Objective To explore the effects of expressing human ciliary neurotrophic factor (hCNTF) mediated by retroviral vector in olfactory ensheathing cells(OECs) on the survival and neurite outgrowth of cultured neurons. Methods S\|hCNTF fragment was digested with endonucleases(Kpn I and Xba I) from pcDNA\-3\|S\|hCNTF plasmid and cloned into pRev\|TRE vector.The harvested pRev\|TRE\|hCNTF was identified and transfected with pRev\|Tet\|On into ecotropic Ecopack\|293 cells,resulting in 2 retroviral supernatants(pRev\|TRE\|hCNTF and pRev\|Tet\|On).Primarily cultured rat olfactory ensheathing cells(OECs) were co\|infected with the 2 retroviruses,and induced to secrete hCNTF with different concentrations of doxycline.The secreted hCNTF in OEC culture supernatant was detected with Western\|blot.Dorsal root ganglion (DRG) from a postnatal rat of 2 days was co\|cultured with CNTF\|modified OECs,and the supernatant was used to culture retinal ganglion cells(RGCs).Following ?\|tubulin immunocytochemical staining,the length of DRG neurites were measured,while the numbers of surviving RGCs were counted. Results 1.Individual 630bp and 400bp fragments were digested from pRev\|TRE\|S\|hCNTF expression vector with endonucleases(Hind Ⅲ and BamH Ⅰ),and respected direction and integration of hCNTF cDNA which inserted pRev\|TRE vector were identified; 2.The expression of 24kD CNTF proteins in CNTF\|modified OEC culture supernatant was positively\|correlated with the concentration of doxycline,while no such protein expression was detected in the control groups; 3.The number of surviving RGCs in CNTF\|modified OECs group(41\^34?5\^4) was significantly higher than those in unmodified OEC(23\^15?4\^7),OECs(24\^55?5\^8) and blank(16\^8?6\^5) groups;and 4\^The neurites of DRG were longer (660?67?m) and denser in CNTF\|modified OECs group,as compared with unmodified OECs(418?45?m),Mock+OECs(400?65?m) and blank (0?m) control groups.No process migrated and grew from the tissue mass in blank group.Conclusion\ hCNTF can be expressed in OECs with a doxycline concentration\|dependent manner after transfected via pRev\|TRE\|S\|hCNTF vector,and possesses a marked enhancing effect on the survival and neurite outgrowth of cultured neurons.[

China ; Congresses as Topic ; Fatty Liver ; Humans

AIMTo explore the differentially expressed proteins between hypobaric hypoxic delayed preconditioning (HHDP) and normal mouse hippocampus.

METHODSAfter the animal model of HHDP was constructed, hippocampal proteins were obtained by a series of abstraction with lysis solution containing high concentration urea. As soon as isoelectric focusing and SDS-PAGE was performed. The resolved proteins in the 2-DE gels were visualized by Coomassie blue R-250. The gels were scanned, and the images were processed with PDQuest software. Differential proteins were exactly excised from the gels, destained and digested with trypsin. The peptides were isolated and sent for MALDI-TOF-MS testing. Database searching was performed using peptide masses obtained from MALDI-TOF-MS.

RESULTSAverages of 481 +/- 38 and 477 +/- 21 protein spots were detected in control gels and preconditioning gels, respectively. 169 +/- 6 protein spots were matched between these two types of gels. Among the matched spots, while the quantities of 21 +/- 12 spots in control gels increased by above 2 times than that in preconditioning one, the quantities of 33 +/- 10 spots in preconditioning gels increased by the same times than that in control one. The correlation coefficient between these two patterns were 0.7748 +/- 0.0267. 12 spots in preconditioning gels significantly increased compared with the control (P < 0.05, n = 4). Among 12 spots excised from the gels, perfect peptide mass fingerprinting spectrums of 8 spots were acquired. The results showed that one protein was fructose biphosphate aldolase A. Three proteins matched nothing might be new proteins. The other four proteins just matched the partial sequences of the proteins of database were no coincidence to it's isoelectric point and molecular weight. So they might be homological proteins.

CONCLUSIONMany proteins, for example fructose biphosphate aldolase A, has been differentially expressed in hippocampus of mice during HHDP. This may be one of the molecule mechanisms of HHDP.

Adaptation, Physiological ; Animals ; Electrophoresis, Gel, Two-Dimensional ; Hippocampus ; metabolism ; Hypoxia ; metabolism ; Hypoxia, Brain ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Proteins ; isolation & purification ; Proteome ; analysis

OBJECTIVETo study the influence of HBV/P22 protein on the induced apoptosis of HepG2 cells.

METHODSIn vitro, two HepG2 strains were transfected with pcDNA3.1+ and pcDNA3.1+HBV/P22 respectively and the cells were exposed to Act D and TNF alpha for 6h and then the induced apoptosis was detected by flow cytometry (FCM) and TUNEL technique. Supernatant HBeAg was detected by Abbott reagent. The intracellular expression of HBV/P22 protein was measured by Western blot and immunochemistry. In vivo, three cell groups were inoculated into nude mice by subcutaneous injections. After two weeks, Act D and TNF alpha were injected into the tumors and then the induced apoptosis in the tissues was detected by TUNEL technique. The expression of HBV/P22 protein in the tumor tissues was detected by immunochemistry.

RESULTSIn vitro, in HepG2- pcDNA3.1+HBV/P22 cells, supernatant HBeAg was positive and intracellular HBV/P22 protein was positively expressed. The apoptosis proportion of HepG2-pCDNA3.1+HBV/P22 cells was markedly lower than HepG2 and HepG2-pcDNA3.1+ cells (P < 0.05). In vivo, HBV/P22 protein was expressed in the tumor tissues, and the apoptosis proportion in the group injected with HepG2-pcDNA3.1+HBV/P22 cells was markedly lower than those injected with HepG2 and HepG2-pcDNA3.1+cells (P < 0.05).

CONCLUSIONHBV/P22 protein could inhibit the induced apoptosis of HepG2 cells both in vitro and in vivo.

Animals ; Apoptosis ; Female ; Hep G2 Cells ; Hepatitis B Core Antigens ; genetics ; Hepatitis B e Antigens ; metabolism ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Transfection ; Viral Core Proteins ; genetics

OBJECTIVETo generate the skeletal muscle-specific transforming growth factor beta receptor II (TbetaR II) gene knockout mice for the research on the function of the TbetaR II gene in skeletal muscles.

METHODSTbetaR II (flox/flox) mice were generated using embryonic stem cell technology. The MCK-Cre mice were engineered containing Cre recombinase under the control of creatine kinase (MCK) muscle-specific promoter. TbetaR II (flox/flox) mice were crossed with MCK-Cre mice generating TbetaR II (flox/flox)/MCK-Cre double Tg mice. And then, TbetaR II (flox/wt) /MCK-Cre(+) double Tg mice were crossed with TbetaR II (flox/flox) mice to generate TbetaR II (flox/flox)/MCK- Cre(+) mice genetically ablating TbetaR II in cre-expressing skeletal muscle cells.

RESULTSAs predicted, mice lacking TbetaR II by gene targeting in skeletal muscles were generated first in the world using Cre/loxP system. TbetaR II null mutant mice were viable, fertile and showed apparently normal development.

Animals ; Mice ; Mice, Knockout ; Mice, Transgenic ; Muscle, Skeletal ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Receptors, Transforming Growth Factor beta ; genetics ; metabolism ; Recombination, Genetic ; Signal Transduction

OBJECTIVETo investigate the behaviors of the children suffered from the secondary deformity after the repair of the cleft lip.

METHODSWith the application of the PCPI, eighty patients with the secondary deformity after the repair of the cleft lip were selected in this study and 134 normal children was used for the control.

RESULTSIn the age between 6 and 11 years, there were no significant difference of the behaviors between the children suffered from secondary deformity of cleft lip and the normal children,but in the age from 12 to 16, the children with the deformity showed more behavior problems with the social withdraw and the poor social relationships, compared with the normal children.

CONCLUSIONThe children with the secondary deformity after cleft lip repair in adolescence could have the tendency to suffer from the behavior problems, especially showing the social withdraw and the poor social relationships.

Adolescent ; Adolescent Behavior ; psychology ; Behavior ; physiology ; Child ; Child Behavior ; psychology ; Child, Preschool ; Cleft Lip ; surgery ; Female ; Humans ; Lip ; abnormalities ; surgery ; Male ; Postoperative Complications ; psychology ; Social Behavior Disorders ; etiology ; Surveys and Questionnaires

OBJECTIVETo study the diagnostic value of the signal intensity on T1-weighted images of MRI and proton magnetic resonance spectroscopy (1H MRS) for neonatal hypoxic-ischemic encephalopathy (HIE).

METHODSThirty full-term neonates with HIE admitted into the Department of Neonatology of the First Affiliated Hospital of Medical College, Xi'an Jiaotong University between January, 2007 and December 2009 were enrolled. Ten normal neonates born at the same period served as control group. Cerebral MRI and 1H MRS examinations were performed within 15 days after birth.

RESULTSIn the HIE group, the signal intensity of the posterolateral lentiform nucleus was higher than or equal to that of the posterior limb of internal capsule, but in the control group, the results were opposite, namely, the signal intensity of the postero-lateral lentiform nucleus was lower than that of the posterior limb of internal capsule. The ratios of lactic acid/creatinine and glutamate/creatinine in the basal ganglia and the frontal lobe shown by 1H MRS increased significantly in the HIE group compared with controls (P<0.05 or 0.01). The differences of the signal intensity between the posterolateral lentiform nucleus and the posterior limb of internal capsule were positively correlated with the ratios of lactic acid/creatinine and glutamate/creatinine shown by 1H MRS (P<0.05).

CONCLUSIONSThe comparison of the signal intensity between the posterolateral lentiform nucleus and the posterior limb of internal capsule on T1-weighted images of the cerebral MRI is valuable for the diagnosis of neonatal HIE and the accuracy of diagnosis can be improved when combined with 1H MRS.

Female ; Humans ; Hypoxia-Ischemia, Brain ; diagnosis ; Infant, Newborn ; Magnetic Resonance Imaging ; methods ; Magnetic Resonance Spectroscopy ; methods ; Male

BACKGROUNDTumor necrosis factor a receptor 1 (TNFalphaR1) plays an important role in the signal pathway of apoptosis. The objective of this study was to investigate the effects of TNFalphaR1 knockout on the up-regulation of erythropoietin receptor (Epo-R) and the coordinated anti-apoptosis functions during myocardial ischemia-reperfusion injury in mice.

METHODSThe ischemia-reperfusion injury model for cardiomyocytes was performed by ligating the left circumflex branch artery of TNFalphaR1 knockout (P55(-/-)) C17 B6 mice, as well as wild-type (P55(+/+)) C17 B6 mice. Triphenyltetrazolium chloride (TTC) staining was performed to observe the damaged area of the heart. TUNEL staining and DNA fragmentation were used to identify apoptosis. Mitochondrial Bcl-2 and Bax as well as expression of Epo-R and its downstream genes (Jak-2, stat-5, Akt, IkB-alpha, HIF-1alpha) were measured by Western blotting. The gene knockout mice were assigned into those undergoing the apoptosis surgical model group (KO group), and those subjected to sham operation (KOs group). Similarly, wild-type mice were either exposed to the surgical model (WT group) or subject to a sham operation (WTs group).

RESULTSThe myocardial damage ratio of the wild-type group after the operation was significantly higher than that of the knockout group, (50.5 +/- 6.4)% vs (36.9 +/- 6.9)%, P < 0.01. Similarly, TUNEL positive ratio of the wild-type group was significantly higher than that of the knockout group, (63.1 +/- 5.6)% vs (42.1 +/- 4.7)%, P < 0.01. The gray value ratios of Epo-R, Jak-2, stat-5, Akt, IkB-alpha, HIF-1 and mitochondrial Bcl-2 in the KO group were significantly higher than those of the WT group, P < 0.05; however, mitochondrial Bax was significantly lower than that of the WT group significantly (P < 0.05).

CONCLUSIONSUsing the ischemia-reperfusion injury model in mice, cardiomyocytes of TNFalphaR1 knockouts exhibited anti-apoptotic characteristics. This information could be used to coordinate the prevention of myocardial apoptosis by up-regulating and activating the Epo-R pathway.

Animals ; Apoptosis ; Blotting, Western ; Disease Models, Animal ; I-kappa B Proteins ; metabolism ; In Situ Nick-End Labeling ; In Vitro Techniques ; Janus Kinase 2 ; metabolism ; Male ; Mice ; Mice, Knockout ; Myocardial Reperfusion Injury ; genetics ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; NF-KappaB Inhibitor alpha ; Oncogene Protein v-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Receptors, Erythropoietin ; metabolism ; Receptors, Tumor Necrosis Factor ; genetics ; metabolism ; STAT5 Transcription Factor ; metabolism ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.
Animals ; Chickens ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Newcastle disease virus ; immunology ; Plasmids ; Salmonella typhimurium ; genetics ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

OBJECTIVETo explore the effects of hypobaric hypoxia on the apoptosis of germ cells in male rats.

METHODSAdult male Wistar rats were randomly divided into four groups: a control group raised at sea level; a 5 d, a 15 d and a 30 d hypoxic group raised in a hypobaric chamber simulating 5000 m altitude for 5 days, 15 days and 30 days respectively. Flow cytometry and TUNEL were used to evaluate the apoptosis of germ cells in the testis. Bax and Bcl-2 in the testis were measured by Western blot.

RESULTSSeminiferous tubules with apoptotic germ cells were significantly more in the hypoxic groups than in the control (P < 0.01). Most apoptotic germ cells were spermatogonia and spermatocytes. Compared with the control group, apoptotic germ cells detected by PI flow cytometry were significantly increased in the hypoxic 15 d and 30 d groups (P < 0.05); Bax was significantly higher (P < 0.05), and so was the ratio of Bax to Bcl-2 in the hypoxic 30 d group (P < 0.01).

CONCLUSIONHypoxia promotes apoptosis of testicular germ cells in male rats. Chronic hypoxia increases Bax expression in the rat testis.

Animals ; Apoptosis ; Hypoxia ; metabolism ; pathology ; In Situ Nick-End Labeling ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Spermatozoa ; cytology ; Testis ; metabolism ; pathology ; bcl-2-Associated X Protein ; biosynthesis