50 000 bp)was extracted from frozen gastric cancer tissue and their corresponding normal samples and used for RLGS assay.The genome DNA was digested by methylation-sensitive restriction enzyme Not Ⅰ, and labeled by radioisotope ~(32)p,then separated by two-dimensional electrophoresis and autoradiography. Experimental conditions for each step were optimized step by step.DNA fragment sequences for the dots on scanning profile were identified according to the human RLGS database.Results RLGS assay was set up and used to get the RLGS profiles of the representative tested samples successfully.These profiles can display more than 1 200 available dots averagely,the profiles of high quality DNA sample can display more than 1 800 dots which is the average level at an excellent RLGS lab,discrepant dots which had weaken or enhanced signals and their sequence information were obtained.The result can be reproduced.Conclusion The RLGS assay is established,stabilized for detection of DNA methylation of tissue samples.

Objective To understand the attitude of health workers towards people living with HIV and AIDS(PLWHA) and AIDS knowledge and desire for related training among them in selected hospitals in Beijing.Method Convenience sampling was used to select health workers from 32 hospitals of different levels in 4 districts within Beijing.Anonymous self-administrated questionnaires were used to collect the information regarding AIDS knowledge and desire for relevant training.Results A total of 1 292 health workers completed the questionnaire.They demonstrated a moderate level of general AIDS knowledge,but a low level of knowledge related to AIDS diagnosis and therapy.Of them 39.7% had received specialized AIDS training and 96% had strongly desire for such specialized training.Most health workers sympathized with PLWHA and understood their situation,while some of them were afraid of PLWHA and wanted to have minimal contact with PLWHA.91.3% of the health workers expressed that PLWHA should have the same rights to medical services.At the same time,49.2% of the health workers stated that they would not like to work in hospitals or departments which accepted PLWHA.80% of the health workers expressed concern over acquiring HIV infection in the workplace.52.7% of them thought that they had a risk of becoming infected in the workplace.Conclusion The level of AIDS knowledge among health workers in Beijing needs to be improved.Specialized training to enhance and develop health workers knowledge of prevention,diagnosis and treatment of AIDS is required.Most of the health workers do not discriminate against PLWHA,but they express concern over infection in the workplace.

OBJECTIVETo study the overexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells for repairing articular cartilage injury in vivo.

METHODSRabbit bone marrow mesenchymal stem cells (BMSCs) were transduced with lentivirus vector containing Sox9 gene and then cartilage specific molecule was detected by RT-PCR in vitro. Total 48 knee joints of 24 mature New Zealand white rabbits were randomly divided into 3 groups according to different defect treatment. After animals anesthesia,a full-thickness cylindrical cartilage defect of 4 mm diameter and 3 mm deep was created in the patellar groove using a stainlesssteel punch. Meanwhile, the transfected cells were implanted to repair the rabbit model with full-thickness cartilage defects. Cartilage defects tissue was observed with light microscope, electron microscope, HE and immunohistochemistry staining to assess the repair of defects by the complex at 6 weeks or 12 weeks after the implantation.

RESULTSAt 3 days after the transfection, Sox9 gene expression was highest and Sox9 gene expression decreased with the increase of time. At 3 days after the transfection, the expression of collagen type II began and reached the peak at 14 days. It showed that the bone marrow mesenchymal stem cells went into chondrogenic differentiation after transfected by Sox9 gene. Histological observation showed that at 6 weeks after the operation, the defects in the experimental group was filled with hyaline like cartilage tissue, 12 weeks after operation,the defects of cartilage and subchondral bone had satisfactory healing. Both at 6 and 12 weeks postoperatively, the defects were filled with fibrous tissues in control groups. Meanwhile, immunohistochemical staining of sections with type II collagen antibodies showed the proteins in the regenerated tissue stained positive for type II collagen and stronger than the control groups. The histological scoring system indicated that the cartilage repair of experiment groups were better than the two control groups with statistical significances.

CONCLUSIONOverexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells (BMSCs) promote the repair of cartilage defect.

Animals ; Bone Marrow Cells ; metabolism ; Bone Marrow Transplantation ; Cartilage, Articular ; injuries ; metabolism ; Cell- and Tissue-Based Therapy ; Female ; Genetic Vectors ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; metabolism ; Osteoarthritis ; genetics ; metabolism ; therapy ; Rabbits ; SOX9 Transcription Factor ; genetics ; metabolism ; Tissue Engineering

OBJECTIVETo explore the effect of drug-containing serum of Chinese herbal compounds [Xiongshao Capsule (XS, for activating blood) and Huanglian Capsule (HL, for dispelling toxin)] on tumor necrosis factor-alpha (TNF-alpha)-induced adherence between human umbilical vein endothelial cells (HUVECs) and polymorphonuclear neutrophils (PMN), inflammatory reaction and expression of related proteins in mitogen-activated protein kinase (MAPK) pathway.

METHODSThirty-two rats were randomly divided into four groups (8 in each group) using random digit table: the blank control group treated with distilled water, the test group I treated with Chinese herbal compound of XS (0.135 g/kg), the test group II treated with Chinese herbal compound of HL (0.135 g/kg), and the test group Ill treated with Chinese herbal compound of XS (0.135 g/kg) and HL (0.135 g/kg). All medication was given by gastrogavage once a day for a week. Rats' blood serum was harvested 1 h after the last administration to prepare drug-containing serum. HUVECs were exposed to TNF-alpha (100 ng/mL) to induce cell injury model and incubated with corresponding drug-containing serum (10%) for 24 h. Normal rats' serum was given to cells in the blank control group and the model group, while XC + HL containing serum was given to cells in the rest 3 groups. The adherence of HUVECs and PMN cells was detected by using rose bengal strain. Levels of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and interleukin-1beta (IL-1P) in the supernatant of cultured HU-VECs were determined by ELISA. Protein expressions of mitogen-activated protein kinases p38 (p38MAPK) and extracellular signal-regulated kinase 1/2 (ERK 12) were determined by Western blot.

RESULTSCompared with the blank control group, HUVECs were seriously injured; PMN adherence amount significantly increased; levels of E-selectin, ICAM-1, and IL-1beta increased; expression levels of p-p38MAPK and p-ERK 1/2 in the supernatant of HUVECs significantly increased in the model group (all P < 0.01). Compared with the model group, HUVECs-PMN adherence amount decreased (P < 0.05); levels of E-selectin, ICAM-1, and IL-1 beta in the supernatant of HUVECs decreased (P < 0.01, P < 0.05); expression levels of p-p38MAPK and p-ERK 1/2 of endothelial cells decreased in the test group I, II, and III (P < 0.01).

CONCLUSIONSDrug-containing serums of activating blood, activating blood and dispelling toxin could attenuate TNF-alpha induced injury of HUVECs, inhibit HUVECs-PMN adherence and the release of adhesion factors. Its mechanism might be involved with protein phosphorylation of p38MAPK and ERK 1/2 in the MAPK pathway.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; E-Selectin ; Endothelial Cells ; physiology ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; Mitogen-Activated Protein Kinase 3 ; Neutrophils ; Rats ; Serum ; Tumor Necrosis Factor-alpha ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective To compare and quantify the determinants in quantitative electroencephalogram(q-EEG) and heart rate variability power spectrum analysis(HRV-PSA) of ketamme(KTM) anesthesia for preschoolers. Methods Seventy four cases were selected and assigned into 3 groups named A(4-5 years), B(5-6 years), C(6-7 years), 22,28,24 cases in every group respectively. All cases were induced with KTM 5 mg /kg intramuscularly and changes of determinants were recorded continuously. If body movement happened, KTM would be injected with 1 mg/kg. Results On pre- anesthesia, BIS in group A was the least among 3 groups, while LF/HF and HRVI were the largest(P

Objective To explore the temporal and spatial expression pattern of tenascin-c(tnc) and tenascin-w(tnw) in zebrafish early development,to further explore the role of tenacsin in zebrafish embryo development,and the association between them.Methods Zebrafish embryos at 2 hours post fertilization(hpf),4 hpt,8 hpt,10 hpf,24 hpt,48 hpr,72 hpf and 7 days post fertilization(dpf) were collected to extract RNA for reverse transcription-polymerase chain reaction(RT-PCR) and fix the embryos at different stages for in situ hybridization.Temporal and spatial expression pattern of tnc and tnw on different stages of zebrafish early development was observed.Results tnc and tnw all expressed in zebrafish from 24 hpf to 7 dpf,but did not expressed from 2 hpf to 10 hpf.Tnc expressed at pharyngeal arch,notochord,somite in 24 hpf,then weakly expressed at somite,but highly expressed at otic vesicle,pectoral fin and hindbrain in 48 hpf,and tnc was expressed at hindbrain,pharyngeal and notochord and disappeared at somite and pectoral.tnw expressed at hindbrain,midbrain and otic vesicle in 24 hpf,expressed at somite,notochord,hindbrain,otic vesicle and pharyngeal in 48 hpf.In 72 hpf,tnw expressed weakly at somite and notochord.Conclusions Zebrafish tnc and tnw have special temporal and spatial expression pattern,and share partial overlapping expression pattern.

Objective To explore the association of Toll-like receptor 4 TLR4 and lipopolysaccharide receptor CD14 gene polymorphisms with Kawasaki disease (KD) susceptibility.Methods Three-color fluorescent staining flow-cytometry was used to detect the expression of TLR4 in peripheral blood white blood cell of 76 KD children and 118 healthy control group.The gene of TLR4 (-896A/G), (-1196C/T) and CD14 (-260C/T) polymorphisms was identified by polymerase chain reaction-restriction fragment length polymorphisms; and the relationship between genotype and KD was analyzed.Results 1.The values of mean fluorescence intensity (MFI) of TLR4 in peripheral blood white blood cell of the KD groups and the healthy control groups were 2.87?0.96, 10.55?4.87, 23.36?8.28 and 3.26?0.65, 7.55?1.21, 25.41?6.97, respectively; There was a gradual increase of these values on lymphocyte, neutrophilic leukocyte and mononuclear cell in both groups.2.(-896A/G), (-1196C/T) polymorphisms of TLR4 gene were not found in both groups.3.The frequency of each genotype of CD14 gene (-260C/T) was 35.5%CC, 30.3%CT, 34.2%TT in KD group and 38.1%CC, 47.5%CT, 14.4%TT in healthy control group.The frequency of each genotype was significantly different in 2 groups(?2=11.62 P

Objective To explore the expression pattern of tenascin-c(tnc)gene in zebrafish embryo abnormal development which was induced by ethanol,and to further understand the function of tnc gene in embryo develepment.Methods Zebrafish were treated with ethanol at different concentration from 100 to 500 mmol/L,and embryos at 24 and 48 hours were collected and fixed,then tnc expression pattern was observed by in situ hybridization and reverse transcriptase-polymerase chain reaction(RT-PCR).Results The result of RT-PCR showed that ethanol at 100 and 200 mmol/L could increase the expression of tnc,while the result of in situ hybridization showed that,while ethanol at 300 mmol/L and above decrease the expression of tnc in presumptive position at 24 hours,and ethanol at 100 mmol/L and above caused increase expression of tnc in zebrafish heart.Conclusions tnc is increased when treated with 100 and 200 mmol/L ethanol and is presented in the abnormal development of hearts of zebrafish,which can promote the normal development of embryos in some degrees.The expression pattern of tnc in pathologic state is highly conserved in all vertebrate,and in adult and embryos as well.

AlM:To investigate the changes of retinal histology and oxidative stress in diabetic retinopathy and its reversal by pyruvate in diabetic rats.
METHODS: Eighty Wistar rats were divided into 3 groups:control group ( 20 rats ) , model group ( 30 rats ) and treatment group ( 30 rats ) . After streptozotocin ( STZ) induced diabetes mellitus in the model group and the treatment group, the treatment group received 2%pyruvate in diet and drinking. The changes of body weight and blood glucose were observed and the changes of glutathione peroxidase ( GSH-PX ) , malonie dialdehyde ( MDA) , and Na+-K+-ATPase levels of retinal tissue and retinal ultrastructure were investigated in three groups at 12wk after occurrence of diabetes.
RESULTS: Compared with control group, the body weight of the model group were significantly decreased, the activities of GSH-PX and ATP in the retina of diabetic rats were significantly lower, the MDA was signigicantly higher and significant changes occurred in retinal ultrastructure. Compared with model group, the blood glucose of the treatment group had no significant changes. However, the activities of GSH and ATP in the retina of diabetic rats were higher, the MDA was lower and the retinal ultrastructure was comparatively mild.
CONCLUSlON:Pyruvate can alleviate oxidatie stress reaction, improve the energy metabolism of retina, and delay the development of retinopathy.

Objective To analyze comparatively the outcome of modified Manipal and DeVega tricuspid annuloplaaty.Methods From Oct.2001 to Aug.2004,the consecutive 123 patients operated with modified Manipal tricuspid annuloplasty for tricuspid re- gurgitation at the time of left cardiac valve replacement(group A)were elected in this study.The other 174 patients operated with De Vega tricuspid annuloplasty at the time of left cardiac valve replacement were elected randomly for control(group B).There were no significant differences of the patient data before surgery between two groups.Results There were overall 11 early deaths(4 in group A,7 in group B),The overall in-hospital mortality rate was 3.7 %.215 of the 286 surviving patients were followed;the overall follow up was 75.2 %.Mean follow-up was(28.4?9.1)months(range from 13 to 49 months).There were ten late deaths and the late mortality rate was 4.7%.There was no significant differences in the patients with 3+～4+ tricuspid regurgitation at follow-up 18,30 and 42 months in group A;but there was an increase of the patients with 3+～4+ tricuspid regurgitation with an incremental follow up term in group B(P