Objective To study the effect of overexpression of TRPC6 on Ang Ⅱ-induced apoptosis of mouse podocytes in vitro and to explore the possible mechanisms. Methods Mouse TRPC6 cDNA eukaryotie expression vector pEGFP-NI-mTRPC6 was transfected to conditionally immortalized routine podocyte cell line by liposome. The fluorescent microscopy was used to examine the expression of EGFP after 24 hours. The change of TRPC6 protein expression was observed by Western-blot. Podocytes were treated by different concentrations of Ang Ⅱ. The podocyte intracellular calcium concentration was measured with laser-scanning con_focal microscope. The expression of Bax and Bcl-2 mRNA was assessed by RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot. The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst staining. Results About 35% of the cells expressed EGFP. An up-regulation of protein expression of TRPC6 was detected in podocytes when transfected with pEGFP-N1-mTRPC6 (P<0.01). The overexpression of TRPC6 promoted the Ang Ⅱ-induced influx of extracellular calcium and elevated the expression of Bax but decreased the expression of Bcl-2 (P<0.01, P<0.05). The apoptotic ratio of podocyte was (2.50±0.72)% when treated by low-dose Ang Ⅱ (10-10 mol/L), and it was increased to (4.33±0.45)% when transfected with pEGFP-N1-mTRPC6 (P <0.05 ). Transfection with pEGFP-NI-mTRPC6 increased apoptosis rate from (15.46± 1.40)% to (18.33±0.87)%(P<0.01) by high-dose Ang Ⅱ (10-6 mol/L). Conclusion TRPC6 plays an important role in the Ang Ⅱ-induced apoptosis of podocytes by promoting the influx of extraeellular calcium, which leads to the apoptosis cascade initiation.

Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside (PAN)-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN treatment+shRNA transfection group, and PAN treatment+ negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.

OBJECTIVETo study HBV preS2/S gene expression effects in mammalian cells transferred with recombinant adenoviral vector.

METHODSThe replication-deficient recombinant adenoviral vector (Ad-HBs) carrying HBV preS2/S gene were constructed by homologous recombination in bacteria. The 293 cells, Vero cells, HepG2 cells and mesenchymal stem cells (MSCs) were infected with adenoviruses. The expressions of enhanced green fluorescent protein (EGFP) were observed with fluorescence microscope and the expressions of HBsAg were detected by RT-PCR and ELISA in vitro.

RESULTSMore than 90% of 293 cells, Vero cells, HepG2 cells or MSCs expressed EGFP after transfection at the MOI of 20 and the titers of HBsAg were more than 3.229 (A value) in culture supernatant.

CONCLUSIONThe HBV preS2/S gene was not only expressed efficiently in immortalized cells, but also expressed efficiently in stem cells with the recombinant adenoviruses vector.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cell Line, Tumor ; Cercopithecus aethiops ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Humans ; Microscopy, Fluorescence ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vero Cells

OBJECTIVETo explore whether hepatitis B virus (HBV) S gene-modified dendritic cells (DCs) might induce a specific cytotoxic T lymphocyte (CTL) response.

METHODSThe recombinant adenoviruses carrying HBsAg genes were prepared and used to transfect DCs generated from cord blood. The efficacy of transfection was observed through the expression of enhanced green fluorescent protein (EGFP) in DCs and the expression of HBsAg was detected by ELISA. HBV S gene-modified DCs were co-cultured with T cells from cord blood and T cells stimulating activities were detected using mixed lymphocyte reaction (MLR). The CTL assay was carried out to assess the ability of CTL lines to lyse target cells of HepG(2)22.1.5 by measuring lactate dehydrogenase (LDH) release.

RESULTSThe results showed that HBV S genes were expressed in DCs with high efficacy by recombinant adenoviral vector. DCs had a normal shape after transfection. The result of MLR showed that HBV S gene-modified DCs could effectively stimulate naive T cells to proliferate. The induced specific CTL lines could lyse target cells of HepG(2)22.1.5.

CONCLUSIONSHBV S gene-modified DCs enhanced the function to induce a specific CTL effect, showing its promise for developing anti-viral vaccine in future.

Cell Line ; Cells, Cultured ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; virology ; Hepatitis B ; immunology ; virology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Cytotoxic ; immunology

Objective To investigate the variance of cost-effectiveness when treat acute myocardial infaretion of different severe extents with different pattern.Methods Acute myocardial infarction patients were selected from emergency eommand center of Guangzhou from October 2003 to December 2005.These patients wew assigned by the center to First-Class Hospitals at Grade 3 and First-class Hospital at Grade 2,and were followed up after 6 months after post-discharge.Cost in hospital and mortality in hospital were registered.The health of all patients were quantificated using SF-36.According to the assigned hospitals,the patients were divided into single infarction group and complex infarction group.Cost in hospital,mortality in hospital,short-term quality of life were compared between the them.Results Compared with and First-Class Hospital at Grade 2 (101 cases),the single infarction patients in First-Class Hospitals at Grade 3 had higber costs in hospital (P=0.016),better society function,affection role,mental health and health status (P

Objective To construct a recombinant adenoviral vector carrying HBcAg-HSP70 chimeric gene by homologous recombination in bacteria and to detect its expression in vitro. Methods Heat shock protein 70 gene from Mycobacterium tuberculosis were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV-HBsAg. Then the resultant pAdTrack-CMV-HBsAg-HSP70 was eotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBsAg-HSP70 gene (pAd-HBsAg-HSP70) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells (293 cells and Vero cells) were infected with adenoviruses and the expression of HBsAg-HSP70 was detected by RT-PCR and ELISA in vitro. Results The adenoviral plasmids pAd-HBsAg-HSP70 were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac Ⅰ analyses. The recombinant adenoviruses Ad-HBsAg-HSP70 were packaged successfully in 293 cells. The titer of Ad-HBsAg-HSP70 was up to 2 × 1012 pfu/L after the second passage of proliferation in 293 cells. HBsAg and HSP70 were expressed efficiently in mammal cells after infection. Conclusion The recombinant adenoviruses expressing HBsAg and HSP70 were constructed successfully which can be used further in study of gene therapy for HBV.

Objective To test the infeciton efficiency of recombinant adenovixal vector carrying HBsAg-HSP70 chimeric gene and to abserve its biological characteristics. Methods Peripheral blood mononuclear cells (PBMC) were separated from healthy blood donor and they were infected by Ad-HSP70-HBsAg on the first day of isolation. DCs were induced in medium with cytokines IL-4, GM-CSF and TNF-α in vitro. The biological characteristics of DC induced were analyzed by inverted fluorecent microscope, RT-PCR, flow cytometer(FACS), and mixed lymphocyte reaction (MLR). Results The traced gene-GFP were abserved in DCs by inverted fluorecent microscope and HSP70-HBsAg gene mRNA expression was detected by RT-PCR after the Ad-HSP70-HBsAg infection. FACS analysis shown that the expression of CD1a,CDSO,CD86 and HLA-DR on surfece of two groups of DCs were similar. MLR showed that there are not a statitie difference of stimulated index (SI) between two groups.Conclusion Results indicated that Ad-HSP70-HBsAg can effectively infected DCs without affecting its biological characteristics.

Objective To discuss the effect and nursing of oxygen nebulization and compressed air nebulization on capillary bronchitis. Methods According to check-in time, 129 children with capillary bronchitis were divided into oxygen nebulization group and compressed air nebulization group. Treatment effect and nursing was observed and compared. Results Effective rate was 96.87 ％ in the oxygen nebulization group and 98.46％ in the compressed air nebulization group. There was no significant difference between the two groups (X2 = 0. 457, P ＞ 0. 05). Conclusions The two types of nebulization could both treat capillary bronchitis. However, compressed air nebulization shows obvious advantages considering its safety, economy,simple operation and convenient carrying, thus presents strong practicability.

OBJECTIVETo establish a sequential antiviral regime and evaluate its efficacy in patients with chronic hepatitis B using a controlled trial.

METHODSSeventy-four patients with chronic hepatitis B were divided into 3 groups: 30 cases were enrolled in the sequential antiviral group in which patients received eight-week treatment with thymosin alpha1 (1.6 mg/time, subcutaneous injection, 2 times/week), six-month treatment with interferon (500 MU/ times, muscle inject, every other day) begun in the fifth week of the therapeutic course, and lamivudine treatment (100 mg/days) begun 2 months later after HBeAg seroconversion or just after the withdrawal of interferon to more than eighteen months. Fourteen cases were enrolled in combination group in which patients received six-month treatment with interferon and thymosin alpha1 simultaneously in the same manner as in sequential antiviral group. Thirty cases were enrolled in lamivudine group in which patients received more than eighteen-month treatment with lamivudine.

RESULTSThe temporary rates of HBeAg seroconversion and normalization of alanine aminotransferase (effective rate) in sequential antiviral group, combination group and lamivudine group were 76.7%, 78.6% and 13.3%, respectively. The effective rates of sequential group and combination group were very similar, and significantly higher than that of lamivudine group (P less than 0.01). Long-term efficacy rates were 76.7%, 57.1% and 16.7% among the three groups, respectively. The long-term effective rate of sequential group was relatively higher. The rate of liver damage sensitive period in sequential antiviral group and combination group was 47.7%. The time of onset was from 2 to 8 weeks after the treatment begun, earlier than that from 6 to 8 weeks after the beginning of interferon alone in the literature.

CONCLUSIONSequential antiviral therapy had much higher rates of long-term HBeAg seroconversion, undetectable HBV DNA and normalization of alanine aminotransferase with good cost-effectiveness. Its mechanism to promote the antiviral effect might be dependent on the immunoregulatory action of thymosin alpha1 in the earlier period and the specific inhibition of HBV DNA replication by lamivudine in the later period of the therapeutic course.

Adjuvants, Immunologic ; administration & dosage ; Antiviral Agents ; administration & dosage ; China ; Drug Therapy, Combination ; Hepatitis B, Chronic ; drug therapy ; Humans ; Interferon-alpha ; administration & dosage ; Lamivudine ; administration & dosage ; Thymosin ; administration & dosage ; analogs & derivatives ; Treatment Outcome

Objective: To evaluate the effects of dietary behaviors on the risk of hypertension, diabetes and cardiovascular diseases. Methods: A total of 12 208 subjects aged 18-60 years old were investigated by questionnaires to collect demographic data, dietary behaviors and lifestyle information, when they did health examination in a tertiary hospital in Beijing from 2014 to 2019. During the observation period of five year, the incidence of hypertension, diabetes and cardiovascular diseases were collected through health examination files every year. The multivariate logistic regression model was employed to analyze the associations of dietary behaviors with hypertension, diabetes and cardiovascular diseases. Results: The study included 6 218 ( 50.93% ) males and 5 990 ( 49.07% ) females. The cumulative incidence rates of hypertension, diabetes and cardiovascular diseases were 7.75%, 2.72% and 3.49%, respectively. The multivariate logistic regression analysis indicated that the high-sodium diet ( OR=1.422, 95%CI: 1.191-1.697 ) , eating fast ( OR=1.457, 95%CI: 1.102-1.974 ), eating more refined grain ( OR=1.251, 95%CI: 1.050-1.490 ) and drinking milk less than once a week ( OR=1.316, 95%CI: 1.022-1.697 ) were risk factors for hypertension. The high-sodium diet ( OR=1.344, 95%CI: 1.048-1.725 ), eating fast ( OR=1.733, 95%CI: 1.046-2.871 ), eating more meat ( OR=1.651,95%CI: 1.263-2.158 ) were risk factors for diabetes. High-sodium diet ( OR=1.501, 95%CI: 1.192-1.889 ) was risk factors for cardiovascular disease. Conclusion The diet with high sodium, more meat and refined grain as well as eating fast can increase the risk of hypertension, diabetes and cardiovascular diseases.