for evaluating renal function in preterm neonate.Methods 89 neonates in our NICU were selected,divided into three groups:the asphyxial preterm group (18 cases),the no-asphxial preterm group (25 cases),and the control group (46 term infants without asphyxia).All objects were detected the urinary RBP,NAG and creatinine(Cr).The levels of RBP/Cr and NAG/Cr and blood urea nitrogen(BUN),Cr were observed within 48h after birth after birth.The fluctuations of urinary RBP/Cr and NAG/Cr in no-asphxial preterm group also were observed in 0~48h,~96h,~168h after birth respectively.Results The urinary RBP/Cr levels in asphyxial preterm group [(0.951±0.629)g/mol] were higher than those in no-asphxial preterm group[(0.389±0.281)g/mol] and the control group[(0.119±0.081)g/mol](P<0.05).The urinary RBP/Cr levels in no-asphxial preterm group were also significantly higher than those in the control group(P<0.05).The levels of urinary NAG/Cr in the asphyxial and no-asphxial preterm groups were both higher than those in the control group(P<0.05),but there was no difference betwteen the former two groups(P>0.05).The levels of serum Cr and BUN were no significant difference in the three groups(P>0.05).The urinary RBP/Cr level had non-linear correlation with either postnatal or gestational age in no-asphyxial preterm group.While the urinary NAG/Cr levels negative correlated with the gestational age(r=-0.625,P<0.05).And the correlation between the urinary NAG/Cr and postnatal age was postive(P<0.05).Conclusion The determination of urinary NAG/Cr and RBP/Cr provides a sensitive and reliable method to evaluate the renal function of neonates,especially in preterm infants.The RBP/Cr is affected by asphyxia more than NAG/Cr,which is rather correlated with gestational age.

Objective To investigate the molecular epidemiology as well as the subtypes of HIV in intravenous drug abusers, which can help pursue the source of infection in Zhejiang province and predict the epidemic strain in the future. Methods Such as ELISA, Western blot, nest PCR, DNA and sequencing technologies were used to analyze HIV subtypes of 15 strains isolated from intravenous drug abusers(IVDU) from Xinjiang autonomous region. Results All of the 15 IVDUs were infected with HIV 1, 2 of which were infected by subtype E and the others were infected by subtype C. Conclusions Subtype C is main HIV subtype infecting IVDUs. The epidemic subtype may have changed in China.

Objective To investigate the role and mechanism of retinoblastoma protein-associated proteins 48 (RBBP4) in HIV-1 latency.Methods CEM-Bru cells latently infected with HIV-1 were stimulated with 25 ng/ml of tumor necrosis factor alpha (TNF-α) in combination with 10 ng/ml of interleukin-2 (IL-2).Chromatin immunoprecipitation (ChIP) was performed to detect the changes in RBBP4 and in histone deacetylases 1 and 2 (HDAC1/2) binding to long terminal repeat (LTR).Binding activities of HDAC1/2 and RNA polymerase Ⅱ (RNA Pol Ⅱ) to LTR and acetylated histone H3 at LTR region were detected by ChIP after partially interfering the expression of RBBP4 in CEM-Bru cells with electroporation.Initiating and elongated transcripts were measured by RT-PCR.Results The binding activities of RBBP4 and HDAC1/2 to LTR in HIV-1 latently infected cells were enhanced significantly as compared with those in TNF-α and IL-2 co-stimulated cells.Fewer RBBP4 and HDAC1/2 bound to LTR following the interference of RBBP4 expression, which was accompanied with enhanced histone acetylation and strengthened binding activity of RNA Pol Ⅱ to LTR.Moreover, more initiating transcripts were detected in HIV-1 latently infected cells after the RBBP4 expression was interfered by electroporation.Conclusion RBBP4 contributes to the maintenance of HIV-1 latency, in which HDAC1 and HDAC2 might be involved.

Objective To optimize the PCR primer sets for Simian virus 40 (SV40) detection and establish an assay method for SV40 which is of high sensitivity, strong specificity, broad applicability. Methods Two pairs of PCR primers were designed of based on 21 different SV40 strains genome by Primer Premier 5.00 software, and the features of two pairs of PCR primers were analyzed by Oligo software (version 6.71), conservative nucleotide of two pairs of PCR primers and the PCR amplification product were analyzed by DNAMAN software (version 6.0.40). Two pairs of new-built PCR primers were compared with those derived from China pharmacopoeia (Clip) in these aspects. The detection sensitivity of four pairs of PCR primers were analyzed using different SV40 DNA diluent as PCR template. The detection specificity of four pairs of PCR primers were analyzed using sterile water, Vero cell DNA, SV40 DNA as PCR template, respectively. Results The sequences of the new PCR primer sets VP1 and T are conservative for 21 Strains. The sequences of PCR primer sets GCVP1 and GCT are conservative for SV40 strains whose accession No. is J02400, NC_001669, AF316139 and AF316141. As far as the same diluent SV40 DNA template is concerned, the PCR amplification efficiency of PCR primer set VP1 and T is higher than that of PCR primer set GCVP1 and GCT. There are non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets GCVP1 and GCT, whereas there are no non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets VP1 and T. Conclusion The new assay method for SV40 nucleic acid sequence has many better qualities than those in Chp such as high sensitivity, strong specificity, broad applicability, conservation of primers and their amplification products and so on.

Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites (TFBS) in activity of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) pro-moter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/En-hancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% (293) and 10% (Hacat), which was 40%-50% with enhancer. The activity of DC-SIGN pro-moters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-1 TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter.

DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is specific receptor on Dendritic cells, and plays a pivotal role on antigens presentation. Uptodate, the clear regulation mechanisms for DC-SIGN expression are not available.IL-4 is one of the most important cytokines inducing DC-SIGN production, while, NF-κB is an important transcription factor controlling signaling transduction. Both IL-4 and NF-κB are closely related to DC-SIGN regulation. NF-κB and IL-4 actions on DC-SIGN promoter activity, DC-SIGN expression as well as interactions between IL-4 and NF-κB were investigated in THP-1 cell. It was found that the mutation of NF-κB binding site in DC-SIGN promoter results in DC-SIGN promoter activity decrease about 50%.NF-κBp50 stimulates DC-SIGN expression in THP-1 cells. IL-4 upregulates DC-SIGN expression on THP-1 cells as well as NF-κB production. These data reveal that NF-κB is associated with IL-4 induced DC-SIGN expression.

Objective To explore the mechanism of latent human immunodeficiency ciency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC).We hypothesized that DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 may activate HIV-1 provirus.Methods We generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and a HIV-1 5'long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 and VSV-G-pNL4.3 pseudotype virus ( without gp120 protein).CEM-Bru cells were transfected with the DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein.Then HIV-1 replication was detected.The involvement of the ERK, p38 and NF-κB pathways signaling in this response were determined by inhibiting the pathways specifically and detecting the phosphorylation of the signaling kinase.Results The HIV-1 5'LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells.After HIV-1 gp120 protein stimulation of the mold of CEM-Bru cells, the increasing expression of HIV-1 Tat mRNA and HIV-1 p24,which implies early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation.HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.Conclusion HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.

This study was aimed to explore the action mechanism of massage treatment in combination with small needle knife to cervical spondylotic vertebral arteriopathy (CSA). A total of 400 CSA patients were randomly divided into four groups, which included group A (medicine treatment group), group B (small needle knife therapy group), group C(massage treatment group) and group D (small needle knife combined with massage treatment group). The observation was given on the average blood flow velocity (Vm), pulsatility index (PI) and treatment efficiency of ver-tebral artery(VA) and basilar artery (BA) before and after treatment among 400 cases using transcranial doppler (TCD). The results showed that TCD detection of Vm of VA and BA in group B, C and D were obviously improved after treatment; and the PI of them was close to normal. The total effective rate of group A was 66.0%, 78.6% in group B, 80.2% in group C, and 96.0% in group D. It was concluded that the treatment by massage in combination with small needle knife therapy of vertebral artery type of cervical spondylosis had a clear effect. Its mechanism may be related to the improvement of Vm and PI of vertebral basilar artery.

Objective To explore the relationship between the genetic polymorphism and serum concentration of mannan binding lectin (MBL)and the clinical manifestation of the hand-foot-mouth disease (HFMD) children infection by human enterovirus 71 (HEV71).Methods One hundred and thirty-eight children diagnosed as HFMD infected by HEV71 (including 80 mild cases and 58 severe cases) and 40 healthy,symptom-free children were investigated.The concentrations of serum MBL were measured in 40 healthy controls,80 mild HFMD cases and 56 severe HFMD cases at both acute and convalescent phases by a sandwich enzyme immunoassay with a human MBL ELISA kit.And the genomic DNA of all cases were extracted from blood according to standard phenol-chloroform procedure.Six SNPs in the MBL gene(-550G/C,-221G/C and +4C/T of the promoter,CGT52TGT,GGC54GAC,and GGA57GAA of the exon 1) were analyzed by a sequencing-based typing method.Results The MBL serum level of the severe HFMD circulatory respiratory failure group in acute phase was significantly increased compared with severe HFMD encephalitis group,the mild cases and the control,but in the convalescence phase it significantly decreased compared with them.The frequencis of type B/B mutation (+230 of the exon 1),type P/P mutation (+4C/T of the promoter),and type H/H mutation (-550G/C of the promoter) were a significant difference among mild group,severe group and the control(P=0.006,0.043,0.028,respectively).The frequencies of LYPB/LYPB genotype and HYPA/HYPA genotype were a significant difference among mild group,severe groupand the control (P=0.028,0.014,respectively).Conclusion Low MBL protein level as a result genetic polymorphism seems to be correlative with clinical manifestation of HFMD disease.The MBL gene mutation and low MBI.protein level may be used as one of the evaluation method of HFMD severeity.