The present study was to investigate the role of dopamine D1 receptors and its relationship with glutamate, N-methyl-D-aspartic acid (NMDA) receptor and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor in depression induced by chronic unpredictable mild stress (CUMS). CUMS-induced depression model was established in Sprague-Dawley rats, and intrahippocampal microinjections of D1 dopamine receptor agonist SKF38393, non-competitive NMDA receptor antagonist MK-801 and AMPA receptor antagonist NBQX were respectively adopted by rat brain stereotaxic coordinates. The behavioral observations were conducted by measurement of weight changes, sucrose preference test, open-field test and tail suspension test. The concentration of glutamic acid and the expression of its receptors' subunits were detected by HPLC and Western blot, respectively. The results showed that, compared with control group, CUMS rats showed depression-like behavioral changes, higher concentration of glutamic acid, lower expressions of NMDA receptor (NR1) and AMPA receptor (GluR2/3) in hippocampus. Pretreatment with injection of SKF38393 could rescue such depression effect of CUMS, decrease the concentration of glutamic acid, and increase the expressions of NMDA receptor (NR1), AMPA receptor (GluR2/3) in hippocampus. Pretreatment with MK-801 could enhance the antidepressant effect of SKF38393, while NBQX weakened. These results suggest that agonists of D1 dopamine receptor could reduce the concentration of glutamic acid in hippocampus, and its antidepressant effect may be mediated by AMPA receptor partially.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine ; pharmacology ; Animals ; Depression ; etiology ; physiopathology ; Dizocilpine Maleate ; pharmacology ; Excitatory Amino Acid Antagonists ; Glutamates ; metabolism ; Hippocampus ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; metabolism ; Receptors, Dopamine D1 ; agonists ; physiology ; Stress, Physiological ; physiology

AIMTo Investigate the effect of glutamate (Glu) and gamma-aminobutyric acid(GABA) in orbitofrontal cortex (OFC) on regulation of gastric motility.

METHODSUsing microinjection in OFC,together with lesion of related nucleus,and recording the intragastric pressure(IGP).

RESULTS(1) Microinjection of Glu in OFC caused a significant reduce of the amplitude of gastric motility, this effect could be reverse by lesion of amygdala, while lesion of LC had no influence on the effect of Glu. (2) microinjection of GABA in OFC could increase the amplitude of gastric motility significantly,and lesion of LC could abolish this effect,while lesion of amygdala could enhance the effect of GABA more.

CONCLUSIONMicroinjection of Glu in OFC may enhance the normal inhibitory effect of amygdale on gastric motility, and the effect of microinjection of GABA in OFC on gastric motility is closely related with LC.

Amygdala ; physiology ; Animals ; Female ; Frontal Lobe ; metabolism ; physiology ; Gastrointestinal Motility ; drug effects ; Glutamic Acid ; pharmacology ; Locus Coeruleus ; physiology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; pharmacology

Objective To analyze CYP17A1 gene mutation in a patient with 46,XY disordered sex development and to explore the possible influence on the phenotype of the patient.Methods Eight exons of CYP17AI gene in the patient and her parents were amplified and directly sequenced.In order to construct Mini-gene system,PCR fragments containing wildtype and mutant splicing sites were inserted in expression vector,and then transfected into cells.RT-PCR was used to observe the influence of splicing site mutation.Wildtype and aberrant splicing CYP17A1 cDNA expression plasmids were constructed and transfected into cells respectively,and CYP17A1 enzyme activity was tested in vitro.Results Mutation analysis revealed compound heterozygous CYP17A1 mutations,with Y329fs in one allele and a synonymous substitution( c.1263G＞A:GCG＞GCA) in another allele.In vitro analysis showed that the synonymous substitution induced a novel splicing site,which resulted in aberrant splicing of CYP17A1 mRNA and lacked six or seven amino acids after 415 in splicing product.In vitro transfection and enzyme activity experiment showed that the aberrant splicing product abolished the enzyme activity completely.However,this mutation did not completely influence splicing.The patient also had a part of normal splicing product,which was a coincidence to the phenotype of the patient.Conclusion This is the first description of an exonic splicing mutation in CYP17A1 relevant to the 17ot-hydroxylase deficiency phenotype.The functional study of the aberrant splicing variant has been initiated.

OBJECTIVETo explore the damage of respiratory system in the traffic policemen induced by automobile tyre colloidal particle and its allergenicity.

METHODSThe respiratory system symptoms in 445 traffic policemen working outside their offices and 243 controls were investigated and their pulmonary ventilation function index such as FVC, FEV(1.0), MMF and V(50) were determined. The specific IgE antibody of automobile tyre colloidal particle of their serum was determined and the skin-prick test of automobile tyre colloidal particle antigen was performed. Sixty-six traffic policemen working outside their offices and 5 controls with the positive of IgE antibody among them were detected by nasal mucosa provocation test. Sixty-six traffic policemen working outside their offices with the positive of IgE antibody were determined by Terbutaline inhalation test.

RESULTSThe positive rate of respiratory system symptoms of traffic policemen such as cough, stethocatharsis, short breath, nasal obstruction, sneeze and nose running was 38.02%, 27.03%, 20.00%, 23.08%, 27.47%, 32.09% and 34.95% respectively and significantly higher than those of the control with significant difference (P < 0.01) or (P < 0.05). The positive rate of specific IgE antibody of automobile tyre colloidal particle, skin- prick test and nasal mucosa provocation test was 14.51%, 23.73% and 54.55% respectively with significant difference (P < 0.01) and (P < 0.05). The percentage, the actual figure compared with the prediction figure, of the index of pulmonary ventilation function (FVC, FEV(1.0) MMF and V(50)) of traffic policemen were significantly lower than those of the control. Terbutaline inhalation test in 66 positive subjects of specific IgE antibody of automobile tyre colloidal particle was positive in 44 subjects, accounting for 9.67% in all policemen investigated.

CONCLUSIONThe automobile tyre colloidal particle is one of etiological factors that induce pulmonary ventilation function damage and could result in allergic asthma of traffic police.

Adult ; Allergens ; analysis ; Automobiles ; Humans ; Immunoglobulin E ; blood ; Male ; Middle Aged ; Occupational Exposure ; adverse effects ; Police ; Respiratory System ; drug effects ; Rubber ; adverse effects ; Young Adult

This study was purposed to culture murine compact bone-derived mesenchymal stem cell (MSC) and analyze the immunological and trilineage differentiation potential. Tibia and femur were extracted. Bone marrow cells were flushed out and compact bone fragments were digested with collagenase. The digested cells were cultured in 6-well plates. The immunophenotype, immunosuppressive function and trilineage differentiation potential were analysed by flow cytometry, mixed lympocyte reaction and Oil red O, von Kossa and alcian blue straining, respectively. The results indicated that the pure compact bone MSC could be isolated with in 3 weeks. The resulting MSC had trilineage differentiation potential and immunosuppressive effect on mixed lymphocyte reaction. The count per minute (CPM) value in control group of BALB/c T cells cocultured with irradiated C57BL/6 T cells was (2.56 ± 0.31) × 10(4), while CPM values of mixed lymphocyte cocultured with C57BL/6 compact bone MSC at ratios of 100:1 and 10:1 were (0.47 ± 0.12) × 10(4) and (0.28 ± 0.09) × 10(4). The CPM value of control group was higher than those of MSC cocultured group (P < 0.001). Compact bone-MSC had an immunosuppressive effect on mixed lymphocyte reaction in a dose dependent manner. It is concluded that murine compact bone has rich MSC and the primary MSC is contaminated with less hematopoietic cells. Murine compact bone-MSC have immunosuppressive effect on mixed lymphocyte reaction and trilineage differentiation potential. Compact bone-MSC have promising experimental study value.
Animals ; Bone Marrow Cells ; cytology ; immunology ; Bone and Bones ; cytology ; Cells, Cultured ; Female ; Immunophenotyping ; Lymphocyte Culture Test, Mixed ; Mesenchymal Stromal Cells ; cytology ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

Objective:To investigate the value of autoantibodies and serum levels of IgG4 and CA19-9 in the diagnosis of IgG4 associated cholangitis (IgG4-SC).Methods:Detect the serum IgG4 and CA19-9 of 41 clinical cases of IgG4-SC patients,162 clinical cases of non IgG4-SC patients and 40 healthy human serum samples by immunoassay and direct chemiluminescence methods, also detect the antinuclear antibodies (ANA),anti neutrophil antibody (ANCA),anti smooth muscle antibody (SMA) and anti mitochondrial antibody (AMA) of the above serum samples by indirect immunofluorescence and analyze the detection results.Results:①The positive rates of ANA,ANCA,SMA and AMA in patients with IgG4-SC were 41.46%,7.32%,0 and 2.44%.Among them,the positive rate of ANA was significantly different from that of the normal control group(P<0.01),and the positive rate of SMA and AMA was significantly different from that of non IgG4-SC group(P<0.01),and so as the positive rate of ANCA do with that of PSC group.②The number of serum IgG4 and CA19-9 increased samples were significantly compared with the normal control group (P<0.01);the area under the ROC curve (AUC) was 0.979 and 0.646,respectively,and P<0.05.Conclusion:The high level of serum IgG4 and CA19-9 and autoantibody detection are of great accuracy and important clinical value in the differential diagnosis of IgG4-SC.

Objective To understand the characteristics of sexual network of 321 HIV positive MSM and HIV infection status among sexual partners in Zhejiang Province. Methods A cross-sectional study selected HIV positive MSM diagnosed between September, 2015 and September, 2016 were conducted in Hangzhou, Ningbo, Wenzhou in Zhejiang Province using self-administrated questionnaire. Information related to socio-demographic characteristics, risky behavior, size of sexual network were collected. Results A total of 321 HIV positive MSM were recruited in this study. 71.76％ aged 20 to 40 years old and 51.71％ were native. The rate of self-reported sexual behavior with stable homosexual partners, casual homosexual partners, spouse and other heterosexual partners at investigation were 37.78％, 45.00％, 36.56％ and 66.67％. The rate of never using condom with stable homosexual partners, casual homosexual partners, spouse were 27.91％, 16.36％ and 44.00％. HIV positive rate among stable homosexual partners, casual homosexual partners, spouse and other heterosexual partners were 28.26％ , 3.77％, 11.43％ and 0.00％. Conclusion The size of sexual network was large, especially in casual sexual network. Unsafe sexual behavior, like consistent condom use was low and multiple sexual partners . HIV infection was high among stable homosexual partners and spouse.

OBJECTIVEUtilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment.

METHODSEukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method.

RESULTSThe eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition.

CONCLUSIONAntisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.

Bone Neoplasms ; metabolism ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; Luciferases ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Promoter Regions, Genetic ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.

METHODSEukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.

RESULTSThe eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.

CONCLUSIONSAntisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Bystander Effect ; Cell Hypoxia ; Cell Line, Tumor ; Cell Proliferation ; DNA, Neoplasm ; biosynthesis ; Ganciclovir ; pharmacology ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; Oligodeoxyribonucleotides, Antisense ; Osteosarcoma ; metabolism ; pathology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo detect the expression of protein 4.1 family members in mouse melanoma cell lines and evaluate their effect on cell proliferation.

METHODSPCR and Western blot were used to detected to the expression of protein 4.1 family members (4.1R, 4.1B, 4.1G, and 4.1N) at the mRNA and protein levels in B16 and B16-F10 cell lines. The expression plasmid vector pEGFP-N1-EPB41L3 carrying 4.1B gene sequence amplified from genomic RNA of mouse embryo fibroblasts was constructed and transiently transfected into mouse melanoma cells. The change in cell proliferation was assessed using MTT assay.

RESULTSThe mRNA and protein expressions of all the protein 4.1 family members, with the exception of 4.1B, were detected in both B16 and B16-F10 cells. Transfection of cells with the eukaryotic expression vector pEGFP-N1-EPB41L3 markedly inhibited cell proliferation as compared with the non-transfected cells.

CONCLUSIONThe eukaryotic expression vector carrying EPB41L3 sequence is capable of inhibiting the proliferation of mouse melanoma B16 and B16-F10 cells.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; Neuropeptides ; metabolism ; Plasmids ; Transfection