Objective To investigate the function and possible action mechanisms of microRNA hsa-mir-634 in Vero cells.Methods The binding sites for hsa-mir-634 in the 3' UTR of cyclin D1 (CCND1) were predicated by bioinformatics methods.Then,the 3'UTR sequence of CCND1 containing the binding sites for hsamir-634 was amplified by PCR.Site-directed mutagenesis was used to create mutations in the binding sites.The wild and mutant 3' UTR sequences of the CCND1 gene were ligated into the psi-CHECK2 vector separately to construct dual-luciferase reporter vectors,including CHECK2-CCND1 wild,CHECK2-CCND1 mut 1,CHECK2-CCND1 mut 2 and CHECK2-CCND1 mut 3.Then,293T cells were transfected with the four constructed plasmids,and luciferase activity was measured 48 hours after the transfection.Vero cells were transfected with hsa-mir-634 mimics and negative control separately,and harvested after additional culture for different durations; the Vero cells remaining untreated served as the blank control.Subsequently,fluorescence-based quantitative PCR and Western blot were performed to detect the mRNA and protein expressions of CCND1 respectively in,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay to evaluate the proliferation of,and flow cytometry to detect the apoptosis in,Vero cells.Results The binding sites for hsa-mir-634 in the 3'UTR of CCND1 were successfully predicated.Sequencing results showed the successful construction of dual-luciferase reporter vectors.As the luciferase assay revealed,the overexpression of hsa-mir-634 could significantly inhibit the CCND1 3'UTR-mediated luciferase activity.Compared with the negative control,the hsamir-634 mimics markedly decreased the protein expression of CCND1,but had no obvious effect on the mRNA expression of CCND1 in Vero cells.The proliferation of Vero cells transfected with hsa-mir-634 mimics was significantly restrained compared with those transfected with the negative control,and the strongest restraining effect was observed on day 4 after the transfection.In addition,the overexpression of hsa-mir-634 also induced the apoptosis of Vero cells,with the apoptosis rate being 8.03％,7.96％ and 17.33％ in the blank control group,negative control group and mimics group respectively.Conclusion Hsa-mir-634 may regulate the proliferation and apoptosis of Vero cells via influencing the expression of CCND1.

Objective To study the relation between the clinical feature and glycoprotein D gene sequence analysis of a wild strain of HSV 2 isolated from one relapsed patient with genital herpes. Methods The partial glycoprotein D gene sequence of the above mentioned strain was amplified and cloned with PCR. Results The comparison of the amino acid sequence of gD gene between the wild strain and HSV 2G strain published showed that there was a mutation at site80 and site159. Conclusion In order to develop effective vaccine which is suitable for China, it is necessary to investigate the differences of the gene structure and function of gD among HSV isolated from China and other countries.

The herpes simplex virus(HSV) Us3 gene encodes a ser/thr protein kinase(PK).As an accessory gene,it plays an important role in the regulation of the apoptosis of infected cells and virus release.Us3-induced alterations in the host cytomorphology are associated with enhanced intercellular virus spread,suggestive of a previously undescribed aspect of alphaherpesvirus spread.

Objective To observe the prophylactic effects of a HSV-2gD2DNA vaccine in guinea pigs challenged with HSV-2strains.Methods Female guinea pigs were divided into3groups with10each,which was immunized intramuscularly with100?g of pc-gD plasmids(recombinant HSV-2DNA vac-cine),or with pcDNA3blank plasmids,with normal saline as control,respectively.Two booster injections were given on day7and day21.Sera were collected for virus neutralization test on day0,day28,and day56.The animals were challenged with HSV-2strain sav intravaginally,and lesions induced on the external genital skin were scored between day1and day21after challenge.Results The titer of neutralizing anti-body to HSV-2was much higher in the sera from animals immunized by pc-gD plasmids than that from ani-mals immunized by pcDNA3blank plasmids or normal saline.Furthermore,the lesion scores on external genital skin were significantly decreased in pc-gD group than those in other two groups with either primary or recurrent infections.Conclusion The constructed gD2vaccine can efficiently protect guinea pigs from genital infection and reduce recurrent infection induced by latent herpes simplex virus.

Objective To investigate whether systemic lupus erythematosus (SLE) T cell function disorder is related to abnormal biochemical pathways.Methods After cross linking of anti CD3 mAbs to sheep anti mouse IgG and stimulating T cells,the changes of free calcium ion within T cells and these changes under interference of Thapsigargin and EGTA were observed respectively for 10 minutes with an adhesion cytometry.The relation between [Ca 2+ ]i response in SLE T cells and expression of CD3 molecules,or InsP 3 levels was evaluated.Results The base [Ca 2+ ]i response in T cells of SLE patients was similar to that of normal control ( P =0 105).Peak and plateau [Ca 2+ ]i responses were significantly higher in the group of SLE patients ( P

Objective To screen for genes associated with low-dose UVA irradiation-induced adaptation reaction in cultured human melanocytes, and to explore the molecular mechanism of the adaptation.Methods Cultured human melanocytes of fifth to tenth passage were divided into two groups, experimental group was subjected to an irradiation with UVA at 7.2 J/cm2 once daily for 4 times and an additional irradiation at a lethal dose of 86.4 J/cm2 6 hours after the above 4-session irradiation, and control group subjected to a single irradiation with UVA at 86.4 J/cm2. A human genome-wide oligonucleotide chip was used to screen for differentially expressed genes between the two groups of cells followed by a functional classification based on international standard. Moreover, a part of these genes were analyzed and identified by real-time fluorescent quantitative PCR. Results In the adaptation reaction induced by low-dose UVA irradiation in cultured melanocytes, there were 129 differentially expressed genes, including 81 up-regulated genes and 48 down-regulated genes. These genes were found to be mainly involved in metabolism, transport, signal transduction, apoptosis, DNA synthesis and repair, and some of them were oncogenes or anti-oncogenes. Real-time PCR confirmed some of the differentially expressed genes. Conclusions The whole genome-wide oligonucleotide chip could screen with high efficiency for differentially expressed genes in low-dose UVA irradiation-induced adaptation reaction in cultured melanocytes.

Objective Up to now, there has been no sure cure for genital herpes (GH), and vaccine seems a most promis-ing approach to the prevention and treatment of herpes simplex virus Ⅱ(HSV-2) infection.In this study, we investigated the feasibili-ty of preparing a dendritic cell ( DC) vaccine modified by the adenovirus-mediated HSV-2 gD gene. Methods We subcloned the HSV-2 gD gene into the vector Shuttle-2 and constructed the recombinant adenovirus pAdeno-HSV-2 gD following identification by en-zyme digestion and DNA sequence analysis .We isolated DCs from the mouse bone marrow , analyzed their phenotypes by flow cytome-try after transfection with the recombinant adenovirus pAdeno-HSV-2 gD, and determined the expression of HSV-2 gD by immunohisto-chemistry, RT-PCR, SDS-PAGE, and Western blot. Results Based on HSV-2 DNA, the corresponding target fragments were am-plified with the gD gene primers.Agarose gel electrophoresis showed the correct size of the PCR product (1182 bp) as predicted.The recombinant adenovirus pAdeno-HSV-2 gD was obtained by transfecting the 293 cells with pAdeno-gD DNA, which had an activity of 4 ×1010 IU/mL.The contents of CD40, CD80, and CD86 were (74.2 ±3.9), (73.9 ±4.1), and (76.1 ±5.5) % in the mature DCs and (81.3 ±3.1), (80.4 ±2.9), and (83.7 ±3.9) % in the pAdeno-HSV-2 gD DCs, significantly increased as compared with those in the immature DCs ([9.7 ±0.5], [7.5 ±1.2], and [5.2 ±1.1] %) (P<0.01).No statistically significant differ-ences were found between the expression of the surface molecules in the pAdeno -HSV-2 gD DCs and that in the cytokine stimulation-in-duced mature DCs (P>0.05).RT-PCR and immunohistochemistry confirmed the expression of HSV-2 gD in DCs.SDS-PAGE and Western blot of the expressed protein showed a new band with an apparent molecular mass corresponding to the predicted size (43000). Conclusion The results of our study have paved the ground for the successful preparation and identification of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene.

Objective:To assess the periodontal diseases as a possible risk factor for preterm and low birth weight. Methods: A population-based, cross-sectional study was carried out in Hubei. The samples were chosen randomly by the rule of admittance. Socio-demographic information relating to health and maternal habits was collected through a questionnaire. The clinical parameters were debris index-simplified (DI-S), sulcus bleeding index (SBI), probing pocket depth (PD) and clinical attachment loss (CAJ). The birth time and weight were recorded. The result was analyzed with chi-square test and logistic regression analysis.Results: The incidence of preterm low birth weight was 12.3%. The gingivitis and periodontitis were diagnosed in 86.6% and 64.2% of preterm low birth weight cases. The odds ratio of having gingivitis and preterm low birth weight was 1.30(95%confidence interval: 0.53-3.22). The odds ratio of having periodontitis and preterm low birth weight was 2.69(95%confidence interval: 1.37-5.27). Conclusion: The periodontal diseases of pregnancy can be regarded as an important risk factor for preterm low birth weight.

Objective To study the expression and signficance of the osteopontin (OPN) in epithelial ovarian cancer tissue and serum. Methods Immunohistochemistry method and ELISA were used to detect the expression of OPN in 64 cases of epithelial ovarian cancer tissue and serum, 20 cases of ovarian benign tumors and 10 cases of ovarian nomal tissues. Results The OPN expression was associated with the clinical staging and histological grading of epithelial ovarian cancer tissue and serum (P < 0.01). The level of OPN in epithelial ovarian cancer tissue was significantly higher than that in benign ovarian tumor and normal control groups (P < 0.01). Conclusion OPN is remarkably correlated with the carcinogenesis and the development of epithelial ovarian cancer.

Objective To assess the efficacy and safety of 308 nm excimer laser plus topical pimeevaluated after 15 and 30 times of laser therapy respectively.ResultsExcept for one patient,all patients were able to be evaluated for effiicacy.After 30 times of laser therapy,the response and excellent response rates were 89.6%and 77.1%respectively,in group A，5.0%and 52.1%respectively in group B;both rates were significantly higher in group A than in group B(both P＜0.05).Also,a highler repigmentation rate was obtained in facial lesions in group A compared with group B.Conclusions The 308 nm excimer laser is safe,erective and well-tolerated for vitiligo in childhood,and the combination with topical imecrolimus cream may improve its efficacy in facial vitiligo.