The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells (MSCs) onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically (hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy), biochemically (DNA and 4-hydroxyproline) and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline (P>0.05). However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls (P<0.05). This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.

Adult ; Aged ; Aged, 80 and over ; Coal Mining ; Humans ; Lung Neoplasms ; complications ; microbiology ; Male ; Methicillin Resistance ; Microbial Sensitivity Tests ; Middle Aged ; Pneumoconiosis ; complications ; microbiology ; Staphylococcus aureus ; drug effects ; isolation & purification

OBJECTIVETo screen the proteins interacting with inhibitor of differentiation 1(Id1) using yeast two-hybrid analysis in adult human lung cDNA libraries.

METHODSThe coding sequence of Id1 was amplified by PCR and cloned into the bait plasmid. The recombinant bait vector pHybLex/Zeo-Id1 was verified by restriction endonuclease digestion before transformation into the yeast strain EGY48/pSH18-34, which was tested subsequently for reporter genes Leu2 and LacZ activation. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into the yeast strains and screened to obtain Leu2(+) and Leu2(+)LacZ(+) clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.

RESULTSSuccessful construction of pHybLex/Zeo-Id1 was confirmed by enzyme digestion. After transformation of pHybLex/Zeo-Id1 into EGY48/pSH18-34, no specific reporter genes Leu2 and LacZ activation was found. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into yeast strain, and 198 Leu(+) clones and 19 Leu(+)LacZ(+) double positive clones were obtained. After elimination of the false positive clones, one true positive clone was obtained, whose plasmid analysis by sequencing and blasting indicated high homology (99.5%, 556/559) to AGGF1 (an angiogenic factor with G-patch and FHA domains 1). AGGF1 expression was confirmed in the true positive yeast cells by Western blotting.

CONCLUSIONAGGF1 is confirmed to interact with Id1 by yeast two-hybrid analysis for screening adult human lung cDNA libraries.

Adult ; Angiogenic Proteins ; genetics ; metabolism ; Cell Proliferation ; Endothelial Cells ; cytology ; metabolism ; Gene Library ; Humans ; Inhibitor of Differentiation Protein 1 ; metabolism ; Lung ; cytology ; Plasmids ; genetics ; Protein Binding ; Two-Hybrid System Techniques

OBJECTIVETo investigate the distribution of estrogen receptor beta (ERbeta) gene polymorphisms between the Uygurs and the Hans in Urumqi and the association of the polymorphisms with intrahepatic cholestasis of pregnancy (ICP).

METHODSICP cases and controls from a hospital were recruited from April 2008 to April 2011,and a total of 105 ICP patients in Uygurs case group and 105 ICP patients in Hans case group were randomly selected, meanwhile, 105 Uygurs and 105 Hans healthy pregnant women were recruited as control group. The distribution of Rsa I and Alu I of ERbeta gene polymorphism were analyzed by PCR amplification and restriction and other molecular biology approaches. Data were analyzed by chi2 and Fisher exact probability.

RESULTSIn Uygurs case group, the genotype frequencies of rr, Rr,RR,aa, Aa and AA were 39.0% (41 cases), 50.5% (53 cases), 10.5% (11 cases), 62.7% (66 cases), 30.5% (32 cases), 6.8% (7 cases). In Uygurs control group, the frequencies were 21.0% (22 cases), 56.2% (59 cases), 22.8% (24 cases), 80.0% (84 cases), 18.1% (19 cases), 1.9% (2 cases). In Hans case group, the genotype frequencies of rr, Rr, RR, aa, Aa and AA were 40. 0% (42 cases), 45.7% (48 cases), 14.3% (15 cases), 66.7% (70 cases), 29.5% (31 cases), 3.8% (4 cases). In Hans control group,the frequencies were 29.5% (31 cases), 57.2% (60 cases), 13.3% (14 cases), 74.2% (78 cases), 23.8% (25 cases), 2.0% (2 cases). The genotype frequencies were not statistically significant between the two case groups and between the two control groups (all P values > 0.05), and between two Hans groups (P > 0.05). The frequencies of RRaa in the Uygur case group was lower(4. 76% ,5 cases)than control group (13.33%, 14 cases) (P <0.05), while the frequencies of rrAa in the Uygur case group was significantly higher (14. 29% ,15 cases)than control group (2.86%, 3 cases) (all P values < 0.05).

CONCLUSIONThe distribution of ERbeta gene polymorphism is of no significant difference between the Uygurs and Hans, ERbeta gene polymorphism may correlate with pathogenesis of ICP in the Uygurs other than in the Hans, and rrAa might be risk factor for ICP in the Uygurs.

Alleles ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; China ; epidemiology ; Cholestasis, Intrahepatic ; ethnology ; genetics ; Estrogen Receptor beta ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Minority Groups ; Polymorphism, Genetic ; Pregnancy ; Pregnancy Complications ; ethnology ; genetics ; Risk Factors

To identify the active constituents in vitro and blood-absorbed ingredients in vivo from Yin Chen Hao decoction provides scientific evidence for probing its prevention and treatment mechanism on acute liver injury. An ultrahigh performance liquid chromatography quadrupole-time of flight-mass spectrometry (UPLC-QTOF/MS) method was applied for analysis of Yin Chen Hao decoction and the serum samples of mice with con-A induced acute liver injury after preventive oral administration for 14 days (the use of all laboratory animals in this study was approved by the Ethics Committee of the Naval Medical University, 19YF1459400). A total of 90 chemical constituents were identified from Yin Chen Hao decoction, mainly were flavonoids, terpenoids, tannins, quinones. 5 prototype compounds were identified in the serum, including chrysophanol, deoxyrhapontin-8-O-gallate, mussaenosidic acid, herniarin, emodin. The established UPLC-QTOF/MS method could efficiently and sensitively identify the constituents in vitro and blood-absorbed ingredients of Yin Chen Hao decoction, primarily clarify the material basis of its hepatoprotective effect, and provided a scientific basis for the quality marker selection and the pharmacodynamic material basis research on the decoction.

OBJECTIVEIn order to study the feature of T cell TCR-CD3 complex-mediated gene in lead poisoning patients.

METHODSReal-time PCR with SYBR Green I technique was used for determination of the expression levels of CD3 genes in peripheral blood mononuclear cells of 46 cases lead poisoning patients (11 cases in observation group and 35 cases in mild lead poisoning group) and 31 cases in control group.

RESULTSThe median expression levels of CD3γ gene in observation group and mild lead poisoning group (6.89%, 5.87 %) were higher than the control group (P < 0.05). The median expression levels of CD3δ gene in observation group and mild lead poisoning group (0.54%, 0.70%) were lower than the control group (P < 0.05). The median expression levels of CD3ε gene in observation group and mild lead poisoning group (10.22%, 6.08%) were higher than the control group (P < 0.05). A significant Positive correlation was found between CD3γ, CD3ε and seniority in lead poisoning patients. A significant negative correlation was found between CD3ε and blood ZPP, urea δ-ALA (r = -0.358, P < 0.05; r = -0.385, P < 0.05), but there was no significant correlation between them after controlling for blood lead, urea lead. The expression levels of CD3 genes prove to be a descending order of CD3γ, CD3ε, CD3δ in control group, while it was changed for CD3ε, CD3γ, CD3δ in the observation group as well as in mild lead poisoning group.

CONCLUSIONExpression of T cell TCR-CD3 complex-mediated gene was changed in lead poisoning patients, it might be related to the body immunodeficiency. The expression level of CD3ε gene can be used as sensitive immune function screening indicator in Lead poisoning patients.

Adolescent ; Adult ; Aged ; Female ; Humans ; Lead Poisoning ; immunology ; Male ; Occupational Diseases ; immunology ; Receptor-CD3 Complex, Antigen, T-Cell ; metabolism ; Young Adult

OBJECTIVETo investigate possible mechanism of angiogenesis in brain tissue of neonatal rat hypoxic-ischemic encephalopathy (HIE).

METHODSForty seven-day old neonatal rats were randomly assigned to hypoxic-ischemic (Model group) or sham treatment (Sham group), each group had 20 rats. Five rats from each group were sacrificed on days 1, 3, 7 and 14 after hypoxia-ischemia. Paraffin sections of the brain were stained with anti-endothelial cell, anti-proliferating cell nuclear antigen (PCNA) or anti-vascular endothelial growth factor (VEGF) by using single or double immunohistochemistry. The brain capillary density index (BCDI), brain proliferating capillary density index (BPCDI) and the expression of VEGF were analyzed under the microscope. The expression of VEGF and hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA in hypoxic-ischemic side of the brain was measured by RT-PCR.

RESULTSBCDI around infarct brain tissue in the model group began to rise on day 3 and remained higher than that of the sham group from day 3 to day 14 [day 3: (9.80 +/- 1.05)/HPF vs. (4.90 +/- 0.66)/HPF, P < 0.01;day 14: (13.29 +/- 3.90)/HPF vs. (6.08 +/- 1.50)/HPF, P < 0.01]. Occasional proliferating capillary was found in brain tissue of normal neonatal rats. The density of proliferating brain capillary on day 3 and day 7 of Model group [(0.54 +/- 0.15)/HPF vs. (0.90 +/- 0.25)/HPF] were significantly higher than those of Sham group [(0.12 +/- 0.05)/HPF vs. (0.13 +/- 0.07)/HPF, P < 0.01]. VEGF was mainly expressed in the cytoplasm of neurons, capillary endothelial cells and pial cells. Viable neurons and endothelial cells in the infarct areas also expressed VEGF. The expression of VEGF mRNA in hypoxic-ischemic brain tissue was significantly higher than that of normal control (P < 0.01) and temporally preceded angiogenesis. The expression of VEGF mRNA at 12 hours of HIE model was significantly higher than that of normal control (1.56 +/- 0.27 vs. 0.95 +/- 0.21, P < 0.05). It reached its peak on day 1 and day 3 (1.85 +/- 0.31 vs. 1.86 +/- 0.39), significantly higher than that of normal control (P < 0.01), and decreased by day 7 and day 14, without significant difference compared with normal control (P > 0.05). The expression of HIF-1alpha mRNA was also up-regulated after hypoxic-ischemic treatment. The expression of HIF-1alpha mRNA (1.07 +/- 0.21) was significantly higher than that of normal control (0.64 +/- 0.28, P = 0.048) at 3-hour of HIE model, reached its peak on day 1 (1.73 +/- 0.42, P < 0.01), remained at high expression level on day 3 (1.44 +/- 0.36, P < 0.05) and began to decline by day 7 and day 14 when it was not significantly different from normal control.

CONCLUSIONSAngiogenesis exists in the brain tissue of neonatal rat HIE model. Up-regulation of VEGF expression mediated by HIF-1 may play an important role in the process of angiogenesis.

Animals ; Animals, Newborn ; Brain ; blood supply ; Brain Diseases ; etiology ; genetics ; metabolism ; Disease Models, Animal ; Hypoxia-Inducible Factor 1, alpha Subunit ; Hypoxia-Ischemia, Brain ; complications ; Immunohistochemistry ; Neovascularization, Pathologic ; etiology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; analysis ; genetics ; Vascular Endothelial Growth Factor A ; analysis ; genetics

The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.
Animals ; Cell Line ; Chickens ; Chorioallantoic Membrane ; blood supply ; Disease Models, Animal ; Genetic Therapy ; Humans ; Male ; Myocardial Infarction ; metabolism ; pathology ; therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

Aged ; Biopsy, Needle ; methods ; Coal Mining ; Humans ; Lung ; pathology ; Lung Neoplasms ; complications ; diagnosis ; Middle Aged ; Pneumoconiosis ; complications

OBJECTIVETo study the inhibitory effects of celecoxib, a cyclooxygenase-2 inhibitor, on the proliferation of hepatocellular carcinoma cells.

METHODSThe in vitro inhibitory effects of celecoxib at different concentrations and for different treatment time lengths on human liver cancer cell line SMMC-7,721 were observed with MTT assay, and flow cytometry was performed to detect the cell cycle changes. The in vivo tumor inhibition effect of celecoxib was evaluated in Kunming mice bearing transplanted tumor derived from liver cancer cell line H22 transplantation.

RESULTCelecoxib significantly inhibited the in vitro growth of human liver cancer cell line SMMC-7721 in a time- and dose-dependent manner. A 36-hour celecoxib treatment (40 micromol/L) resulted in decreased SMMC-7721 cell proliferation and an increase of the cell percentage in G1 phase from 44.7% to 49.9% with decreased cell percentage in S and G(2)/M phases from 55.4% to 50.1%. In the mice bearing H22 transplanted tumor, celecoxib showed significant inhibitory effect on the growth and local metastasis of the transplanted tumor.

CONCLUSIONCelecoxib can inhibit the proliferation of different liver cancer cell lines both in vitro and in vivo, and therefore may serve as an important candidate drug for prevention and treatment of hepatocellular carcinoma.

Animals ; Carcinoma, Hepatocellular ; drug therapy ; physiopathology ; Celecoxib ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 Inhibitors ; pharmacology ; therapeutic use ; Female ; Humans ; Liver Neoplasms ; drug therapy ; physiopathology ; Mice ; Neoplasm Transplantation ; Pyrazoles ; pharmacology ; therapeutic use ; Sulfonamides ; pharmacology ; therapeutic use