Objective To investigate the mechanisms for cytopathic effect(CPE) of wide-type rubella virus(RV)in ECV304 endothelial-like cells.Method ECV304 cells were cultured in MEM supplemented with 10% fetal calf serum,glutamine,sodium bicarbonate,penicillin and streptomycin at 37℃ with 5% CO-2 in a humidified incubator,and then grown in maintenance medium with 2% fetal calf serum to avoid overstimulating cell growth.Infected cells and control cells were harvested at day 2,4, and 6 after infection for following experiments.RT-PCR and indirect immunofluorescence were used to detect RV infection.Phase-contrast and electron microscopy were performed to analyze morphology changes in infected and uninfected groups.TUNEL assay was carried out to study wide-type RV-induced apoptosis.Result In RV-infected ECV304 cells,cytopathic effects were first observed approximately at 2-3 days post-infection.The majority of cells with CPE changes were of detachment from the monolayer,rounding of cells,cell shrinkage and membrane blebbing were seen.At 4-6 days post-infection,the detached cells increased clearly;the CPE regions on the monolayer were extended,and the infected cells in the CPE regions showed in spindly form with wide spacing.RT-PCR and indirect immunofluorescence results indicated RV infection in ECV304 endothelial-like cells.Using electron microscopy,RV particles and its cytopathic effects including the condensed chromatin,fragmented nuclei and clustering of mitochondria with dramatic changes around nuclear were further examined.TUNEL results showed that the difference of apoptosis index between the infected group and uninfected group was significant(P

Aim To further investigate the protective effect and mechanisms of total paeony glycoside (TPG) on apoptosis in experimental myocardial ischemic rats.Methods The myocardial ischemic model of rats was established by subcutaneous injection of isoprenaline (ISO), and early apoptosis rate of myocardial cells and the expression of apoptosis related genes Bax, Bcl-2 and proteins in myocardial tissues were analyzed and contrasted among five groups, including normal control, ISO injury, TPG prevention and treatment, TPG treatment and positive control.Results Compared with ISO injury group, all of indexes had improved in various degrees in TPG prevention and treatment group and TPG treatment group, such as the decrease of early apoptosis rate,expression of Bax gene and protein, the increase of Bcl-2 gene and protein and the ratio of Bcl-2 protein/Bax protein.Conclusion TPG has protective effect on apoptosis in ischemic myocardium induced by ISO.The pharmacological mechanism may relate to the activation of the Bcl-2 gene and protein expression, the inhibition of Bax gene and protein expression, as well as the increase of Bcl-2/Bax ratio.

BACKGROUND: It is demonstrated in modern pharmacologic study that total paeony glycoside (TPG) provides extensive pharmacologic activities,such as inhibiting aggregation of platelets and erythrocytes, anticoagulation,antithrombsis, anti-arterial sclerosis, protecting heart and liver, anti-tumor,etc.OBJECTIVE: Neonatal rat cardiomyocytes were cultured in vitro and by the changes of superoxide dismutase (SOD) activity, malondialdehyde (MDA) and nitric oxide (NO) contents in cell culture solution, the protection of TPG on injured cardiomyocytes was analyzed.DESIGN: Controlled observation was designed.SETTING: Bioengineering Department in School of Life Science and Technology of Xi 'an Jiaotong University and Institute of Bone Diseases in Medical School of Xi'an Jiaotong University.MATERIALS: The experiment was performed in Bioengineering Department in School of Life Science and Technology of Xi 'an Jiaotong University from February to June in 2003, in which, 44 SD neonatal rats aged 1-3 days were employed. The 48-hour-cultured cardioryocytes were prepared in 42 bottles and randomized into 6 groups, named normal control (normal group), medicated-injury group (injury group), TPG 0.625 mg/L group,TPG 3.125 mg/L group, TPG 15.625 mg/L group and positive control, 7 bottles in each group.METHODS: Cardiomyocytic primary culture was performed under aseptic condition. No any drug was used in normal group, isoprenaline was added in injury group to terminate the concentration at 100 mg/L, in TPG 0.625, 3.125 and 15.625 mg/L groups, 30 minutes after isoprenaline added, RGP at dosages of 0.625, 3.125 and 15.625 mg/L were added respectively; in positive control, 30 minutes after isoprenaline added, coenzyme Q10 was used to terminate the concentration as 100 mg/L.Afterwards, the assay of every index was performed. Xanthine oxidase (XOD) method was used to assay SOD activity, thiobarbituric acid (TBA)method was to assay MDA content and nitrate reductase (NR) method was to assay NO content.cell culture solution in each group.Compared with normal group, the levels of total SOD, CuZn-SOD and MnSOD were reduced remarkably in injury group (P ＜ 0.05 or P ＜ 0.01).The above-indexes in every TPG group and positive control were improved to different extents (P ＜ 0.05 or ＜ 0.01), in which, the protection of TPG 15.625 mg/L group was near to or superior to positive control [(79.50±10.67), (80.30±13.50); (48.24±13.26), (49.73±10.23); (31.26±10.22),in cell culture solution in each group: Those in injury group were higher remarkably than normal group (P ＜ 0.01). MDA and NO contents were all reduced in every TPG group and positive control and dose dependence presented in TPG protection, the higher the dose was, the stronger the action of TPG on protection was, in which, in high-dose group, MDA content was near to normal group [(5.41±1.81), (4.48±0.94) μmol/L, P ＞ 0.05] and NO content was similar to positive control [(81.83± 9.08), (82.41±12.37) mol/L,P ＞ 0.05].CONCLUSION: TPG protects myocardial injury induced by isoprenaline,indicating dose-dependence relationship, which is probably associated with enhanced anti-oxidation of cell, reduced injury of cellular membrane induced by free radical and lipid oxidant.

AIM: To explore the expression of nuclear factor-?B(NF-?B) in asthmatic guinea pigs, and the effect of erigeron breviscapus, a protein kinase C(PKC) inhibitor, on the expression of nuclear factor-?B(NF-?B). METHODS: 48 guinea pigs were randomly divided into 6 groups ( n= 8). Airway resistance and eosinophilic inflammation of airway wall were examined, the expression of NF-?B in the lung tissue was detected by immunohistochemical staining. RESULTS: The expression of NF-?B was mainly found in airway epithelium, all the asthmatic animals showed significantly higher optical densities than that of the normal control group( P

Aim To investigate the antioxidative effect of total paeony glycoside(TPG) on cardiomyocytes injured.Methods The ischemia and hypoxia injuy model of cultured neonatal rat cardiomyocytes was induced by adding isoprenaline(ISO), and superoxide dismutase(SOD), malonalde hyde(MDA) and nitric oxide(NO) in the culture solution of normal control group; ISO injure group, CoQ 10 positive control group as well as protective group s with high,middle or low-dose TPG were respectively analyzed and compared. Results Compared with normal control group,the enzyme activity o f total SOD, CuZn-SOD and Mn-SOD decreased obviously, and the content of MDA and NO increased markedly in injury group, but in TPG and CoQ 10 groups all of detective indicators had improvement in varying degrees, and the protective effect was better than or close to positive control group in high-dose TPG grou p.Conclusion TPG has protective action on injured cardiomyocyte s induced by ISO in dose-dependent manner. The mechanism relates to the enhance ment of antioxidative effect in cells, and the reduction of membrane damage caus ed by free radical and lipid peroxide.

Objective:To understand the status of outpatients′medical care and their impact on the doctor -pa-tient trust in Beijing .Methods:Three tertiary hospitals were selected due to most concentrated high -quality medi-cal resources , the largest number of patient visits , universalism and particularism trust could be compared , taking field observations , personal interviews and questionnaires methods .Results:The main factors affecting clinical doctor-patient trust are time, the doctor skills, medical ethics, medical and prescription drug division , etc.are appropriate and effective .Conclusion:Play the role of pyramidmedical system′s overall effectiveness , and es-tablish a patient-centered system for outpatient care as soon as possible a comprehensive grasp of the overall con -struction team doctor , continue to strengthen the standardized management of medication .

OBJECTIVETo explore the role of nuclear factor-kappa B (NF-kappa B) in the signal conduction of protein kinase C (PKC) regulated proliferation, apoptosis and expression of Th2 cytokines -- interleukin-4 (IL-4) and interleukin-5 (IL-5) of T lymphocytes in the bronchial alveolus lavage fluid (BALF).

METHODST lymphocytes were isolated and purified from BALF of asthmatic guinea pigs in normal and asthmatic groups, and were stimulated with PKC agitator phorbol 12-myristate 13-acetate (PMA) and NF-kappa B inhibitor pyrrolidine dithiocarbamate (PDTC), respectively. The expressions of NF-kappa B, IL-4 and IL-5 mRNA and protein, the proliferation and apoptosis of T lymphocytes were observed by immunohistochemistry, in situ hybridization, ELISA, MTT and TUNEL, respectively.

RESULTSThe activation of NF-kappa B, proliferation response, and expression of IL-4 and IL-5 mRNA and protein in T lymphocytes stimulated by PMA were significantly higher than those of their blank control (P < 0.01), while those indexes of T lymphocytes stimulated by PMA and PDTC simultaneously were significantly lower than those stimulated by PMA alone (P < 0.01). The apoptotic index of T lymphocytes stimulated with PMA were significantly lower than that of their blank control (P < 0.01), and the apoptotic index of asthmatic guinea pig T lymphocytes stimulated with PMA and PDTC simultaneously were significantly higher than that stimulated by PMA alone (P < 0.01). The significant positive correlations were found between the activation of NF-kappa B and the proliferation (r = 0.64, P < 0.001), and the expression of IL-4 and IL-5 mRNA and protein of T lymphocytes, respectively (r = 0.55 - 0.68, P < 0.001). There was also significant negative correlation between the activation of NF-kappa B and apoptosis of T lymphocytes (r = 0.62, P < 0.001).

CONCLUSIONSNF-kappa B may participate in the signal conduction of PKC regulated proliferation, apoptosis and expression of IL-4 and IL-5 of T lymphocytes in asthma. The activation of NF-kappa B in PKC signal conduction pathway of T lymphocytes may play an important role in the pathogenesis of asthma.

Animals ; Apoptosis ; drug effects ; Asthma ; metabolism ; pathology ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Division ; drug effects ; Cell Nucleus ; metabolism ; Disease Models, Animal ; Guinea Pigs ; Immunohistochemistry ; In Situ Hybridization ; Interleukin-4 ; genetics ; metabolism ; Interleukin-5 ; genetics ; metabolism ; Male ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Protein Kinase C ; metabolism ; Pyrrolidines ; pharmacology ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Signal Transduction ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology ; Thiocarbamates ; pharmacology

OBJECTIVE: To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-). METHODS: Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors. RESULTS: The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰. CONCLUSION A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.
Humans ; Blood Group Antigens/genetics* ; Genotype ; Genotyping Techniques ; Heterozygote ; Alleles ; Blood Donors ; Rh-Hr Blood-Group System/genetics*

AIM:To explore the effects of CD38 on lysosome reformation and cholesterol efflux in macro-phages.METHODS:Bone marrow-derived macrophages from low-density lipoprotein(LDL)receptor knockout(LDLr-/-)mice were cultured as cell model.Live cell imaging system was applied to evaluate the effect of nicotinic acid adenine di-nucleotide phosphate(NAADP)on lysosome number.ELISA was conducted to measure NAADP level in macrophages.After the cells were treated with nicotinic acid(NA),RT-qPCR was conducted to detect CD38 mRNA expression,and Western blot was conducted to observe CD38 protein expression and phosphorylated transcription factor EB(TFEB)level.Laser scanning confocal microscopy was applied to evaluate the influence of CD38/NAADP signaling on lysosome number and cholesterol egression.RESULTS:NAADP remarkably increased lysosome number(P<0.05),and this effect was significantly inhibited by NAADP antagonist NED-19,Ca2+ chelator BAPTA,and calcineurin inhibitor CsA(P<0.05).CD38 markedly enhanced NAADP synthesis in macrophages(P<0.05).NAADP synthetic substrate NA prominently ele-vated the expression of CD38 mRNA and protein(P<0.05).NA significantly decreased the phosphorylated TFEB level;this effect was also attenuated by NED-19,BAPTA and CsA(P<0.05).Disrupting CD38/NAADP signaling pathway markedly inhibited NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux in macrophages(P<0.05).NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux abolished in LDLr/CD38 DKO macrophages(P<0.05),whereas these effects induced by NA were recovered after CD38 gene rescue.CONCLUSION:CD38 triggers lysosome reformation via TFEB and consequently pro-motes the efflux of lysosomal free cholesterol and cytosol cholesterol ester.