To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.

In order to investigate the pathogenesis of Alzheimer disease (AD) and study the enzymatic progress of amyloid precursor protein (APP), the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP (which was named C99 containing APP717 mutation), together with the fragment encoding yellow fluorescence protein (which was named YFP) were amplified by PCR. The two fragments (YFP and C99) were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β (Aβ) was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells. Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.

The effects of all-trans-retinoic acid (ATRA) administration on the concentration of retinoids (RA and vitamin A) in liver, oxidative stress and the hepatic injury in a rat model of common bile duct ligation (CBDL)-induced liver injury were investigated. Female rats were subjected to a sham (n=5) or CBDL (n=48). Two weeks after operation, rats undergoing CBDL were randomized to receive treatment with either ATRA at three different doses (0.1, 1.5, 7.5 mg/kg) dissolved in bean oil or only bean oil every day over a 4-week experimental period. Rats were killed and blood samples were collected from the heart for determination of the serum transaminase. The contents of retinoids in rat liver were detected by using HPLC. Malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) levels in liver were determined by a spectrophotometric method according to the instruction of the kits. Liver pathologic changes were observed under the light microscopy and electron microscopy. The results showed that compared with sham-operated group, the levels of retinoids in the liver tissue were significantly decreased in the CBDL group (P<0.01). ATRA (0.1 mg/kg) administration in CBDL rats partially restored the contents of retinoids (P<0.05). Liver RA and vitamin A contents in CBDL group were significantly increased after ATRA (1.5 and 7.5 mg/kg) supplementation as compared with sham-operated group (P<0.05). However, in ATRA-treated CBDL group, hepatic GSH level and SOD activity, depressed by CBDL, and hepatic MDA level, increased by CBDL were returned to those in sham-operated group (P<0.05). The histologic observation of liver tissues indicated that ATRA treatment notably alleviated hepatocellular swelling, steatosis, the swelling of mitochondria and proliferation of smooth endoplasmic reticulum (SER). Treatment with ATRA could reduce levels of serum transaminase as compared with sham-operated group, more greatly in 1.5 and 7.5 mg/kg ATRA-treated groups than in 0.1 mg/kg ATRA-treated group. It was concluded that ATRA treatment can recover MDA and GSH levels and SOD activity in CBDL rat liver through restoring RA and vitamin A contents, and eventually ameliorate liver injury.

In order to investigate the clinical value of vascular endothelial growth factor (VEGF) combined with interferon-γ (IFN-γ) in diagnosing malignant pleural effusion and tuberculous pleural effusion, 42 cases of malignant pleural effusion and 45 cases of tuberculous pleural effusion in Tongji Hospital, from March 2004 to May 2005, were included. The carcinoembryonic antigen (CEA), VEGF and IFN-γ levels of pleural effusion were detected by using ELISA, and adenosine deaminase (ADA) activity was determined by using enzyme kinetic analytical method. The sensitivity, specificity, accuracy and area under the curve (AUC^ROC) of CEA and VEGF, VEGF/IFN-γ ratio, ADA and IFN-γ were measured by receiver operating characteristic curve (ROC). The results showed that CEA, VEGF levels and VEGF/IFN-γ ratio were significantly higher and the ADA and IFN-γ levels were significantly lower in malignant group than those in tuberculous group (P<0.01). The sensitivity, specificity, accuracy and AUC^ROC of VEGF/IFN-γ ratio (88.7%, 99.8%, 94.4%, 0.96 respectively) were higher than those of CEA (67.8%, 96.1%, 82.4%, 0.78 respectively) and VEGF (81.5%, 84.3%, 82.9%, 0.79 respectively). The sensitivity, specificity, accuracy and AUC^ROC of IFN-γ (85.7%, 96.4%, 90.9%, 0.94 respectively) were higher than those of ADA (80.2%, 87.6%, 83.8%, 0.81 respectively). It was concluded that VEGF/IFN-γ ratio and IFN-γ could be used as valuable parameters for the differential diagnosis of malignant pleural effusion and tuberculous pleural effusion.

The expression of interleukin-17 (IL-17) in lung and peripheral blood of asthmatic rats and the influence of dexamethasone, and the role of IL-17 in the pathogenesis of asthma were investigated. Thirty Sprague-Dawley (SD) adult rats were randomly divided into three groups (n=10 in each group): normal group, asthmatic group, and dexamethasone-interfered group. Rat asthmatic model was established by intraperitoneal (i.p.) injection of 10% ovalbumin (OVA) and challenge with 1% OVA via inhalation. Rats in dexamethasone-interfered group were pretreated with dexamethasone (2 mg/kg, i.p.) 30 min before each challenge. The expression of IL-17 protein in serum and bronchoalveolar lavage fluid (BALF) was detected by ELISA. The expression of IL-17 mRNA in peripheral blood mononuclear cells (PBMC) and BALF cells was semi-quantitatively detected by RT-PCR. The expression of IL-17 protein in serum and BALF of asthmatic rats was significantly elevated as compared with normal rats and dexamethsone-interfered rats (P<0.01), and there was significant difference between normal rats and dexamethsone-interfered rats (P<0.05). The expression of IL-17 mRNA in PBMC and BALF cells of asthmatic rats was markedly increased as compared with normal rats and dexamethsone-interfered rats (P<0.01), and significant difference was found between normal rats and dexamethsone-interfered rats (P<0.05). It was concluded that the expression of IL-17 was increased significantly in asthmatic rats and could be inhibited partly by dexamethasone, suggesting that IL-17 might play an important role in the pathogenesis of asthma as an inflammation regulation factor.

In order to investigate the effect of interleukin-18 (IL-18) on airway inflammation in asthmatic murine models and its mechanisms, BALB/C mice were randomly divided into three groups (n=10 in each group): group A (control group); group B (asthmatic model group); group C (IL-18-treated group). The asthmatic model was established in groups B and C by respiratory syncytial virus (RSV) killed by ultraviolet. Saline solution (0.1 mL) and IL-18 (0.1 mL, 1 μg) were intraperitoneally injected respectively in groups B and C at 7 time points (day 1, 2, 7, 8, 9, 21, 22). The number of eosinophils (EOS) and plasmacytes in the airway was observed. The levels of interferon gamma (IFN-γ) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The results showed that symptoms of asthma in group C were more severe than in groups A and B. In group A, there were no EOS and plasmacytes in the airway submucosa. The number of EOS [15±3 (average cell counts per microscopic visual field, the same below)] and plasmacytes (10±2) in group B were increased significantly. However, the number of EOS and plasmacytes in group C (6±2 and 2±1, respectively) was decreased significantly as compared with group B (both P<0.05). The levels of IFN-γ in groups A, B and C were 31±3, 40±5 and 63±5 pg/mL respectively, and those in group C were significantly higher than in groups A and B (both P<0.05). It was suggested that the mechanism by which IL-18 inhibited the airway inflammation in asthmatic mice might be contributed to the fact that IL-18 could induce the induction of IFN-γ.

In order to explore the roles of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) in the pathogenesis of pulmonary emphysema, male Wistar rats were randomized into group A₁, group A_2.5 and group A₄, each with smoke exposure for 1 month, 2.5 months or 4 months, respectively. Group B₁, group B_2.5 and group B₄ were used as non smoking controls at corresponding time points. TNF-α in bronchoalveolar lavage fluid (BALF) and expression of VEGF in lung tissue was determined by ELISA or by SABC immunohistochemistry assay either. Lung slices were stained with hematoxylin and eosin (HE). Results showed that in animal with smoke exposure the mean linear interceptor (Lm), an index of pulmonary emphysema and the content of TNF-α in BALF increased gradually, on contrary, the expression of VEGF in lung tissue decreased (P<0.05). This phenomenon was not obvious in animals without smoke exposure. Lm was negatively correlated to the VEGF expression (γ=−0.81, P<0.01) and positively correlated to TNF-α concentration (γ = 0.52, P<0.004), which implies that smoke exposure decreased the expression of VEGF and increased the expression of TNF-α. It is plausible to speculate that the imbalance of TNF-α and VEGF may play an important role in the pathogenesis of smoke-induced pulmonary emphysema.

This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor (VEGF) and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concentrations (12.5–200 μmol/L) for different time lengths (12–48 h). The proliferation of the U937 leukemic cells was determined by MTT assay. Apoptosis was observed by Annexin-V-FIFC/PI double staining and flow cytometry (FCM). Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose-and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resveratrol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGF, induce apoptosis and suppress the proliferation of U937 cells.

In order to investigate the antitumor effect and molecular mechanism of interferon-α (IFN-α) on human acute myeloid leukemia cell line U937 cells in vitro, the proliferation of U937 cells was determined by MTT assay, the apoptosis rate was analyzed by flow cytometry (FCM), and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR. The results showed that IFN-α could inhibit the proliferation of U937 cells significantly in a dose-and time-dependent way (P<0.01), and induce the apoptosis of U937 cells also in a dose-and time-dependent manner at the concentration of 1000 4000 U/L (P<0.01). The apoptosis rate of U937 cells was even over 50% when cultured with IFN-for 36 48 h at the concentration of 2000 4000 U/L. Moreover, the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-, and the inhibition was time-dependent (P<0.01). It was concluded that the anti-leukemia mechanism of IFN-α might be correlated with its antiproliferative and apoptotic inducing effects, and the down-regulation of the cyclin E expression might be one of its molecular mechanisms.

Intracellular Ca²⁺ and Ca²⁺-dependent signaling molecule play an essential role in the genesis of long-QT (LQT) syndrome-related ventricular arrhythmias. The effect of calcium-channel antagonist verapamil on repolarization heterogeneity of ventricular myocardium was assessed in an in vitro rabbit model of LQT syndrome. By using the monophasic action potential (MAP) recording technique, MAPs of epicardium, mid-myocardium and endocardium were simultaneously recorded by specially designed plunge-needle electrodes across the left ventricular free wall in rabbit hearts purfused by Langendorff method with standard Tyrode’s solution. Bradycardia was induced by complete ablation of atrioventricular node. A catheter was introduced into the right ventricle to pace at the cycle lengths (CLs) of 1500, 1000, and 500 ms, successively. Quinidine (2 μmol/L) prolonged QT interval and ventricular MAP duration (MAPD), and increased transmural dispersion of repolarization (TDR) in a reverse rate-dependent fashion in isolated rabbit heart. No polymorphic ventricular tachycardias were induced under this condition. The effective free therapeutic plasma concentrations of verapamil (0.01–0.05 μmol/L) used in this experiment had no effect on quinidine-induced changes of QT interval, MAPD and TDR. This study demonstrated that, in this model of LQT syndrome, blockade of calcium-channel with verapmil had no effect on quinidine-induced changes of repolatiation heterogeneity of ventricular myocardium.

In order to investigate the association of G+1688A (Ser563Asn) polymorphism of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene with myocardial infarction (MI) in the Chinese Han population, the G+1688A polymorphism in PECAM-1 gene was detected by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method among 502 subjects, including 218 patients with MI and 284 controls. The results showed that there was significant difference in AA frequencies of genotype G+1688A polymorphism between case and control groups (39% vs 24%, P<0.001). A similar trend was observed on the allele frequencies (A/G: 62% vs 49%, P<0.001). Among the subjects with high serum total cholesterol level or high systolic blood pressure level, the variant AA genotype was associated with high risk of MI (adjusted OR, 2.13; 95% CI, 1.08–4.41 and adjusted OR, 2.53; 95%CI, 1.63–3.63). The single nucleotide polymorphism (SNP) at position +1688 in the exon 8 of PECAM-1 gene was associated with MI and the allele A might be a risk factor for MI in the Chinese Han population.

The function of CD4⁺CD25⁺ regulatory T lymphocytes (Treg) in patients with acute coronary syndrome (ACS) and the effects of atorvastatin were investigated. Forty-eight patients with ACS were randomly divided into two groups: group C receiving conventional therapy (n=24), and group C+A receiving conventional therapy+atorvastatin (10 mg/day, n=24). T lymphocytes from ACS patients (before and 2 weeks after the treatment) or 18 healthy subjects were separated and the flow cytometry was used to measure the percentage of Treg. The inhibitory ability of Treg on effector T cells was determined by mixed lymphocyte reaction (MLR). ELISA was used to measure the serum levels of cytokines (IL-10, TGF-β1 and IFN-γ) before and after treatment. The results showed that as compared with normal control group, Treg percentage was decreased significantly (P<0.01), the inhibitory ability of Treg on the T lymphocytes proliferation was reduced (P<0.01), IFN-γ levels were increased and IL-10 and TGF-β1 levels were lowered in ACS patients. After treatment with atorvastatin, Treg percentage and the inhibitory ability of Treg on T lymphocytes proliferation were significantly increased in ACS patients. Serum IFN-γ was decreased significantly, while IL-10 and TGF-β1 were elevated significantly as compared with the non-atorvastatin group. The number of Treg was positively correlated with serum TGF-β1, but negatively with serum IFN-γ and CRP. It was concluded that ACS was associated with decreased number and defected function of Treg, which may play an important role in initiating immune-inflammatory response in ACS. The inhibitory effects of atorvastatin on inflammation in ACS may be due to its beneficial effects on Treg and restoration of immune homeostasis.

The preventive effects of nitroglycerine (NG) on glucocorticoid-induced osteoporosis in growing rats were studied. Three-month-old female Wistar rats were randomly divided into control group (CON), dexamethasone group (DXM), DXM plus a low dose NG group (NG-L), DXM plus a middle dose NG group (NG-M) and DXM plus a high dose NG group (NG-H), 8 rats in each group. The rat model of osteoporosis was developed by intramuscular injection of dexamethasone twice a week. NG 0.2, 0.4 and 1.0 mg/kg was administered by oral gavages to the treatment groups every day for 12 weeks. Rats in CON group and DXM group were treated with normal saline of the same amount. After the treatment, the bone mineral density (BMD) and bone metabolism-associated biochemical markers were determined. Compared with CON group, BMD of lumbar spine and femur in DXM group was decreased significantly (P<0.05 and P<0.01 respectively), blood BGP levels and NO levels reduced (both P<0.01), and TRAP level increased (P<0.05). As compared with DXM group, BMD, serum BGP and NO were increased, and TRAP decreased in NG-L group and NG-M group, but had no significant difference in comparison to CON group. All the markers other than serum NO and TRAP levels had no significant difference between NG-H group and DXM group. It was concluded that low or middle doses of NG could prevent glucocorticoid-induced bone loss in growing rats, but high dose of NG could not. Supplement with NO donor could be considered as a preventive treatment for glucocorticoid-induced osteoporosis in a developing skeleton.

Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy (TEM). PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established. Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S. HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfectionw as added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes. The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system, TEM displayed the nanoparticles self-assembled into large network aggregates. It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.

The recombinant plasmid pCI-TSLC1 carrying TSLC1 gene was stably transfected into human hepatocellular carcinoma cell line HepG2. Cell proliferation was analyzed by MTT assay. The ability of migration was determined by transwell and FACSort flow cytometry was used to detect the cell cycle distribution and apoptosis. Western blotting revealed that H4 expressed higher amounts of TSLC1 protein than H15 and H0 did. The growth of TSLC1-transfected cells was significantly suppressed in vitro, and the ability of migration was reduced as well. The re-expression of TSLC1 could induce cell apoptosis. It was concluded that TSLC1 strongly inhibited the growth and ability of migration of HepG2 cell line in vitro and also induced apoptosis, suggesting that TSLC1 could reduce the tumorigenicity of human hepatocellular carcinoma cell line HepG2 in vitro, which provided a basis for further exploring the roles of TSLC1 in hepatocellular cellular carcinoma.

Differential gene expression profiles in Balb/cJ mouse model of acute hepatic failure infected with MHV-3 virus intervened by anti-hepatic failure compound (AHFC) and the changes of cytokines regulated by genes were investigated. The Balb/cj mice were divided into AHFC-intervened group and control group randomly. Acute hepatic failure model of Balb/cJ mice infected with MHV-3 virus was established. The survival rate in the two groups was observed. It was found that the survival rate in the AHFC-intervened group and control group was 90% and 50% respectively 48 h after intraperitoneal injection of MHV-3 (P<0.05). Before and after the experiment, the cytokines in peripheral blood of the survival mice were determined, and RNA was extracted from survival mouse liver tissue for the analysis of the differential gene expression by a 36 kb mouse oligonuleotide DNA array. In all the genes of microarray there were 332 genes expressed differently in the two groups, in which 234 genes were up-regulated and 78 genes down-regulated. Through clustering analysis, the differential expression of immune related genes, including TNF receptor superfamily, Kctd9, Bcl-2, Fgl2, IL-8, IL-6, IFN-γ, TNF-α etc. might be related with the curative effectiveness of AHFC. It was suggested that AHFC can balance the immune state of mouse model of acute hepatic failure infected with MHV-3 virus mainly through regulating the expression of immune related genes, decrease the immune damage and inhibit liver cell apoptosis of mouse acute hepatic failure model obviously so as to increase the survival rate of mouse models of acute hepatic failure.

The effects of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on the proliferation of MDA-MB-435S cells and the expression of tumor suppressor gene maspin were investigated. Human breast cancer cell line MDA-MB-435S was treated with 5 μmol/L 5-Aza-CdR, a specific demethylating agent for 0 to 8 days. The growth of MDA-MB-435S cells was observed by MTT assay before and after 5-Aza-CdR treatment, respectively. The expression of maspin mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The cell cycle of MDA-MB-435S cells was analyzed by flow cytometry. The results showed that the growth of MDA-MB-435S cells treated with 5-Aza-CdR for 8 days was significantly suppressed as compared with the control groups, and the inhibition rate increased sharply from 5 day to 8 day (35.42% to 71.29%). Flow cytometry showed that 5 μmol/L 5-Aza-CdR could induce G₂/M cell cycle arrest and decrease the percentage of mitosis cell number in this cell line. Maspin mRNA was expressed in MDA-MB-435S cells after 5-Aza-CdR treatment, but it was weakly detectable before the treatment. It was concluded that Maspin gene might be transcriptional silencing by hypermethylation and the re-expression of maspin gene by 5-Aza-CdR can inhibit the proliferation and induce the G2/M arrest of MDA-MB-435S breast cancer cells.

In order to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the proliferation, apoptosis of pancreatic cancer cell line SW1990 cells and the expression of cyclin E mRNA, the SW1990 cells were treated with different concentrations of EPA or DHA (20, 40, 60 μg/mL) for 0, 12, 24, 36 and 48 h respectively. By using MTT method, the inhibitory effects of EPA or DHA on the cell growth were assayed. Real time PCR was used to detect the expression changes of cyclin E mRNA after the SW1990 cells were treated with 40 μg/mL EPA or DHA for different time. Flow cytometry was used to test the changes of apoptostic rate in the SW1990 cells treated with different concentrations of EPA or DHA for 24 h. The results showed that EPA and DHA could inhibit the growth of SW1990 cells in a time-and concentration-dependent manner (P<0.01). EPA or DHA could also significantly inhibit the expression of cyclin E mRNA in a time-dependent manner (P<0.05). EPA or DHA could induce the apoptosis of SW1990 cells in a concentration-dependent manner (P<0.01). It was concluded that ω-3 fatty acid could inhibit the proliferation of pancreatic cancer cell line SW1990 cells and promote their apoptosis. The down-regulation of the cyclin E expression by ω-3 fatty acid might be one of the mechanisms for its anti-tumor effect on pancreatic cancer.

The suppressive effect of anti-KDR antibody against VEGF on proliferation of hemangioma-derived vascular endothelial cells (HVECs) was investigated. HVECs from one case of hemangioma in proliferative phase were cultured. Both primary culture and sub-culture were conducted in M199 medium. The HVECs of passage 3 were divided into 4 groups based on the concentrations of anti-KDR antibody. Cell count was performed and inhibitory rate of HVECs was measured before and 9 days after interference. The results showed that the number of HVECs in the anti-KDR antibody-treated groups was significantly decreased and the inhibitory rate of HVECs by anti-KDR antibody (50, 10 and 2 μg/mL) was 84%, 63% and 39% respectively at 9th day after interference, with the difference being significant. In the control group, the number of HVECs was increased significantly. In was concluded that the anti-KDR antibody could suppress the activity of VEGF through blocking the KDR, indicating the potential clinical applications of anti-KDR antibody in the treatment of hemangioma.

In order to explore the method to prepare hypoxia UW solution and the stability and preservation of hypoxia UW solution, UW solution was purged by argon or air for 15 min or 60 at a flow rate of 0.8 or 2 L/min, and the oxygen partial pressure of UW solution was detected. The hypoxia UW solution was exposed to the air or sealed up to preserve by using different methods, and the changes of oxygen partial pressure was tested. The results showed that oxygen partial presure of 50 mL UW solution, purged by argon for 15 min at a flow rate of 2 L/min, was declined from 242±6 mmHg to 83±10 mmHg. After exposure to the air, oxygen partial pressure of hypoxia UW solution was gradually increased to 160±7 mmHg at 48 h. After sealed up by the centrifuge tube and plastic bad filled with argon, oxygen partial pressure of hypoxia UW solution was stable, about 88±13 mmHg at 72 h. It was concluded that oxygen of UW solution could be purged by argon efficiently. Sealed up by the centrifuge tube and plastic bag filled with argon, oxygen partial pressure of UW solution could be stabilized.

By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 μmol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.

The effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and osteogenic revulsants alone or in combination at different time points and in different dosages on proliferation and osteogenesis of bone marrow stromal cells (BMSCs) in SD rats were investigated. Rat BMSCs were cultured in vitro and induced by rhBMP-2 in different dosages (10, 50, 100 and 200 μg/L) alone or in combination with osteogenic revulsants. MTT colorimetric assay was used to evaluate The proliferation, activity of alkaline phosphoric (ALP) and osteocalcin were measured at 3rd, 6th, 9th, 12th day respectively. The results showed that rhBMP-2 and osteogenic revulsants could promote the differentiation of BMSCs towards osteoblast phenotype. The proliferation of BMSCs could be enhanced by rhBMP-2 in a dose-dependent manner. The expression of osteoblast phenotype was significantly higher by using both of them than by using them alone, which was verified by the activity of ALP and osteocalcin. It was suggested that the combined use of rhBMP-2 and osteogenic revulsants could promote the proliferation and simultaneously induce and maintain the expression of osteoblast phenotype of BMSCs in rats.

In order to provide us new clues to induce some endogenous protective molecular mechanisms, the changes in gene expression profile induced by ischemia-reperfusion in pulmonary tissues of rats were investigated and the dynamic mechanism of pulmonary ischemia-reperfusion injury was elucidated. Thirty male Wistar rats were randomly divided into 6 groups: 5 ischemia-reperfusion (I/R) groups (I/R 0-h, I/R 1-h, I/R 3-h, I/R 6-h, I/R 24-h) and control group (n=5 in each). An in situ ischemia-reperfusion lung injury rat model was established by occluded hilus of lung. The RatRef-12 Expression Beadchip (22 226 gene probes per array) was used to analyze the pattern of gene expression in all groups. The results showed that 648, 340, 711, 1279 and 641 genes were differentially expressed in I/R 0-, 1-, 3-, 6-and 24-h groups respectively. The differentially expressed genes were classified as following 7 functional categories: cytokine, adhesion molecule, growth factor and apoptosis-related factor, oxidation and antioxidation molecule, metabolic enzyme, ion channel and aquaporin, signal transduction molecule. It was suggested that gene chip technology was an effective and quick method for screening differentially expressed genes. Many differentially expressed genes with different functions interacted each other to result in pulmonary ischemia-reperfusion injury.

The neural stem cells in Wistar rats were cultured in vitro, purified, and transplanted into C6 glioma model in order to observe their biological characters and provide a basic foundation for treatment of neurological diseases by neural stem cell transplantation. The cells at hippocampal area from gestation 15-day rats were cultured in vitro, and frozen and preserved in liquid nitrogen. C6 tumor-bearing models (n=25) and neural stem cells transplantation models (n=35) were established. When the tumor grew to 3 to 4 weeks, 5 rats in each group were randomly selected for MRI examination. At different intervals, the rats were perfused and sampled for HE staining, GFAP and BrdU immunohistochemical staining. The results showed that after resuscitation of neural stem cells at 1–4 passages, the cell viability was 40%–63% with the difference being not significant. The cells could proliferate, passage, and most cells transplanted into glioma model survived. The mean survival time in neural stem cell transplantation group and control was 4.28 and 3.88 weeks respectively, and the average tumor size in the former was smaller than in the latter. It was concluded that embryonic neural stem cells in rats could proliferate and differentiate, and after resuscitation the biological characteristic and viability of the cells were not influenced. Neural stem cells had inhibitory effects on the growth of glioma cells and could prolong the survival of rat model.

The expression levels and changes of endogenous acid fibroblast growth factor (aFGF) in microwave burn wound tissues were detected in order to investigate how to get better therapeutic effects by using the exogenous aFGF for repairing trauma. A burnt-wound animal model was established by NS-F multifunction spectrum therapeutics equipment, and reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry assay were applied to detect the expression levels of endogenous aFGF mRNA in microwave burn wound tissues. The expression level of endogenous aFGF mRNA was significantly increased in the burn wound tissues 12 h after burn, reached the peak at 48 h, and gradually deceased 96 h after burn. The expression of endogenous aFGF mRNA after tissue damage was reversible, and its intensity was in accordance with the repair process of tissue damage, suggesting endogenous aFGF may take part in the cell metabolism and proliferation, and then promote the repair of the burn wound.

To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10–100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantion blastocysts.

In order to screen the genes differentially expressed in two human prostate cancer cells with different metastasis potentials, suppression subtractive hybridization (SSH) was done twice on human prostate cancer cell line with high potential of metastasis PC3M-1E8 and its synogenetic cell line PC3M-2B4 with low metastasis potential. In the first subtraction PC3M-2B4 was used as tester and PC3M-1E8 as driver and the forward subtractive library was constructed. In the second on the tester and driver were interchanged and the reverse subtractive library was constructed. The screened clones of both libraries were sequenced and Gene Bank homology search was performed. Some clones were confirmed by quantitative real-time PCR. The results showed that two subtractive libraries containing 238 positive clones were constructed. Analysis of 16 sequenced clones randomly picked from two libraries showed that 4 differentially expressed gene fragments were identified as new EST with unknown functions. It was concluded that two subtractive libraries of human prostate cancer cell lines with different metastasis potentials were constructed successfully.

The hypoxic model to simulate hypoxic microenvironment in solid tumors was established and the effect of hydrocamptothecin (HCPT) on the hypoxia-induced over-expression of HIF-1α and VEGF genes was explored. Human cervical cancer SiHa cells were cultured in vitro under hypoxic conditions (37°C, 5% CO₂, 1% O₂) and treated with different concentrations of HCPT for 24 h. The mRNA and protein expression levels of HIF-1α, VEGF and Glut1 in SiHa cells were detected by semi-quantitative RT-PCR and Western blot respectively. Normoxic control groups were exposed to normoxic conditions for 24 h. Under normoxic conditions, HCPT had no obvious effects on the HIF-1α and VEGF gene expression. Hypoxia induced the up-regulation of HIF-1α protein and downstream VEGF gene, and HCPT showed a dose-dependently inhibitory effect on the hypoxia-induced over-expression of HIF-1α protein and VEGF gene expression in SiHa cells, whereas HCPT had no significant effect on the HIF-1α mRNA expression. No difference in HCPT cytotoxicity was observed between hypoxic groups and normoxic control groups. It was suggested that HCPT could inhibite the expression of HIF-1α protein and downstream VEGF gene in hypoxic SiHa cells in a dose-dependent manner, and the inhibitory effect was not related with HCPT cytotoxicity.

The effect of the trace elements on retinopathy of prematurity (ROP) were studied. Thirty preterm infants who had potential high risk factors of ROP were selected as observation group and 18 normal infants as control groups. By using atom spectrophotometer, the contents of serum trace elements (Mg, Cu, Zn, Mn, Se) were measured and analyzed statistically. The contents of serum Zn, Cu and Se in observation group were 0.75±0.22, 0.41±0.20 and (134.07±71.57)×10⁻³ mg/L respectively, and 0.55±0.12, 0.65±0.194 and (202.92±44.71)×10⁻³ mg/L in control group respectively (P<0.01). The contents of Cu and Se were obviously lower and that of Zn higher in observation group than those in control group. The same results were obtained between the infants with ROP and controls (P<0.01). However, there was no significant difference in the contents of serum Mg and Mn between two groups (P>0.05). It was concluded that the contents of serum Cu and Se in preterm infants who had high risk factors of ROP were obviously lower than in the controls. The contents of serum Cu and Se in the ROP infants were also much lower while contents of Zn much higher. Attention should be paid to the detection of the trace elements in preterm infants in order to prevent the deficiencies of Cu and Se. Only in this way can we prevent the deficiencies of Cu and Se, so as to decrease the ROP risk factors and prevent the disease.

The diagnosis and treatment of the lacrimal passage obstruction with lacrimal endoscope was investigated and its subsidiary surgical procedures were evaluated. Ninety-three patients (109 eyes) with lacrimal passage obstruction, including presaccal canalicular obstruction (PSCO) and nasolacrimal duct obstruction (NLDO), were examined under a lacrimal endoscope, and the obstructions were treated with laser or micro-drill. All patients were followed up after the operation for 3–6 months. The difference between the laser and the micro-drill treatment was observed. During the period of follow-up, the curative rate was 82.57%. The healing rate in PSCO group and NLDO was 80.36% and 84.91% respectively (P>0.05). After treatment with the laser, the healing rate was 93.33% in the PSCO group and 66.67% in the NLDO group respectively (P<0.05). After treatment with the micro-drill, the healing rate in PSCO and NLDO groups was 65.39% and 94.28% respectively (P<0.01). The lacrimal passage obstruction can be observed and treated directly through the lacrimal endoscope. Choosing different surgical procedures in operation according to the locations of the obstruction is helpful to improve the effectiveness.

In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats (rMSCs) and the possibility of rMSCs differentiation into retinal neural cells, the bone marrow-derived cells in SD rats were isolated and cultured in vitro. The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium. The cultured rMSCs were induced to differentiate by two steps. Immunofluorescence method and anti-nestin, anti-NeuN, anti-GFAP and anti-Thy1.1 antibodies were used to identify the cells derived from the rMSCs. The results showed that the in vitro cultured rMSCs grew well and expanded quickly. After induction with two conditioned media, rMSCs was induced to differentiate into neural progenitor cells, then into retinal neural-like cells which were positive for nestin, NeuN, GFAP and Thy1.1 detected by fluorescence method. The findings suggested that rMSCs could be culture and expanded in vitro, and induced to differentiate into retinal neural-like cells.

In order to explore the role of TGF-β1 in scleral remodeling and the possible mechanism, the influence of high level TGF-β1 on scleral thickness and the expression of MMP-2 and TIMP-2 was investigated in a TGF-β1 transgenic mouse model. Alb/TGF-β1 (Cys^223,225Ser) TGF-β1 transgenic mice were used as experimental subjects and non-transgenic littermates as controls. Plasma levels of TGF-β1 were determined by ELISA. TGF-β1, MMP-2 and TIMP-2 levels in sclera were detected by using Western blot. The thickness of posterior sclera was measured by computerized image analysis of a midsagittal section. Mean difference was analyzed with independent t-test. The results showed plasma levels of TGF-β1 in transgenic mice were 1.68 times as much as that in the controls (P<0.01). TGF-β1 levels in the sclera of transgenic mice were 2.68 times of the controls (P<0.01). Posterior scleral thickness in transgenic mice were significantly thicker than in the controls. There was no significant difference in the MMP-2 levels between transgenic mice and controls, but the TIMP-2 levels were increased significantly in transsgenic mice as compared with those in the controls. It was suggested that high levels of TGF-β1 in transgenic mice could result in the increased scleral thickness by inducing the expression of TIMP-2 to suppress the activity of MMP-2, finally inhibiting the degradation of collagen.

In order to study the expression of interleukin-22 (IL-22) and S100A7, A8, A9 mRNA in the skin lesions of patients with psoriasis vulgaris and their relationship, the biopsies were taken from skin lesions in 35 patients with psoriasis vulgaris and the skin of 16 normal controls, and the expression levels of IL-22 and S100A7, A8 and A9 mRNA were detected by semi-quantitative RT-PCR. The results showed that (1) IL-22 and S100A8, A9 mRNA were positively expressed in the psoriatic skin lesions but negatively expressed in the normal controls; The expression level of S100A7 was (1.133±0.040) in the psoriatic skin lesions, significantly higher than that in the normal controls (0.744±0.037, P<0.01). (2) There were significantly positive correlations between the expression of IL-22/S100A7 mRNA, IL-22/S100A8 mRNA, IL-22/S100A9 mRNA in the psoriasis vulgaris (r₁=0.543, r₂=0.774, r₃=0.621, P<0.01). It was concluded that IL-22 and S100A7, A8, A9 might play important roles in the occurrence and progression of psoriasis.

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria Gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.

In order to investigate the expression of endothelin receptor B (ETR-B) in human malignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a concentration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.

The possible mechanism of inhalation anesthetics on the internal auditory impairment of the rat was investigated by determining the effect of nitrous oxide (N₂O) and isoflurane on the total RNA yield from the cochlea of the rats. Thirty healthy Wistar rats were randomly divided into 3 groups: group C (control group, n=10) with a 3-h unremitting inhalation of 50% O₂, group N (experiment group, n=10) with a continuous inhalation of 50% N₂O+50% O₂ for 3 h, and group I (experiment group, n=10) with a 3-h sustained inhalation of 2.5% isoflurane. The TRIzol in combination with RNeasy was used to respectively extract the total RNA from cochlea of rats in the 3 groups. Spectrophotometry was used to detect total RNA yield and electrophoresis to detect the quality. The total RNA extracted from the cochlea of the rats in the groups C and N was 7.69 and 6.51 μg, respectively. There was a 15% decrease in the N group as compared with group C. The total RNA from the rats in the group I was 7.32 μg, and there was hardly any change in the group as compared with the group C. The value of A₂₆₀/A₂₈₀ in groups C, N and I was 2.07, 2.04 and 2.04, respectively, showing a very high RNA purity. The result of gel electrophoresis suggested that there was no degradation in the total RNA. It was suggested that the interference of N₂O on the cochlear RNA yield might be one of the reasons which cause an injury of the ear. The isoflurane shows no harm on the hearing.

In order to observe the feature of age-related marrow conversion and maturation of epiphyseal cartilage and analyze the distribution of red and yellow marrow in the proximal femur at STIR MR imaging, STIR and T₁ weighted MR imaging of the proximal femur in 52 subjects, aged 4 months to 25 years old, were retrospectively analyzed for the distribution and appearance of red and yellow marrow. The subjects with no known bone marrow abnormalities were divided into 6 age groups. The signal intensity of the marrow in the proximal epiphysis, proximal metaphysis, proximal diaphysis, distal diaphysis and greater trochanter was compared with the signal intensity and homogeneity of surrounding muscle and fat and graded by two observers. The results showed that the conversion of hematopoietic marrow in the proximal femur followed a well-defined sequence, occurring first in the proximal epiphysis, followed by the distal diaphysis, and then greater trochanter and metaphysis. STIR in combination with T₁-weighted imaging could display clearly the origin of ossification center and the course of conversion from red to yellow marrow in proximal epiphysis and greater trochanter. STIR imaging showed that the marrow conversion in proximal metaphysic began below epiphyseal plate and intertrochanter. The site of red yellow was distributed in weight-bearing axis by 20 years of age. The marrow conversion of diaphysis was from distal end to proximal end, and the consequence of conversion was that distal diaphysis contained yellow marrow but proximal diaphysis partly red marrow connected with the red marrow of metaphysic. The epiphyseal cartilage had different characters of signal-intensity with age in STIR sequence. The distribution of red marrow in STIR imaging was more close to that of anatomy than T₁-weighted imaging. It was concluded that STIR could dynamically display the feature of morrow conversion and the development of epiphyseal cartilage and accurately reveal the age-related distribution of red and yellow marrow on STIR imaging in the proximal femur.

The neonatal burst suppression is a severe EEG pattern and always demonstrates serious damage of nerve system. But the outcome of these patients depends on the different etiology. A total of 256 cases of video EEG recordings were analyzed in order to summarize the etiology and outcome of burst suppression. The results showed that some patients in all 17 cases of burst suppression showed EEG improvement. The etiology was the dominant factor in long term outcome. It was suggested that effective video EEG monitoring is helpful for etiologic study and prognosis evaluation.