[Assessment and application of a molecular diagnostic method on the detection of four types of diarrheagenic Escherichia coli].

OBJECTIVE: To establish and evaluate a molecular diagnostic method for routine monitoring of four types of diarrheagenic Escherichia (E.) coli (DEC)and to study the distribution of four types of DEC isolated from diarrheal patients in Shanghai. METHODS: DEC-PCR standard operation procedure(SOP)had been developed for DEC detection and isolation, using the Statens Serum Institute (SSI) DEC PCR kits with multiplex PCR technique after verification tests on reference strains. Diarrhea specimens from 3 clinical hospitals in Shanghai were tested from June to September, 2012. RESULTS: Specificity of the PCR kit was 100% by verification on the 26 DEC reference strains. A total number of 218 DEC isolates, including 181 fermented lactose and 37 unfermented lactose were identified from the 1887 stool specimens of diarrhea patients, with positive rate as 11.6%. The most common pathogen(54.1%, 118/218)was enteropathogenic E. coli(EPEC), followed by enterotoxigenic E. coli(ETEC, 41.3%, 90/218), enteroinvasive E. coli (EIEC, 4.1%, 9/ 218) and Shigatoxin-producing E. coli(STEC, 0.5%, 1/218)in addition to 18 Shigella isolates. ETEC dominated in diarrhea patients with foreign residency, as well as 1/3 were perinatal stage of neonatal ETEC of all diarrhea cases under the age of 5, while EPEC dominated in the Chinese diarrhea patients especially among young kids under the age of 2. CONCLUSION: Data was reliable after assessment on this molecular diagnostics and seperation procedures used for the routine monitoring on four types of DEC, while the diagnosis and reference ability of DEC regarding the laboratories net-working on food-borne pathogens need to be built up and improved.

[The fundamental role of stage control technology on the detectability for Salmonella networking laboratory].

OBJECTIVE: To evaluated the fundamental role of stage control technology (SCT) on the detectability for Salmonella networking laboratories. METHODS: Appropriate Salmonella detection methods after key point control being evaluated, were establishment and optimized. Our training and evaluation networking laboratories participated in the World Health Organization-Global Salmonella Surveillance Project (WHO-GSS) and China-U.S. Collaborative Program on Emerging and Re-emerging infectious diseases Project (GFN) in Shanghai. Staff members from the Yunnan Yuxi city Center for Disease Control and Prevention were trained on Salmonella isolation from diarrhea specimens. Data on annual Salmonella positive rates was collected from the provincial-level monitoring sites to be part of the GSS and GFN projects from 2006 to 2012. RESULTS: The methodology was designed based on the conventional detection procedure of Salmonella which involved the processes as enrichment, isolation, species identification and sero-typing. These methods were simultaneously used to satisfy the sensitivity requirements on non-typhoid Salmonella detection for networking laboratories. Public Health Laboratories in Shanghai had developed from 5 in 2006 to 9 in 2011, and Clinical laboratories from 8 to 22. Number of clinical isolates, including typhoid and non-typhoid Salmonella increased from 196 in 2006 to 1442 in 2011. The positive rate of Salmonella isolated from the clinical diarrhea cases was 2.4% in Yuxi county, in 2012. At present, three other provincial monitoring sites were using the SBG technique as selectivity enrichment broth for Salmonella isolation, with Shanghai having the most stable positive baseline. CONCLUSION: The method of SCT was proved the premise of the network laboratory construction. Based on this, the improvement of precise phenotypic identification and molecular typing capabilities could reach the level equivalent to the national networking laboratory.

[Preliminary application of Back-Propagation artificial neural network model on the prediction of infectious diarrhea incidence in Shanghai].

OBJECTIVE: To establish BP artificial neural network predicting model regarding the daily cases of infectious diarrhea in Shanghai. METHODS: Data regarding both the incidence of infectious diarrhea from 2005 to 2008 in Shanghai and meteorological factors including temperature, relative humidity, rainfall, atmospheric pressure, duration of sunshine and wind speed within the same periods were collected and analyzed with the MatLab R2012b software. Meteorological factors that were correlated with infectious diarrhea were screened by Spearman correlation analysis. Principal component analysis (PCA) was used to remove the multi-colinearities between meteorological factors. Back-Propagation (BP) neural network was employed to establish related prediction models regarding the daily infectious diarrhea incidence, using artificial neural networks toolbox. The established models were evaluated through the fitting, predicting and forecasting processes. RESULTS: Data from Spearman correlation analysis indicated that the incidence of infectious diarrhea had a highly positive correlation with factors as daily maximum temperature, minimum temperature, average temperature, minimum relative humidity and average relative humidity in the previous two days (P < 0.01), and a relatively high negative correlation with the daily average air pressure in the previous two days (P < 0.01). Factors as mean absolute error, root mean square error, correlation coefficient(r), and the coefficient of determination (r(2)) of BP neural network model were established under the input of 4 meteorological principal components, extracted by PCA and used for training and prediction. Then appeared to be 4.7811, 6.8921,0.7918,0.8418 and 5.8163, 7.8062,0.7202,0.8180, respectively. The rate on mean error regarding the predictive value to actual incidence in 2008 was 5.30% and the forecasting precision reached 95.63% . CONCLUSION: Temperature and air pressure showed important impact on the incidence of infectious diarrhea. The BP neural network model had the advantages of low simulation forecasting errors and high forecasting hit rate that could ideally predict and forecast the effects on the incidence of infectious diarrhea.

PI3K p110α isoform-dependent Rho GTPase Rac1 activation mediates H2S-promoted endothelial cell migration via actin cytoskeleton reorganization.

Hydrogen sulfide (H(2)S) is now considered as the third gaseotransmitter, however, the signaling pathways that modulate the biomedical effect of H(2)S on endothelial cells are poorly defined. In the present study, we found in human endothelial cells that H(2)S increased cell migration rates and induced a marked reorganization of the actin cytoskeleton, which was prevented by depletion of Rac1. Pharmacologic inhibiting vascular endothelial growth factor receptor (VEGFR) and phosphoinositide 3-kinase (PI3K) both blunted the activation of Rac1 and the promotion of cell migration induced by H(2)S. Moreover, H(2)S-induced Rac1 activation was selectively dependent on the presence of the PI3K p110α isoform. Activated Rac1 by H(2)S thus in turn resulted in the phosphorylation of the F-actin polymerization modulator, cofilin. Additionally, inhibiting of extracellular signal-regulated kinase (ERK) decreased the augmented cell migration rate by H(2)S, but had no effect on Rac1 activation. These results indicate that Rac1 conveys the H(2)S signal to microfilaments inducing rearrangements of actin cytoskeleton that regulates cell migration. VEGFR-PI3K was found to be upstream pathway of Rac1, while cofilin acted as a downstream effector of Rac1. ERK was also shown to be involved in the action of H(2)S on endothelial cell migration, but independently of Rac1.

Neuroprotective effects of water-soluble Ganoderma lucidum polysaccharides on cerebral ischemic injury in rats.

AIM OF THE STUDY: To investigate the neuroprotective effects of water-soluble Ganoderma lucidum polysaccharides (GLPS) on cerebral ischemic injury in rats, and to explore the involved mechanisms. MATERIALS AND METHODS: Two models [middle cerebral artery occlusion (MCAO) in Sprague-Dawley (SD) rats and oxygen and glucose deprivation (OGD) in primary cultured rat cortical neurons] were employed to mimic ischemia-reperfusion (I/R) damage, in vivo and in vitro, respectively. Cerebral infarct area was measured by tetrazolium staining, and neurological functional deficits were assessed at 24h after I/R. Neuronal apoptosis was studied by Nissl staining and DNA fragmentation assay. Neuronal injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage, cell viability was measured by sodium 3'-1- (phenylaminocarbonyl)-3, 4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid (XTT) reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3, -8 and -9 activation and Bcl-2, Bax protein expression were determined by western blot analysis. RESULTS: Oral administration of GLPS (100, 200 and 400mg/kg) significantly reduced cerebral infarct area, attenuated neurological functional deficits, and reduced neuronal apoptosis in ischemic cortex. In OGD model, GLSP (0.1, 1 and 10 microg/ml) effectively reduced neuronal cell death and relieved cell injury. Moreover, GLPS decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspases-3, -8 and -9 and Bax, and inhibited the reduction of Bcl-2 expression. CONCLUSIONS: Our findings indicate that GLPS protects against cerebral ischemic injury by inhibiting apoptosis by downregulating caspase-3 activation and modulating the Bcl-2/Bax ratio.

[Establishing and applying of autoregressive integrated moving average model to predict the incidence rate of dysentery in Shanghai].

OBJECTIVE: To explore the feasibility of establishing and applying of autoregressive integrated moving average (ARIMA) model to predict the incidence rate of dysentery in Shanghai, so as to provide the theoretical basis for prevention and control of dysentery. METHODS: ARIMA model was established based on the monthly incidence rate of dysentery of Shanghai from 1990 to 2007. The parameters of model were estimated through unconditional least squares method, the structure was determined according to criteria of residual un-correlation and conclusion, and the model goodness-of-fit was determined through Akaike information criterion (AIC) and Schwarz Bayesian criterion (SBC). The constructed optimal model was applied to predict the incidence rate of dysentery of Shanghai in 2008 and evaluate the validity of model through comparing the difference of predicted incidence rate and actual one. The incidence rate of dysentery in 2010 was predicted by ARIMA model based on the incidence rate from January 1990 to June 2009. RESULTS: The model ARIMA (1, 1, 1) (0, 1, 2)(12) had a good fitness to the incidence rate with both autoregressive coefficient (AR1 = 0.443) during the past time series, moving average coefficient (MA1 = 0.806) and seasonal moving average coefficient (SMA1 = 0.543, SMA2 = 0.321) being statistically significant (P < 0.01). AIC and SBC were 2.878 and 16.131 respectively and predicting error was white noise. The mathematic function was (1-0.443B) (1-B) (1-B(12))Z(t) = (1-0.806B) (1-0.543B(12)) (1-0.321B(2) x 12) micro(t). The predicted incidence rate in 2008 was consistent with the actual one, with the relative error of 6.78%. The predicted incidence rate of dysentery in 2010 based on the incidence rate from January 1990 to June 2009 would be 9.390 per 100 thousand. CONCLUSION: ARIMA model can be used to fit the changes of incidence rate of dysentery and to forecast the future incidence rate in Shanghai. It is a predicted model of high precision for short-time forecast.

Organized intrasylvian subarachnoid hematoma in post-traumatic child with dyskinesia for 8 years: a case report and review of the literature.

OBJECT: The authors present their experience with an organized intrasylvian subarachnoid hematoma (OISH) in a post-traumatic pediatric patient with dyskinesia for nearly 8 years. METHODS: An 11-year-old Chinese boy was admitted to the authors' hospital because of dyskinesia in his right upper and lower extremities. When he was 18 months old, he fell down from a trolley and then his mouth drooped to a right angle. The brain computer tomography (CT) revealed a space-occupying lesion in his left temporoparietal region. The symptom improved after 20 days of acupuncture therapy in local hospital. Two years later when he was 4 years old, his right lower limb became lame gradually with sensorial deficit. A concealed arteriovenous malformation was suggested by the brain magnetic resonance imaging and magnetic resonance angiography at that time. The child had been treated with ginkgo biloba leaf extract from 2001 to 2007 and the symptom improved gradually during that period. However, the symptom of his right upper and lower extremities deteriorated continually since January 2007. He fell down again when he was walking 1 month before he was admitted to the authors' department in July 2007. An enlarged left pterional craniotomy was performed to remove the lesion. Histopathology diagnosis was compatible with an organized hematoma with remote hemorrhage and gliosis. The child is presently healthy after 1 year's follow-up. CONCLUSION: The rarity of an OISH in a post-traumatic pediatric patient with dyskinesia for nearly 8 years makes this case very peculiar. This is the first reported pediatric case of OISH found in the literature.

Intracranial solitary juvenile xanthogranuloma in an infant.

BACKGROUND: Juvenile xanthogranuloma (JXG) is a disorder of histiocyte proliferation. Most cases present with a solitary cutaneous lesion. JXG with systemic involvement is rare with significant morbidity. Intracranial solitary JXG may be misdiagnosed before operation. METHODS: A 5-month-old boy showed an elevated anterior fontanel but no other abnormalities on admission. Brain MRI showed a large mass in the right parietal region. RESULTS: The tumor was removed with the encroached meninges. A JXG in the right parietal region was diagnosed pathologically. CONCLUSION: Total excision of the tumor may be curative with a prerequisite of ensuring normal vital signs and nervous function.

[Study on the epidemiological characteristics and molecular typing of Salmonella enterica subsp. enterica serovar Senftenberg in Shanghai].

OBJECTIVE: To study the molecular epidemiological characteristics of Salmonella enterica subsp. enterica serovar Senftenberg (Salmonella senftenberg) in Shanghai, from 2006 to 2007. METHODS: A retrospective analysis in 2006 and 2007 was performed to explore the source of food-borne Salmonella senftenberg. The isolates from diarrhea patients between 2006 and 2007 were identified, including biochemical test, hilA and invA gene phenotyping, drug susceptibility, Riboprinter((R)) (RP) and pulsed-field gel electrophoresis (PFGE). RESULTS: Of the diarrhea patients isolates in the monitoring program on non-typhi Salmonella infection in the year of 2006 in Shanghai, number of patients caused by Salmonella senftenberg ranked the third. The stock of Salmonella senftenberg food-born isolates were derived from swine and beef products between 2003 and 2005. All of the strains from diarrhea patients were susceptible to antibiotics except tetracylina (75.6%). With RP and PFGE molecular typing, the two groups (with hydrogen sulfide and hilA, invA gene or without) could be divided into two different independent clone cluster in genetics. 34 strains of diarrhea were divided into 16 PFGE typing-pattern, and among them 12 strains including type 4 (4 strains), type 5 (1 strains), type 6 (6 strains), type 7 (1 strains) and 13 strains including type 11 (3 strains), type 17 (5 strains), type 23 (5 strains) were two different dominant clone cluster. CONCLUSION: The epidemic of Salmonella senftenberg within 2006 might have been the result of a long period of case occurrence in Shanghai. This rare outbreak belonged to a cluster of outbreaks caused by two different PFGE clone clusters. Data suggested that the genetic clone of Salmonella senftenberg might have been unstable and the source of contamination were complicated, with the characteristics as the obvious decreasing number of patients, with no food-borne isolates in 2007.

Biphasic response of endothelial progenitor cell proliferation induced by high glucose and its relationship with reactive oxygen species.

In this study, the effect of high glucose (HG) on endothelial progenitor cell (EPC) proliferation and its relationship with cyclins and reactive oxygen species (ROS) were investigated. Mouse EPCs were isolated from bone marrow using a magnetic activated cell-sorting system and cultured in the presence or absence of HG (30 mmol/l). We found that in the early stage of incubation (3 days), HG promoted cell proliferation, and increased the expressions of cdk2 and cyclin E, while in the late stage of culture (7 days) it inhibited cell proliferation and decreased the expressions of cdk2, cyclin E, and proliferating cell nuclear antigen (PCNA). Moreover, on the third day after incubation, HG significantly inhibited the apoptosis of EPCs, while in the late stage it markedly activated caspase-3 and promoted apoptosis. ROS generation in cells and maleic dialdehyde level in medium were significantly increased in HG group on the seventh day, whereas the expressions of superoxide dismutase and glutathione levels decreased. Tempol, a membrane-permeable radical scavenger, significantly inhibited ROS production in EPCs and partially reversed the HG-mediated inhibition of EPCs proliferation on the seventh day. We hypothesize that in the HG environment, the biphasic response of EPC proliferation may be related to the generation of ROS, which causes modulation of cyclins and cell cycle effect.

Roles of cyclooxygenase-2 in microvascular endothelial cell proliferation induced by basic fibroblast growth factor.

BACKGROUND: The level of basic fibroblast growth factor (bFGF) increases rapidly after cerebral ischemia. However, the molecular mechanisms for the effects of bFGF on cerebral microvascular endothelial cells (cMVECs) have not yet been fully elucidated. In this study, a murine cMVEC line, bEnd.3, was employed to study the effects of bFGF on cyclooxygenase (COX) expression and its downstream effects in cMVECs. METHODS: After treatment with bFGF, RT-PCR and Western blotting analyses were carried out to evaluate the changes in COX-2 mRNA and protein expression, respectively. MTT assays were performed to measure cell proliferation. The prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) concentrations in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: COX-2 mRNA and protein expressions in bEnd.3 cells were induced by bFGF in time- and dose-dependent manners. The bFGF-induced COX-2 upregulation led to enhanced PGE2 production by bEnd.3 cells, and this effect was abolished by the selective COX-2 inhibitor NS-398. bFGF also increased VEGF production by bEnd.3 cells, and this effect was blocked by NS-398 and the EP1/2 (PGE2 receptors) antagonist AH6809. Furthermore, exogenous PGE2 increased VEGF production in bEnd.3 cells, and AH6809 blocked this effect. CONCLUSION: bFGF increases VEGF production in an autocrine manner by increasing COX-2-generated PGE2 in cMVECs and subsequently stimulates MVEC proliferation and angiogenesis.

The novel proangiogenic effect of hydrogen sulfide is dependent on Akt phosphorylation.

OBJECTIVE: Hydrogen sulfide (H(2)S) has been reported to be a gasotransmitter which regulates cardiovascular homeostasis. The present study aims to examine the hypothesis that hydrogen sulfide is able to promote angiogenesis. METHODS: Angiogenesis was assessed using in vitro parameters (i.e. endothelial cell proliferation, adhesion, transwell migration assay, scratched wound healing and formation of tube-like structure) and in vivo by assessing neovascularization in mice. Phosphorylation of Akt was measured using Western blot analysis. RESULTS: Exogenously administered NaHS (H(2)S donor) concentration-dependently (10-20 micromol/l) increased cell growth, migration, scratched wound healing and tube-like structure formation in cultured endothelial cells. These effects of NaHS on endothelial wound healing and tube-like structure formation were prevented by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 (5 micromol/l) or transfection of a dominant-negative mutant of Akt. NaHS increased Akt phosphorylation and this effect was also blocked by either LY 294002 or wortmannin (25 nmol/l). NaHS did not significantly alter the levels of vascular endothelial growth factor, mRNA expression of fibroblast growth factor and angiopoietin-1, or nitric oxide metabolites. NaHS treatment (10 and 50 micromol kg(-1) day(-1)) significantly promoted neovascularization in vivo in mice. CONCLUSION: The present study reports a novel proangiogenic role of H(2)S which is dependent on activation of Akt.

Tight filum terminale syndrome in children: analysis based on positioning of the conus and absence or presence of lumbosacral lipoma.

BACKGROUND: Tight filum terminale syndrome (TFTS) characterized by findings consistent with a tethered cord but with the conus ending in a normal position has only recently been observed in children. In this situation, diagnosis may prove difficult and sectioning of the filum terminale is questionable. MATERIALS AND METHODS: Sixty cases of pediatric TFTS were analyzed by methods including spinal X-ray and magnetic resonance imaging (MRI). Twenty-one patients exhibited a normally positioned conus, 18 a low-lying conus, and 21 a low-lying conus with accompanying lumbosacral lipoma. These three groups were compared preoperatively and postoperatively for lumbosacral cutaneous stigmata, vertebral anomalies, concomitant congenital spinal dysraphisms, lower limb deformities, and sphincter dysfunction. RESULTS: Rates of occurrence of lumbosacral cutaneous stigmata and other concomitant congenital spinal dysraphisms differed significantly among the groups. Differences in other parameters were not observed. All groups responded positively to surgery. CONCLUSIONS: Pediatric TFTS may involve a normally positioned conus. Diagnosis of pediatric TFTS should be based on clinical presentation, physical and radiological examinations, MRI, and pathologic changes in the filum. When neurological signs accompany such changes, early severing of the filum is indicated regardless of conus position.

Dynamic changes in the expression of growth factor receptors in the myocardium microvascular endothelium after murine myocardial infarction.

BACKGROUND: After myocardial infarction, specific growth factors promote cardiac angiogenisis, leading to a therapeutic effect. Although this effect is mediated by specific receptors in the endothelium of the cardiac microvasculature, few studies have investigated dynamic changes in their expression. We explored this phenomenon in a murine model. METHODS: We observed the mRNA expression of receptors by specific angiogenesis gene microarray at day 3 and day 7 after infarction. The vascular endothelial growth factor (VEGF) receptor Flk-1 was observed at the protein level at day 3 and day 7 by immunohistochemistry. The dynamic expression of fibroblast growth factor receptor-1 (FGFR-1) mRNA in the border zone and the noninfarcted zone at day 3, day 7, day 14, and day 42 was investigated by real-time PCR. Statistical significance was analyzed with SPSS 10.0 software using one-way analysis of variance (ANOVA). RESULTS: Three days after infarction, 9 receptors in the border zone and 7 receptors in the noninfarcted zone were down-regulated. Two receptors in the infarct edge and 5 receptors in the distant myocardium were up-regulated. However, at day 7, 11 receptors in the border zone were up-regulated, and only one was down-regulated. In the border zone, Flk-1 levels decreased at day 3 but increased significantly at day 7. Real-time PCR showed that FGFR-1 mRNA decreased markedly in the border zone at day 3 but increased afterward for at least 6 weeks. In the early stage (3 days) after infarction, the expression of receptors had decreased to some extent. However, at day 7, receptor expression was active and had moved from the distant noninfarcted zone to the border zone as a part of the acute repair process. CONCLUSION: Selecting the proper growth factors to target receptors with protective activity, and determining appropriate therapeutic timing may be important to the success of therapeutic angiogenesis.

Surgical management to conjoined twins in Shanghai area.

Conjoined twins are very rare congenital malformation. The aim of this study was to summarize our experiences of surgical separation on seven sets of conjoined twins, and improve the treatment of conjoined twins in the future. A retrospective review of surgical separation included data of prenatal diagnosis, associated malformation, timing of separation, intra- and postoperative management, and follow-up of six sets of conjoined twins at Shanghai Xin-Hua Hospital from 1980 to 2005 and one set at Shanghai Children's Hospital in 2002. Surgical separation was performed on seven sets of conjoined twins; six sets of thoracopagus-omphalapagus (including four sets of xipho-omphalopagus) and one set of ischiopagus. All sets presented varying degrees of severity of congenital cardiac malformations. Four sets were diagnosed prenatally by ultrasonography. Two sets of conjoined twins (case 2 and case 3) required emergent separation within 7 days after birth; both members of case 2 died within 2 days post operation, one member of case 3 died during operation while the other member survived. Five sets had scheduled separation undertaken more than 30 days after birth. One member of a set (case 6B) died 13 days after operation due to severe congenital cardiovascular anomalies. All other members of conjoined twins survived. Case 6A had a severe defect of the anterior thoracic cage and prosthesis of titanium alloy scaffold filled with silicone rubber was used to repair the defects successfully. Following up from 1980 to 2005, one member of a set (case 1A) died 4 years after operation due to pneumonia. Contact was lost to the surviving member of case 3 (ischiopagus). Other survivors of the separations had normal development. (1) Timing of operation and separation plan should be given according to the circumstances and the nature of the organ shared in each individual set of twins. (2) Prosthesis of titanium alloy scaffolds filled with silicone rubber may become one of viable methods for repairing severe defects of the thoracic cage.

Inhibition of microvascular endothelial cell apoptosis by angiopoietin-1 and the involvement of cytochrome C.

BACKGROUND: Angiopoietin-1 (Ang-1) is an endothelial-specific growth factor that can promote angiogenesis. Studies demonstrated that Ang-1 can inhibit apoptosis of umbilical endothelial cells, but so far little is known about its effects on apoptosis of microvascular endothelial cells. With the apoptotic model of murine-cerebral-derived microvascular endothelial cells (bEnd.3) induced by serum-free culture, we attempted to clarify the molecular mechanism of bEnd.3 apoptosis, particularly its relation to cytochrome C (Cyt C). METHODS: The cultured microvascular endothelial cell strain, bEnd.3 cell, was employed. An apoptotic model of bEnd.3 was established by serum-free culture. Flow cytometry after Annexin labeling and PI staining were used to assess the apoptotic effects of Ang-1 on bEnd.3, and the expression of Bax/Bcl-2, caspase 8, caspase 3, and Cyt C were detected with Western blotting and ELISA. RESULTS: The apoptotic rate of bEnd.3 cells after stimulation with Ang-1 (100 ng/L) in serum-free medium was significantly higher than that in control group. Ang-1 inhibited early-stage apoptosis more than late-stage apoptosis provided by propidium iodide (PI) and AnnexinV double staining. The inhibition of Ang-1 on bEnd.3 cell apoptosis was strengthened with the increase in concentration (0 - 400 ng/ml). Ang-1 could decrease the expression of Bax, caspase3 and 8, and increase that of Bcl-2. The results of ELISA indicated that Ang-1 significantly decreased CytC content in cytoplasm and increase that in mitochondria. CONCLUSIONS: Ang-1 could inhibit bEnd.3 apoptosis induced by serum-free medium culture. The apoptosis was associated with decreased Bax expression, increased Bcl-2 expression, which result in Cyt C transferring from mitochondria to cytoplasm, and then caspases activation are reduced and cell apoptosis is suppressed.

Effects of basic fibroblast growth factor on the expressions of angiogenic gene profile and cyclooxygenase-2 in brain microvascular endothelial cells.

The present study aimed to investigate the effects of basic fibroblast growth factor (bFGF) on the expressions of angioge-nesis-related genes in a mouse brain microvascular endothelial cell line, namely bEnd.3, using cDNA microarray. The effects of bFGF (10 ng/ml) on mRNA and protein expressions of cyclooxygenase-2 (COX-2), an angiogenesis bystander molecule, were further investigated. cDNA microarray was employed to study the effects of bFGF on the expressions of angiogenic genes in a high throughput pattern. RT-PCR was used to study the effect of bFGF on COX-2 mRNA expression. Western blot and immunocytochemistry were utilized to study the effect of bFGF on COX-2 protein expression. The results showed that, 2 h after bFGF treatment, pro-angiogenic genes (Adamts1, MMP-9, Ang-1, PDGF B, G-CSF, FGF16, IGF-1, etc.) were significantly upregulated, whereas anti-angiogenic genes (TIMP-2, TSP-3, etc.) were significantly downregulated. The bystander molecule in angiogenic pathway COX-2 mRNA and protein expressions were significantly upregulated after bFGF treatment. It is suggested that triggering angiogensis switch through upregulating pro-angiogenic gene and downregulating anti-angiogenic gene expression is one of the major mechanisms of bFGF-induced angiogenesis. The expression change of COX-2, as a bystander molecule, was observed after bFGF treatment in bEnd.3 cells and the significance was discussed.

[Gene transfer of tumor necrosis factor related apoptosis inducing ligand to endothelial progenitor cell for ovarian cancer therapy].

OBJECTIVE: To express tumor necrosis factor related apoptosis inducing ligand (TRAIL) protein using endothelial progenitor cell (EPC) and observe antitumor activity of secreted TRAIL in cell culture supernatant. METHODS: EPC were isolated by immunomagnetic sorting. Human TRAIL plasmid was transfected into EPC by lipofectamine 2000. The cell culture supernatant was harvested and the secreted protein was measured by immunoassay method. Apoptosis rate was analysed by flow cytometry. RESULTS: EPC could develop from the culture of cord blood CD(133)(+) (cluster of differentiation 133(+)) cells. EPC may be transiently transfected with human TRAIL plasmid. The level of TRAIL in the cell culture supernatant was higher in the TRAIL transfected group than in the non-transfected group and green-fluoro protein (GFP) transfected group (532.8 pg/ml versus 12.4 and 9.2 pg/ml). Apoptosis detection showed that TRAIL had high antitumor activity in 3AO cell, the apoptosis rate of ovarian cancer cell could reach 24.1%. CONCLUSIONS: In vitro, TRAIL EPC gene transfer enhances EPC to secrete TRAIL protein, and induces apoptosis of ovarian cancer cell. There is a good clinical prospect for EPC in the gene therapy of ovarian cancer.

[Dynamic changes in focal adhesion kinase during cell migration induced by bFGF and the significance].

Cell migration plays an important role in repair of injury, angiogenesis, cancer metastasis and so on. In this paper the effects of basic fibroblast growth factor (bFGF) of different concentrations on ECV-304 cell migration, and the dynamic changes in focal adhesion kinase (FAK) were observed. The relationship between FAK and cell migration induced by bFGF was studied. A ECV-304 cell scratch wound model was established and the images of cell migration were quantitatively measured using a computer-assisted videomicroscopic system. The dynamic changes in FAK content (Western blot), FAK activity (immunoprecipitation plus Western blot) and FAK mRNA (RT-PCR) were measured in vitro. The expression of integrin alpha3 was investigated using immunocytochemical staining (ABC method). The results showed that bFGF produced a dual-phase regulatory effect on ECV-304 migration when the cell confluent areas reached 90%-95% in culture. It was found that compared with the control group (0 ng/ml bFGF), the cell migration was stimulated (P<0.05), inhibited (P<0.05) and unchanged when the cultured cells were treated with bFGF at the concentrations of 5, 10 and 15 ng/ml, respectively. The content and activity of FAK protein were markedly up-regulated in 5 ng/ml bFGF group and down-regulated in 15 ng/ml bFGF group, respectively. FAK mRNA expression came to the peak in 5 ng/ml bFGF group after 6 h culture and there was a significant difference compared with the control group. In various experimental groups there were no significant differences in the expression of integrin alpha3 compared with the control group according to the immunocytochemical staining. The results mentioned above suggest that different concentrations of bFGF have a dual-phase effect on the migration of cultured ECV-304 cells, which correlates positively with FAK content, activity and mRNA in cultured ECV-304 cell scratch wound model. The FAK plays an important role in the signal transduction pathway of cell migration induced by bFGF, while bFGF can regulate the content of FAK in ECV-304 cells at gene transcription level.