Objective To investigate the effects of extracellular-signal regulated kinase (ERK) on hypoxic-ischemic brain damage of neonatal rats.Methods The hypoxic-ischemic brain damage model of neonatal Wistar rats was established as following:first the right common carotid artery of the rats was ligated;2 h after operation,the rats began to inhale 8%-oxygen oxygen-nitrogen gas mixture lasting for 2 h.Rats were randomly divided into three groups:sham-group,hypoxic-ischemic group and ERK inhibitor PD98059 group (the rats were injected PD98059 2 mg/kg 10 min before the ligation).Six hours after the models were done,hippocampi and cortex of the ligation side of rats in the three groups were collected,and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured.Apoptosis of neuron was assessed by TUNEL staining.The expression of ERK1,ERK 2,Bcl-2 and Bax were examined by Western blot.The differences among the groups were analyzed with ANOVA and q test.Results Compared with the sham-group,the MDA level [(342.9± 10.8) μmol/L vs (181.5± 17.0) μmol/L,q= 6.35,P＜0.01) and the apoptosis rate of neuron [(18.80±1.37)% vs (3.53±0.34)%,q=6.06,P＜0.01) of hypoxic-ischemic group was higher,and SOD level was lower [(34.8±4.3) U/ml vs (63.4±4.3) U/ml,q=4.99,P＜0.01].While the apoptosis rate of neuron [(15.53±0.64) %] and MDA level [(252.0± 17.1) μmol/L] of PD98059 group were lower than those of hypoxic-ischemic group(q=3.87 and 5.28,P＜0.01respectively),the SOD level [(51.5 ± 3.8) U/ml] was higher than that of hypoxic-ischemic group (q=4.17,P＜0.01).There were no differences of ERK1 and ERK2 expressions among the three groups.The phosphorylated ERK1 and ERK2 levels of hypoxic-ischemic group were higher than those of sham-group (q=3.82 and 4.08,P＜0.01) and PD98059 group (q=4.79 and 5.12,P＜0.01).The expression of Bcl-2 and Bax of hypoxic-ischemic group were higher than those of sham-group (q=3.55 and 3.42,P＜0.01).Compared with hypoxic-ischemic group,Bcl-2 expression (q=3.71,P＜0.01) of PD98059 group was higher,and Bax expression (q=5.86,P ＜ 0.01) was lower.Conclusions ERK is involved in hypoxic-ischemic brain damage of neonatal rats through regulating the expression of apoptosis protein.

ObjectiveTo observe the influence of calcium - sensing receptor (CaSR) on Fas/Fas ligand (Fas L) pathway during anoxia/reoxygenation (A/R)- induced cardiomyocytes apoptosis in neonatal rat. MethodsSingle cells were dissociated from minced hearts of 2 - day - old Wistar rats with a 0. 25％ solution of crude trypsin and then cultured as monolayers at a density of 5 x 104cells/cm2 in DMEM medium equilibrated with humidified air containing 5％ CO2 at 37C. Three days after the cells were seeded, the cultured cardiomyocytes were randomly divided into three groups. ( 1 ) control group: cardiomyocytes were continuously cultured for 26 hours in DMEM medium.(2) A/R group: cardiomyocytes underwent anoxia for 2 hours and reoxygenation for 24 hours.(3) GdCl3 group: 300μmol/L GdCl3 was added to the culture medium at the beginning of reoxygenation. Apoptosis of cardiomyocytes was assessed by flow cytometer and morphological alterations were observed with transmission electron microscope.The expression of CaSR, Fas and Fas L were analyzed by Western blot.Results The result of flow cytometer showed that cardiomyocytes apoptosis was 12. 18％ ± 1.54％ in A/R group,and was higher than that in the control group (P ＜0. 01 ). At the same time, mitochondrial cristae dissolution and disappearance could be detected. Compared with A/R group, GdCl3, a specific activator of CaSR, further enhanced cardiomyocytes apoptosis to 20. 25％ + 2. 87％ ( P ＜ 0. 01 ), along with an increment in CaSR, Fas and Fas L expressions ( P ＜ 0. 05 ). ConclusionCaSR is closely involved in cardiomyocytes apoptosia during anoxia/reoxygenation. CaSR could induce apoptosis of neonatal rat cardiomyocytes through Fas/Fas L receptor pathway.

Objective Calcium-sensing receptor(CaSR)belongs to the family C of G-protein coupled receptors.This study was carried out to observe the influence and mechanism of CaSR on anoxia/reoxygenation(A/R)-induced cardiomyocytes apoptosis.Methods The model of A/R injury was established through anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rats.Cardiomyocytes were randomly divided into three groups:control group,A/R group and GdCl3 group(300μmol/L GdCl3 was added to the culture medium at the beginning of reoxygenation).Apoptosis of cardiomyocytes was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL).The expression of CaSR,cysteine-requiring aspartate protease(caspase)-3,9 and cytochrom c (Cytc)were analyzed by Western blot.Results The TUNEL showed that cardiomyocytes apoptosis was 17%±3% in A/R group,and was higher than that in the control group.At the same time,expression of CaSR in A/R group was markedly increased in response to control group.Compared with A/R group,GdCl3,a specific activator of CaSR,further enhanced cardiomyocytes apoptosis to (28±4)% and decreased the ability of cardiomyocytes to (51.2±6.8)%,along with an increment in CaSR,caspase-3,caspase-9 and Cytc expressions.Conclusion CaSR is involved in anoxia/reoxygenation-induced apoptosis of neonatal rat cardiomyocytes through Cytc-caspase-3 pathway.

Objective Our previous research demonstrated that trkA and p75 receptors of nerve growth factor(β-NGF) are expressed in human pterygium fibroblasts(HPF), and trkA is expressed only in conjunctiva. The purpose of present study was to investigate the effects of β-NGF on proliferation of HPF and analyse the pathogenesis mechanism of pterygium. Methods The HPF specimen was obtained from Union Hospital of Tongji Medical College, Huazhong University of Science and Technology during the surgery. Explant culture technique was used for the primary culture of HPF tissue. The cells of confluenting 80% were collected and digested using 0. 25% tripsin + 0. 02% EDTA (1:1) and the third to fifth generation of cells were utilized in the experiment. Different concentrations of β-NGF was added in medium. Cultured cells were identified using vimentin, keratin and α-SMA. MTT was used to determine the proliferation of HPF after addition of β-NGF. The expression of trkA and p75 in HPF was detected by immumofluorescence method. Cell proliferation also was semi-quantitatively analyzed by detect of expressions of PCNA protein and mRNA in HPF using Western blot and RT-PCR. Results Cultured HPF cells showed the positive responses for vimentin, α-SMA, trkA and p75 but absent reaction for keratin. MTT revealed that the OD value of HPF cells was gradually enhanced with the increase of β-NGF concentration in 12, 24, 48, 72 and 96 hours after β-NGF action with the maximum stimulation at 48 hours. The expression of PCNA protein and mRNA in HPF was significantly different among various concentrations of β-NGF groups(F_(protein) = 24. 980, P = 0. 000; F_(mRNA) = 64. 490, P = 0. 000) and increased from 5 ng/mL β-NGF group through 50 ng/mL β-NGF group in comparison with 0 and 1 ng/mL β-NGF group (P ＜ 0. 05) . Conclusion The findings demonstrate the potential proliferative effect of β-NGF binding to trkA and p75 on HPF.

Objective To investigate the inhibitory effect of downregulation of hepatitis B virus (HBV) core gene (HBcAg) expression by RNA interference and magnetic nanoparticles on both HBV DNA replication and expression in vitro. Methods HepG2 2.2.15 cells were transfected with U6 promoter plasmids coding for small interfering RNA (siRNA) targeting HBV core gene using magnetic nanoparticles. RT-PCR and Western blot were used to assess the mRNA and protein expression HBV core antigen. Real-time PCR was used to evaluate the suppression efficiency of HBV-DNA replication and expression; and radioimmunoassay was used for HBV surface antigen (HBsAg), core antigen (HBcAg), and e antigen (HBeAg) detection. Results We successfully constructed nanoparticles with siRNA plasmid targeting HBV core antigen; HBcAg mRNA and HBV core antigen protein levels were significantly reduced in the transfected cells. HBV-DNA downregulation was estimated at 4-5 logs and the HBsAg and HBeAg levels were also reduced compared with the controls. Conclusion Downregulation of HBV core gene using RNAi technology and magnetic nanoparticles can potentially be used as a therapeutic strategy for Hepatitis B.

Objective To evaluate the application of plasma drug monitoring in pediatric HIV/AIDS patient antiretroviral therapy adherence monitoring.Methods Totally 261 plasma samples and related information were collected from three consecutive follow-up visits of 87 HIV-infected children treated in Shangcai county CDC of Henan province from March to October 2009.The plasma concentrations of antiretroviral drugs were measured by a developed high performance liquid chromatography-mass spectrometry method.Potential adherence influencing factors, such as regimen, age, gender, parent conditions, previous ART exposure and therapy duration, were analyzed by univariate logistic regression.Results Plasma concentration of antiretroviral drugs lower than LLTR (1 000 ng/ml) was the criteria to identify missed dose.The concentrations of 28 plasma samples were lower than LLTR, which meant missing dose.There were 17 patients (19.5%) with their concentrations lower than LLTR at least once in three follow-up visits.Logistic regression analysis of adherence related factors showed that compared with the children whose parents were both alive, the children whose mother and (or) father died were more likely to miss dose.The odds ratio was 4.13(95% credibility interval:1.37-12.46, P values was 0.012).Conclusions HIV-infected children have adherence problems when receiving antiretroviral therapy.Plasma therapeutic drug monitoring can be one of the effective methods to monitor the adherence.

OBJECTIVETo investigate the quantitative analysis based on three-dimensional computed tomography (3D-CT) in contouring surgery of complex craniofacial fibrous dysplasia (FD).

METHODS14 patients with craniofacial FD underwent 3D-CT scan. Axial images of patients with craniofacial FD were reconstructed into 3D model by using Mimics 10.0. Anatomical landmarks were located and the coordinate of the landmarks obtained. The differences between the right landmarks and the left were calculated and analyzed. Quantitative contouring surgery was performed based on the quantitative analysis result.

RESULTSWith the detail data from the 3D-CT analysis, the surgery of contouring was more safe and accurate with less operation time, less bleeding and good results.

CONCLUSIONSThe method of 3D CT quantitative analysis can provide precise information in the diagnosis and treatment planning of craniofacial deformity. Based on the result of 3D-CT quantitative analysis, the operations can be performed more accurately and safely with good symmetric consequence.

Aged ; Craniofacial Abnormalities ; diagnostic imaging ; Facial Bones ; abnormalities ; diagnostic imaging ; Fibrous Dysplasia, Polyostotic ; diagnostic imaging ; Humans ; Imaging, Three-Dimensional ; methods ; Tomography, X-Ray Computed ; methods

OBJECTIVETo compare the photosynthetic characteristics difference of different ploidy Rhodiola sachalinensis germplasm and provide the scientific basis for their cultivation.

METHODLI-6400/XT photosynthesis system was used to measure leaf light response curve and CO2 response curve of diploid and autotetraploid. Biomass, leaf area, stomatal characteristics and chlorophyll content differences were compared in the study.

RESULTStomata of the two germplasms were open during daytime obviously, and stomata conductance responded to the changes of light intensity and CO2 concentration which was not consistent with the characteristics of CAM (crassulacean acid metabolism) plants. Light compensation point of autotetraploid was significantly lower than that of the diploid, and light saturation points of both germplam were close, and their light saturation points were near 500 micromol x m(-2) x s(-1). Quantum efficiency of autotetraploid was significantly higher than the diploid, and the net photosynthetic rate of autotetraploid significantly higher than the diploid when light intensity was higher than 500 micromol x m(-2) x s(-1). Stomata conductance, transpiration rate of autotetraploid was also significantly higher than that of diploid. Biomass, leaf area, stomata diameter and chlorophyll content of autotetraploid were much higher than that of diploid, while the stomata density of autotetraploid was less than diploid.

CONCLUSIONThe results above provide scientific basis for the cultivation of different ploidy Rh. sachalinensi germplasm.

Carbon Dioxide ; metabolism ; Photosynthesis ; physiology ; Ploidies ; Rhodiola ; metabolism

Objectives: To establish an effective method to isolate germinal vesicle frommammalian oocyte and make the isolated germinal vesicle undergo meiotic resumptionin vitro. Methods: The germinal vesicles were isolated from oocytes directly througha physical method by drawing oocyte in and out of fine-bore pipette.The isolatedgerminal vesicles were cultured in cell-free system which was prepared from the celllysate of synchronized HeLa cells.DNA fluorescence dye Hoechest 33342 was used tomonitor the condensation of chromatin during germinal vesicle breakdown. Results:The cell lysate of metaphase HeLa cell induced chromatin condensation while the cellextract of early G2 phase could not. Conclusions: The germinal vesicle can beisolated successfully in such a physical method and it can undergo chromatincondensation in vitro just as the oocyte does in vivo. Natl J Androl,2001,7(2):79～83

OBJECTIVETo compare the clinical characteristics of rifampin-dependent (R-dependent Mycobacterium tuberculosis) and rifampin-resistant (R-resistant Mycobacterium tuberculosis) patients with pulmonary tuberculosis.

METHODSThe clinical data including the demographic data, age groups, course of disease, history of chemotherapy with anti-TB drugs, and results of drug susceptibility test were collected from 61 cases of R-dependent pulmonary tuberculosis and 148 cases of R-resistant pulmonary tuberculosis treated between October, 2008 and January, 2012.

RESULTSMost of the R-dependent and R-resistant patients were between 30 and 44 years of age. The R-dependent patients included 12 receiving the first treatment patients and 49 with previous treatments, and the R-resistant patients included 11 without and 137 with previous treatments. The multi-drug resistant rate was 80.3% in R-dependent group, as compared to 92.6% in R-resistant group.

CONCLUSIONMost of the patients infected with R-dependent Mycobacterium tuberculosis are young or middle-aged, often having serious disease conditions. Detecting rifampin dependence is important for patients with initial treatment failure. Multi-drug resistance therapy guideline should be applied to patients infected with R-dependent Mycobacterium tuberculosis to improve the cure rate.

Adolescent ; Adult ; Aged ; Antitubercular Agents ; pharmacology ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; drug effects ; Rifampin ; pharmacology ; therapeutic use ; Tuberculosis, Multidrug-Resistant ; drug therapy ; microbiology ; Tuberculosis, Pulmonary ; drug therapy ; microbiology ; Young Adult