Objective To explore the changes of genes associated with the nuclear factor of kappa B(NF-?B) signaling pathway in neonatal rats with early hypoxic-ischemic reperfusion brain damage(HIRBD).Methods Twenty-four SD rats at age of 7 days,with male to female of 1212,were randomized into normal control group(group A,n=8),hypoxic-ischemia reperfusion for 2 h(group B,n=8) and hypoxic-ischemia reperfusion for 4 h(group C,n=8).The tissues of hippocampus were taken for complete RNA extraction.Gene chip inspection and biological signal analysis technique were used to detect the expression of 113 involved signal molecules of NF-?B pathway.Results Compared with group A,the up-regulated expression was found in Chemokine(C-C motif) ligand 2,Dual specificity phosphatase 1,FBJ osteosarcoma oncogene(Fos) and Toll-like receptor 9.Whereas the expressions of Caspase-1,8,Mitogen-activated protein kinase kinase 6,Mitogen activated protein kinase 3 and Ras homolog gene family member a from Ras-gene famimly was found down-regulated in group B.The up-regulated expression was in Fos,IL-1? and Toll-like receptor 6,but that of down-regulation was found in Caspase-1,Extracellular matrix protein 1,Lysophosphatidic Acid G-protein-coupled receptor 2,Mucosa associated lymphoid tissue lymphoma translocation gene 1,Inhibitor of kappa B kinase epsilon and Ras homolog gene family member c.Conclusions At the early stage of HIRBD,the Toll-like receptors may induce NF-?B activation,leading to the coordinated induction of multiple genes,which is involved in inflammatory,apoptosis and cell proliferation.Genes induced by NF-?B are responsible for the physiopathological process of early brain damage in neonatal rats with HIRBD.

Adaptation, Physiological ; Animals ; Brain Stem ; Cloning, Molecular ; DNA, Complementary ; Gene Expression ; Gene Library ; Motion Sickness ; genetics ; Rats

Animals ; Disease Models, Animal ; Male ; Motion Sickness ; Rats ; Rats, Sprague-Dawley

AlM: To silent hypoxia inducible factor-1α ( HlF-1α) gene in malignant melanoma of the choroid cell by small interference RNA ( siRNA ) and investigate its effect on the expression of matrix metalloproteinase-2 ( MMP-2 ) in the choroid cell line human uveal melanoma cell (OCM-1) in hypoxia environment.METHODS:OCM-1 cells cultured on culture flask were divided into normal group and hypoxia group. Hypoxia group were divided into five groups: simple hypoxic group, and interference group, and negative control group, and positive control group, and liposome group. Normal group cells were cultured on DMEM culture flask with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin as well as high concentration of glucose. The cells were maintained at 37℃ in a humidified 5% CO2 incubator. Cells in good condition were selected for experiment. For hypoxia group, chemical hypoxia inducer CoCl2 was added into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment. We designed and synthesised siRNA ( siRNA + negative control+positive control ) , the target sequences of the HlF-1α to transfect hypoxic malignant melanoma of the choroid cell. SiRNA including HlF-1α siRNA, β-actin siRNA and negative control group synthesized in vitro transfected hypoxic OCM - 1 cell through Lipofectamine2000. The expression of HlF-1α, MMP-2 gene and the protein were detected by RT-PCR and Western blot. RESULTS: Compared with the normal group, the expression of HlF-1α mRNA was not obviously changed (P>0. 05), but the expression of HlF-1α protein and MMP- 2 mRNA protein was significantly higher ( P<0. 05) . Compared with the other hypoxia groups,β-actin mRNA expression of positive control group decreased ( P< 0. 05 ) , which proved successful transfection. The expression of HlF-1α mRNA and the expression of its protein and both MMP-2 mRNA and its protein was significantly lower ( P < 0. 05 ). The negative control group, liposome control group had no significant difference in the detection of factors (P>0. 05).CONCLUSlON: Hypoxia status may upregulate the HlF-1α in OCM-1 cells by increasing the expression of protein. Hypoxia can also inactivate MMP-2, resulting in upregulation of MMP-2 RNA and the expression of its protein. The expression of HlF-1α and MMP-2 mRNA can be down-upregulated by transfecting OCM-1 with HlF-1α siRNA.

OBJECTIVETo explore the effect of occupational stress on hypertension.

METHODS498 workers whose accumulative length of service was more than two years were investigated with questionnaire by method of cluster sampling from a thermal power plant in Henan province in China; 446 respondents returned qualified questionnaire including 281 male and 165 female Han People. After the patients with secondary hypertension, diabetes patients, and patients with liver or kidney disease were excluded, 84 workers (58 males and 26 females) were diagnosed as hypertension. The occupational stressors, personalities, buffering factors and occupational strain were measured by using the Job Demand-control Model, the Effort-reward Imbalance Model questionnaires and Occupational Stress Measurement Scale. Main risk factors for the development of hypertension such as heredity, body mass index, high salt diet, alcohol use, smoking habit and lack of physical activity were investigated. 498 whole blood samples were collected from workers in field epidemiologic survey. All of the samples were detected TG, CHO and FPG by common biochemistry methods. Multivariate logistic regression analysis were used to determine the relationship between occupational stressors and prevalence rate of hypertension. The difference of morbidity of hypertension between different stress level subjects was analyzed by chi2 test.

RESULTS(1) Logistic regression analysis of the hypertension by all occupational stressors and risk factors of hypertension indicated that not only some common factors such as parents' hypertensive history, BMI, alcohol use and TG, but also responsibility for person, work locus of control and social support were significantly correlated with elevated risks of hypertension. (2) Logistic regression analysis of the hypertension by main dimensions of effort-reward imbalance model and risk factors of hypertension indicated that parents' hypertensive history, BMI, alcohol use, TG, and effort were significantly correlated with elevated risks of hypertension. Logistic regression analysis indicated the risk of hypertension had an effect on the FRI and effort (OR was 1.71 and 2.43 respectively). (3) For the job strain model, results indicated that parents' hypertensive history, UMI, alcohol use, TG, work locus of control and social support were significantly correlated with elevated risks of hypertension. But the main dimensions of job strain model (job demands and decision latitude) didn't enter regression equation. (4) The difference of prevalence of hypertension between high- and low stress level groups in male was statistically significant (OR = 3.13, P < 0.01), but the case was not the same in female (P > 0.05).

CONCLUSIONSOccupational stress might be risk factor of hypertension; The predictive power of effort-reward imbalance model for the development of hypertension would be larger than that of job strain model.

Adult ; Burnout, Professional ; complications ; Chi-Square Distribution ; China ; Cross-Sectional Studies ; Female ; Humans ; Hypertension ; etiology ; Logistic Models ; Male ; Middle Aged ; Risk Factors ; Sampling Studies ; Surveys and Questionnaires ; Young Adult

OBJECTIVETo examine the expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR1, VEGFR2) in transdifferentiated human proximal tubular epithelial (HK-2) cell induced by transforming growth factor beta1 (TGFbeta1).

METHODSThe transdifferentiation of HK-2 cells was detected by evaluation of expression of alpha-SMA by cytoimmunochemistry and RT-PCR. The VEGF mRNA was evaluated with RT-PCR. The secreted VEGF in the culture media was measured with ELISA. The cellular VEGF, VEGFR1, and VEGFR2 were measured with Western blot.

RESULTSThe immunostain of alpha-SMA were positive in HK-2 cell induced by TGFbeta1 at the concentration of 5 and 8 ng/ml for 72 h. The expression of alpha-SMA mRNA was induced by TGFbeta1 in concentration- and time-dependent manners. The expressions of mRNA and protein of VEGF were upregulated by TGFbeta1 at the concentration of 0.1 and 1 ng/ml for 72 h and at the concentration of 8 ng/ml for 12 h and 24 h when compared with the control. But expressions of mRNA and protein of VEGF were downregulated by TGFbeta1 at the concentration of 3, 5, and 8 ng/ml for 72 h and at the concentration of 8 ng/ml for 36, 48, and 72 h, respectively. Meanwhile, Protein levels of VEGFR1 and VEGFR2 were upregulated by TGFbeta1 in concentration- and time- dependent manners.

CONCLUSIONSIncreased expression of VEGFR1 and VEGFR2 and two-phase change in VEGF expression occurred in the process of tubular epithelial transdifferentiation induced by TGFbeta1. Reduced expression of VEGF may contribute to tubular epithelial transdifferentiation in a vicious circle.

Cell Differentiation ; Epithelial Cells ; cytology ; Humans ; Kidney Tubules, Proximal ; cytology ; RNA, Messenger ; metabolism ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factor beta1 ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

Using cDNA from Rehmannia glutinosa leaf as template, a 972 bp fragment of expansin gene which containing a 762 bp ORF that encoded 253 amino acids, was cloned, named RgEXPA10, which GenBank accession number for this gene is KF011918. A 1 207 bp genomic sequence of RgEXPA10 was amplified by PCR with leaf DNA as template, sequencing analysis revealed that three exons and two introns in RgEXPA10 genomic sequence, and which GenBank accession number is KF011919. Molecular and bioinformatic analyses indicated that RgEXPA10 protein have DPBB_1 and Pollen_allerg_1 domain, also including a 26 aa nuclear localization signal and a 19 aa transmembrane region. Phylogenetic analysis revealed that RgEXPA10 showed the highest homology with AtEXPA8 among the 26 α-expansins in Arabidopsis thaliana. However, the RgEXPA10 indicated the highest homology with the expansin from Solanum lycopersicum among 22 plant species. Expression patterns using qRT-PCR analysis showed that RgEXPA10 mainly expressed in unfolded leaf, followed by the tuberous root at stage of expanding period, and rarely expressed in senescing leaf. And RgEXPA10 showed higher expression level in tuberous root at 60 and 90 days after emergence. The transcription level of RgEXPA10 significantly reduced under all the three stresses including continuous cropping conditions, salinity and waterlogging. This study will lay foundations for molecular function in development and regulation of different stresses for R. glutinosa.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Gene Expression Regulation, Plant ; Genes, Plant ; Molecular Sequence Data ; Phylogeny ; Plant Leaves ; Plant Proteins ; genetics ; Plant Roots ; Rehmannia ; genetics

OBJECTIVETo identify the region in PRL-3 gene promoter where the transcriptional factor Snail can bind.

METHODSPRL-3 promoter and the possible binding sites of the transcription factors were analyzed by bioinformatical methods. Chromatin immunoprecipitation and PCR were performed using the antibody specific for Snail to verify the binding of Snail to PRL-3 promoter.

RESULTSAccording to the prediction by TRED, a promoter prediction software, PRL-3 gene promoter was located between -700 bp to 299 bp of PRL-3 gene. Many possible transcription factor binding sites such as for Snail, n-MYC, ARNT, E74A, NF-kappaB, NRF-2 and AML-1 were predicted by Consite, a promoter analysis web system. Interestingly, a 5'-CACCTG-3' core sequence and other related sequences of Snail binding sites were found in the promoter region of PRL-3 genes by Consite software. Two regions in PRL-3 promoter were validated to allow binding of Snail by chromatin immunoprecipitation analysis of SW480 cells.

CONCLUSIONSSnail regulates the activity of PRL-3 gene by binding to the promoter of PRL-3 gene in SW480 cells.

Base Sequence ; Binding Sites ; Cell Line, Tumor ; Computational Biology ; Humans ; Molecular Sequence Data ; Neoplasm Proteins ; metabolism ; Promoter Regions, Genetic ; Protein Tyrosine Phosphatases ; metabolism ; Snail Family Transcription Factors ; Software ; Transcription Factors ; metabolism

OBJECTIVETo construct a lentiviral expression vector for RNA interference of human CDH22 gene, and assess its gene silencing effect in colorectal cancer cells to provide a basis for investigating the role of CDH22 gene in the signaling pathway involved in human colorectal carcinoma metastasis.

METHODSHuman CDH22 gene short hairpin RNA (shRNA) sequence was designed using a software available on-line. After synthesis and annealing, the double-stranded oligonucleotides (dsOligoe) were cloned into the pENTR(TM)/U6 plasmid followed by sequence analysis. A positive clone was subcloned into pLenti6/BLOCK-iT(TM)-DEST vector and transformed into stb13 competent cells, with also verification by sequencing. The recombinant lentivirus was harvested from 293FT cells contransfected with the positive recombined plasmid and lentiviral packing materials. SW480 cells were infected with the recombinant lentivirus and the cells with stable CDH22 knock-down were screened by blasticidin selection. CDH22 expression in the cells was determined by real-time reverse transcription-polymerase chain reaction.

RESULTSA recombinant lentiviral vector expressing shRNAs against CDH22 gene was obtained and confirmed by DNA sequencing. Fifteen clones of SW480 cells infected with the recombinant lentivirus were selected, and clone 11 exhibited substantial knock-down of CDH22 mRNA expression.

CONCLUSIONThe lentiviral shRNA expression vector targeting human CDH22 gene capable of stable CDH22 gene knock-down in SW480 cells has been successfully constructed, which provides a basis for further study of the relationship between human colorectal carcinoma and CDH22 gene.

Base Sequence ; Cadherins ; biosynthesis ; genetics ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Molecular Sequence Data ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo study the clinical and pathological features of drug-induced liver injury (DILI).

METHODSLiver specimens were obtained through needle biopsies from 100 patients with DILI. The histological preparations of the specimens were stained with haematoxylin eosin, several histochemistry methods, and immunohistochemistry stains. The pathological changes of the livers were analyzed together with the patients's clinical data. The patients were divided into two groups, an acute DILI group (n=39) and a chronic DILI group (n=61), based on their clinical courses and histological changes in their livers. In the chronic DILI group, the clinical courses were longer than 6 months and/or fibrosis or cirrhosis occurred in their liver tissues.

RESULTSAmong our cases the leading cause of DILI was Chinese herb medicine, accounting for 21% of the 100 cases; steroids induced cases were 11% of the total. 78% of the patients presented elevated serum transaminases and/or jaundice. The degree of transaminases elevation and the frequency of jaundice happening in the acute group were significantly higher than those in the chronic group (P less than 0.05). The histopathological liver changes in these DILI cases included: (1) necrosis commonly occurred in acinar zone 3, (2) abundant neutrophil and/or eosinophil infiltrations, (3) hepatocytic and/or canalicular cholestasis with little or no inflammation, (4) microvesicular steatosis mixed with macrovesicular steatosis, and (5) presentation of epitheloid cell granuloma. There were no significant differences in liver histopathology between the acute and the chronic DILI groups, except that the fibrosis and the ductular proliferation were different.

CONCLUSIONDILI has become a notable liver disease in mainland China, and the use of Chinese herbal medicine must be improved, standardized and regulated more closely.

Adolescent ; Adult ; Aged ; Chemical and Drug Induced Liver Injury ; pathology ; Child ; Child, Preschool ; Female ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Young Adult