Objective To explore the method to develop a gene vaccine of human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) against its infection. Methods HCMV strain AD169 was propagated in WI-38 cell and viral DNA was extracted as a template for polymerase chain reaction (PCR) amplification of UL83 (pp65), the resulting of PCR was subcloned into pUC118HincII/BAP plasmid and DNA sequence analysis conformed the fidelity of the PCR. The vector pcDNA 5.0 was designed to correctly place CMV promoter sequence, pp65 sequence and secret signal sequence (mouse immunoglobulin kappachain for efficient secretion of recombination protein) into its genomic DNA. Exchanged primers of pp65 sequence, CMV promoter sequence and secret signal sequence to confirm the result by PCR screening. The vector pcDNA 5.0 was transfected into CHO cell, supernatants of transfected cells were extracted and purified. Recombination protein from supernatants was detected by gel electrophoresis and dot blot hybridization of Western- ECL system. Results Compared the sequence of pp65 gene with the standard sequence of pp65 from Medline,it was found that the concordant rate between them was 99.99%,only a nonsense mutation occurrences at 1 455 base.A pcDNA 5.0 Eukaryotic expression vector was established, which including CMV promoter sequence,secretion signal sequence and pp65 sequence. PCR screening and the pp65 protein expressed in CHO cell confirmed it. Extraction from supernatants in transfected CHO cells was recombination protein of pp65, which was detected by gel electrophoresis and dot blot hybridization of Western- ECL system and western blotting.Conclusion Subunit vaccine of HCMV is gained,which is a transfer eukaryotic expression vector pcDNA 5.0 constructed by CMV promoter sequence,secretion signal sequence and pp65 gene sequence.

Objective To explore the neurobehavioral change of mice from dams with human cytomegalovirus(HCMV) infection during first trimester. Methods Eight-week-old fertilized female Kunming mice were randomly divided into infected group and control group.On the 4th gestation day mice in infected group were injected intraperitoneally with 0.5 mL HCMV (1?10-6 50 percent of tissue cultured infective dose),and those in control group were injected intraperitoneally with 0.5 mL supernatant of cultured human fibroblast.Caesarean birth operation was performed on 3 randomly chosen fertilized mice before delivery. Fetuses were observed and their brain tissue were collected and analyzed under light and electron microscope separately.PCR test was used to determine HCMV pp65 antigen of offspring′s sera.Neurobeha-vioral test such as Morris Water Maze and Lashley Ⅲ Water Maze were performed on offspring mice of 6-7 weeks old.Results Compared with control group,the pathological changes such as degeneration,necrosis,and nucleus disappearance of nerve cells and giant cells were found in offspring′s brain of mice in infected group under light microscope. Under electron microscope,swelling of nerve cells and spherical virus particle in the cytoplasm were found in the brain of mice in infected group. HCMV pp65 antigen was detected in 7 offspring mouse′s se-rum in infected group.Offspring′s swimming time and speed were(30.21?12.74) s and(19.10?1.90) cm/s in infected group,while those in control group were (11.87?3.62) s and (23.21?1.02) cm/s by Morris Water Maze test,there were significantly differences between 2 groups (Pa

Objective To explore the correlation of city school-aged children′s intelligence quotients(IQ) with family factors.Methods Picking up 180 healthy children which aged 10-14 and their parents.Children′s IQ were tested with Wechsler intelligence Scale for Children- Revised(WISC-R).Their parents were investigated by using the questionnaire designed by ourselves about some factors of family which includes Eysenck Personality Questionnaire(EPQ), Home Education Index Measuring Scale (HEIMS),and so on. We analyzed the associations between children′s IQ and family factors with the applicable data about 114 only child. Results Multiple stepwise regression analyses show that some factors have significant effects on IQ of children(P

Objective To study the protective effect and mechanism of ganciclovir(GCV) on acute cerebral injury of mice caused by herpes simplex virus(HCV). Methods Mice model of acute cerebral injury caused by HCV were established, morphological changes in the brain tissue of mouse treated with GCV were observed under the electronic microscope, and the mortality were compared. The HSV - I DNA copies of brain tissue were detected by fluorescent quantitative polymerase chain reaction. Results In the infected model group, there were obvious swelling, karyopyknosis and destruction of the structure in the brain cells, as well as myelin sheath solution and vacuolar degeneration in the mitochondrion and crest were destroyed. There were the virions in the nucleolus. With the GCV treatment, the symptoms were improved, the mortality much lowered, the yields of HSV - I DNA much lower. Conclusions GCV may restrain replication of HSV-Ⅰ effectively and lower the mortality of mice with acute cerebral injury caused by herpes simplex virus significantly.

OBJECTIVETo explore the blockade effect of phenylbutyrate (PB), a histone deacetylase inhibitor, on the in vitro biological function of AML1/ETO to reverse its transcription repression and induce Kasumi-1 cells to differentiate and apoptosis.

METHODSKasumi-1 cells were treated with PB at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, morphological changes by light and electron microscopy, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry.

RESULTSPB treatment caused a dose-dependent inhibition of the cell proliferation. The IC(50) was about 2.3 mmol/L. PB treatment led to a progressive decline in the fraction of S-phase cells and increase in G(0)/G(1) cells. PB induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD(11b) and CD(13). A dose-dependent increase in early apoptosis for 2 days treatment, late apoptosis for 3 days treatment. The DNA ladder of apoptosis was observed on agarose gel electrophoresis for 5 days treatment. Morphological features of monocytoid differentiation and apoptosis were seen on Wright-Giemsa staining smears.

CONCLUSIONPB treatment could inhibit proliferation of Kasumi-1 cells, induce partial differentiation, apoptosis and accumulation of cells in G(0)/G(1) phase.

Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Histone Deacetylase Inhibitors ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Phenylbutyrates ; pharmacology

OBJECTIVETo study the efficacy of conductive education combined with Frenkel training in the improvement of balance function in children with cerebral palsy.

METHODSOne hundred and fifteen children with cerebral palsy were randomly administered with conductive education and Frenkel training (study group, n=60) or conventional training (control group, n=55). Activities of daily living (ADL) scale and gross motor function measurement (GMFM) of physical performances were used to assess the balance function.

RESULTSThe scores of ADL scale and GMFM of physical performances in both the study and the control groups increased after training. The study group showed higher scores of ADL scale (37.91+/-10.12 vs 34.18+/-6.13; p<0.05)and GMFM (62.93+/-15.00 vs 54.53+/-14.11) than the control group (p<0.05).

CONCLUSIONSConductive education combined with Frenkel training is more effective for the improvement of balance function in children cerebral palsy.

Activities of Daily Living ; Adolescent ; Cerebral Palsy ; physiopathology ; rehabilitation ; Child ; Child, Preschool ; Disabled Children ; rehabilitation ; Education, Special ; methods ; Female ; Humans ; Infant ; Male ; Motor Skills ; Physical Therapy Modalities ; Postural Balance

OBJECTIVETo investigate the inhibition of COX-2 gene expression and its effects on malignant proliferation of human lung adenocarcinoma A549 cells after interfering at different target sites in vitro.

METHODSThe 3rd, 7th and 10th exon of COX-2 were selected as the targets and three COX-2 siRNA expression vectors with human U6 promoter were constructed. Three siRNA expression vectors and two vacant vectors were transfected into A549 cells expressing COX-2 with lipofectamine, respectively. The transfected cell strains were constructed and the change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A549 cells after interfering at different target sites were studied by cell growth curve and colony formation assay in vitro.

RESULTSThe three siRNAs and U6 promoter were validated by PCR, restriction endonuclease digestion, DNA sequencing and BLAST alignment, and cloned into the pEGFP vector. The cell strains transfected were named as A549-3, A549-7, A549-10, A549-p and A549-pU6, respectively. A549-p cells showed expression of GFP and A549-3, A549-7, A549-10, A549-p and A549-pU6 cells did not show at 24, 48 and 72 hours after transfection. The results of RT-PCR and Western blot showed an inhibition of COX-2 expression after interfering at three target sites (3rd, 7th and 10th exons). In contrast to A549 cells, the levels of COX-2 mRNA of A549-3, A549-7 and A549-10 cells were reduced by 10.6%, 33.4% and 61.2%, respectively. The levels of COX-2 protein of A549-3, A549-7 and A549-10 cells were reduced by 26.7%, 44.7% and 56.2%, respectively. The results of cell growth curve and colony formation assay showed a slowing down of the growth of A549-10 cells and reduction of their colony formation rate. The other two targets had no apparent effect on the growth of A549 cells.

CONCLUSIONThere is a significant inhibiting effect of RNA interference on the malignant proliferation of A549 cells in vitro, and the most striking effect can be seen when the 10th exon of COX-2 is taken as the interference target.

Adenocarcinoma ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cyclooxygenase 2 ; genetics ; metabolism ; physiology ; Exons ; Genetic Vectors ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

Objective To study the colonization rate and antibiotic resistance of group B Streptococcus (GBS) in gravidas during late pregnancy,and to evaluate the effectiveness of GBS screening in late pregnancy and intrapartum antibiotic prophylaxis (IAP) for the prevention of neonatal early-onset GBS disease (EOGBS).Methods A retrospective study was conducted to analyze the colonization rate and antibiotic resistance pattern of GBS in 14 204 gravidas who were screened for GBS at 35-37 gestational weeks during March 2016 to March 2018 in the University of Hongkong-Shenzhen Hospital (HKU-SZH).Differences in the incidence of EOGBS before and after GBS screening and IAP were analyzed using Chi-square or Fisher's exact test.Results Among the 14 204 gravidas,2 027 cases were GBS positive with a colonization rate of 14.27％.Incidence rates of EOGBS before and after GBS screening were 0.6‰ (4/6 356) and 0.07‰ (1/14 403),respectively (Fisher's exact test,P=0.033).GBS isolates were 100％ (2 027/2 027) sensitive to penicillin and vancomycin.Resistance rates to clindamycin and erythromycin were 67.2％(1 363/2 027) and 65.7％ (1 332/2 027),respectively.Conclusions Routine GBS screening in late pregnancy and IAP can significantly decrease the incidence of EOGBS.Penicillin is the optimal choice for prevention and treatment of GBS infection.

OBJECTIVE: To study the phenotypes and genetic features of families with Duchenne muscular dystrophy (DMD). METHODS: Seven children from six families with DMD diagnosed by gene testing were enrolled. The clinical and genetic features of the families were analyzed. RESULTS: There were two new mutations and four maternal inheritance mutations in the six families. The proband of family 1 had one point de novo mutation and one insertion de novo mutation of the DMD gene. Three families had point mutation, one family had fragment deletion of exon, and one family had fragment duplication of exon. The youngest age of onset of the probands was 6 months. All probands had skeletal muscle dyskinesia and significant changes in muscle enzymes, with different severities of clinical phenotypes. Three probands had mild mental retardation. The results of echocardiography were normal for all probands. The mother of the proband in family 6 had mild clinical phenotype. CONCLUSIONS Gene testing can be used for the confirmed diagnosis of DMD. Mental retardation is a frequent clinical phenotype of DMD. The symptoms of myocardial involvement are not obvious in the early stage. Female carriers may have mild clinical symptoms.
Dystrophin ; Exons ; Female ; Genetic Testing ; Heterozygote ; Humans ; Muscular Dystrophy, Duchenne ; Mutation ; Phenotype

Objective:To explore genes related to costimulatory molecule related to the prognosis of bladder cancer,and to construct and evaluate prognosis model based on costimulatory molecule-based signature(CMS).Methods:Gene expression matrix and clinical information of bladder cancer patients were downloaded from TCGA database and GEO database(GSE31684),and costimulatory molecule-related genes were retrieved from the literature.The univariate and multivariate Cox analysis were used to screened prognostic-related genes and constructed prognostic model.Forecast accuracy of model was verified in TCGA training group,TCGA validation data group and GEO group by Kaplan-Meier survival analysis and receiver operating characteristic curve(ROC).Considering risk score and clinical characteristics,we constructed a nomogram and evaluated its performance by consistency analysis and ROC.CIBERSORT algorithm was used to analyze immune cell composition of tumor microenvironment infiltration,and gene set enrichment analysis(GSEA)was performed to explore the potential mechanism.Results:Four prognostic-related CMSs were found:TNFRSF14,CD276,ICOS and TMIGD2,of which three were included in the risk score construction.Multivariate Cox regression results showed that the risk score based on CMS was an independent prognostic factor for bladder cancer patients.Consistency analysis and ROC results showed that the nomogram had ideal prognosis prediction accuracy.Immune infiltration analysis showed that the high risk group was likely to be in immunosuppressive state.GSEA results suggested that genes in high risk group were enriched in extracel-lular matrix(ECM)receptors interaction,cell cycle and other pathways.Conclusion:TNFRSF14,CD276 and ICOS may be potential prognostic biomarkers for bladder cancer patients.CMS-based risk score and nomogram could contribute to early prognosis and choice of personalized treatment.