M2e gene of three copies for H5N1 subtype AIV was synthesized and fused with human mycobacterium tuberculosis hsp70 gene.The fused gene was cloned into the prokaryotic expression vector to get pET-3M2e and pET-3M2e-hsp70.Recombinant protein r3M2e and r3M2e-hsp70 were successfully expressed induced with IPTG and purified with Ni2+-NTA collumn.Following that,the immunity of the recombinant protein was analysized with Western blot.20-day-old AIV non-immunized chickens were vaccined with r3M2e and r3M2e hsp70,at the same time,Trx and KLH-M2e inoculated chickens were served as vector and positive controls.Two weeks after the primary vaccination,every group was boosted with the same vaccine as in the primary vaccination.The humoral immunity of the vaccined chickens was evaluated with antibody detection against M2e,cytopathic suppression test,and indirect fluorescence assay.The cellular immunity was estimated according to lymphocyte subtype analysis with flow cytometry and M2e specific cytokine detection.Four weeks after the boost vaccine,all groups were challenged with 100EID50 AIV of H9N2 subtype,and the virus from swabs was detected with Real-time PCR.Results indicated that r3M2e hsp70 vaccined chicken developed the better humoral and cellular immune response,also,made a better performance compared with r3M2e vaccined group in virus challenge.

To investigate the spatiotemporal properties of epileptiform activity in vitro, 400 microm-thick transverse hippocampal slices were prepared from juvenile rat and planar multi-electrode array (MEA) containing 60 electrodes was used to record the electrical activity induced by bath application of high potassium artificial cerebrospinal fluid (ACSF) on slices. Following successful induction of epileptiform bursts, phenobarbital sodium was applied to test for its inhibitory effects on bursting activity in different regions of slice. Region-specific characteristics of epileptiform activity and anticonvulsant actions of phenobarbital sodium in the hippocampal network were determined by comparing the population activity obtained from MEA. The results showed that: (1) 15 min after high-K+ ACSF application, rhythmic and synchronous epileptiform bursts could be detected from all CA sub-regions. Quantitative analysis indicates that the firing patterns of different CA sub-regions were not statistically different (P>0.05). However, no bursting activity was recorded from granular cells in dentate gyrus, only sparse spikes were observed, with frequency significantly lower than that in CA regions (P<0.05). (2) The high-K+-induced bursting activity could last for more than 40 min with stable bursting activities. (3) Bath application of 60 micromol/L phenobarbital sodium inhibited the bursting activities on hippocampal slice. Bursting activities in CA3c and CA1 were firstly suppressed. 10 min after the phenobarbital sodium application, strong bursting activities persisted only in some of pyramidal cells in CA3a and CA3b. These results show that MEA could be applied for studying the spatial and temporal properties of epileptiform activity in vitro, as well as the region-specific effects of anti-epileptic drugs.
Action Potentials ; physiology ; Animals ; Electrodes ; Electroencephalography ; Electrophysiological Phenomena ; physiology ; Epilepsy ; physiopathology ; Hippocampus ; physiopathology ; In Vitro Techniques ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Processing, Computer-Assisted

OBJECTIVETo study the feasibility of vector-mediated RNA interference for HER-2-positive breast cancer therapy.

METHODSA plasmid vector capable of mediating HER-2 RNA interference was constructed, and HER-2-positive breast cancer cell line SKBR-3 was transfected with this constructed vector. The expression of HER-2 mRNA and protein was analyzed by RT-PCR and Western blotting, and the growth and apoptosis of SKBR-3 cells was analyzed after transfection.

RESULTSThe expressions of HER-2 mRNA and HER-2 protein was downregulated in response to vector-mediated HER-2 RNA interference, which also resulted in tumor cell growth inhibition and increased number apoptotic cells.

CONCLUSIONHER-2 is a good target for RNA interference and RNA interference targeting HER-2 can lead to HER-2 breast cancer cell apoptosis and growth inhibition.

Apoptosis ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, ErbB-2 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo observe the effect of ultrasound microbubble carrying herpes simplex virus thymidine kinase hepatocellular carcinoma in mice.

METHODSKunming mice were inoculated subcutaneously with H22 tumor cells. 40 male mice bearing subcutaneous hepatoma were randomized into 4 groups: PBS (group A), HSV1-TK (group B), HSV1-TK (group C), and microbubble carrying HSV1-TK (group D) were injected into the tail vein every 3 days. Mice in group C and D were exposed to ultrasound. The expression of TK protein was detected by western blot. Ganciclovir (GCV) was intraperitoneally injected at a dose of 100 mg x kg (-1) x d(-1) in group B, group C and group D. The tumor size was measured every 2 days.

RESULTSTK gene could be injected precisely into hepatocellular carcinoma with ultrasound monitor, and the expression of TK protein was found in all 4 groups. Expression in group D was higher than others (P < 0.05). The rate of tumor growth inhibition were 0 in group A, 3.90%+/-1.80% in group B, 22.70%+/-2.86% in group C, 41.25%+/-3.20% in group D (group B vs group C, P < 0.05; group D vs group C, P < 0.05; group D vs group B, P < 0.05).

CONCLUSIONUltrasound microbubble not only improve target gene therapy, but also enhance transfection efficiency.

Animals ; Carcinoma, Hepatocellular ; therapy ; Cell Line, Tumor ; Genes, Transgenic, Suicide ; Genetic Therapy ; Liver Neoplasms ; therapy ; Male ; Mice ; Mice, Inbred Strains ; Microbubbles ; Simplexvirus ; genetics ; metabolism ; Thymidine Kinase ; genetics ; Treatment Outcome ; Ultrasonics

BACKGROUNDPrevention of osteonecrosis (ON) has seldom been addressed. The purpose of this study was to evaluate the effect of resveratrol on preventing steroid-induced ON in rabbits.

METHODSSeventy-two rabbits were divided into four groups: (1) NEC (ON) group: thirty rabbits were treated with lipopolysaccharide (LPS) once, then with methylprednisolone (MPS) daily for 3 days; (2) PRE (prevention) group: thirty rabbits were given one dose of LPS, then MPS daily for 3 days, and resveratrol on day 0 and daily for 2 weeks; (3) RES (resveratrol) group: six rabbits were given resveratrol for 2 weeks but without LPS/MPS; (4) CON (control) group: six rabbits were given alcohol for 2 weeks but without LPS/MPS. Levels of plasma tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), thrombomodulin (TM), vascular endothelial growth factor (VEGF), maximum enhancement (ME) by magnetic resonance imaging, and ON incidence were evaluated.

RESULTSThe PRE group had a lower ON incidence than the NEC group, but with no significant differences at 2 weeks and 12 weeks. The RES and CON groups did not develop ON. TM and VEGF were significantly higher in the NEC group compared with the PRE group at weeks 1, 2, and 4 (TM: 1 week, P = 0.029; 2 weeks, P = 0.005; and 4 weeks, P = 0.047; VEGF: 1 week, P = 0.039; 2 weeks, P = 0.021; 4 weeks, P = 0.014), but the difference disappeared at 12 weeks. The levels of t-PA and PAI-1 were not significantly different between the NEC and PRE groups. The TM, t-PA, PAI-1, and VEGF concentrations in the RES and CON groups did not change over time. Compared to the baseline, ME in the NEC group decreased significantly (P = 0.025) at week 1, increased significantly (P = 0.021) at week 2, and was decreased at week 12. The variance was insignificant in the PRE group.

CONCLUSIONSResveratrol may improve blood supply to bone in a rabbit model of ON of the femoral head via anti-inflammatory effects to protect the vascular endothelium and reduce thrombosis.

Animals ; Disease Models, Animal ; Femur Head Necrosis ; chemically induced ; prevention & control ; Lipopolysaccharides ; toxicity ; Magnetic Resonance Imaging ; Methylprednisolone ; toxicity ; Plasminogen Activator Inhibitor 1 ; blood ; Rabbits ; Stilbenes ; pharmacology ; therapeutic use ; Thrombomodulin ; blood ; Tissue Plasminogen Activator ; blood ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC).

METHODSSGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software.

RESULTSSGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration.

CONCLUSIONSOur results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.

Adenoviridae ; genetics ; Animals ; Apoptosis ; drug effects ; Cisplatin ; pharmacology ; Genes, bcl-2 ; Humans ; Spiral Ganglion ; cytology

OBJECTIVETo study the effect of GMCSF-absence on the rate of wound healing and neovascularization during wound repair.

METHODS30 wild type (WT) mice and 30 GMCSF- absence mice (GMCSF-KO) were obtained. They were received full thickness skin wound (0.8 cm x 0.8 cm) in each side of midline after deeply anesthesia. In the different post-injury time points, the wound sites were digitally photographed to calculate the percentage of wound closure by using computer image analyses software. The wound specimens were also obtained dynamically for immunohistological analysis of CD31 at wound site.

RESULTSThe analysis of the wound closure showed that wound healing in GMCSF-KO mice was delayed significantly comparing with that in WT mice from the day 3 post-wounding. At days 7 and 10 after wounding significantly more numbers of blood vessels were formed in the WT controls compared to the GMCSF-KO mice.

CONCLUSIONSGMCSF-absence inhibits neovascularization during wound repair and leads to the delay of wound healing.

Animals ; Disease Models, Animal ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; deficiency ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Monocytes ; Neovascularization, Physiologic ; Skin ; blood supply ; injuries ; Wound Healing

OBJECTIVETo study the effects of puerarin on pulmonary Vascular remodeling in rats with pulmonary hypertension induced by chronic hypoxia and hypercapnia.

METHODForty male rats (180-220) g of grade two were randomly divided into five groups: normal control group (NC), hypoxia-hypercapnia 1, 2, 3 week groups (LH1, LH2, LH3) and hypoxia-hypercapnia 3-week + puerarin group (LHP3 group, puerarin intraperitoneal injection, 20 mg x kg(-1) x d(-1)). Collagen I, III and their mRNA were observed in pulmonary arterioles by the technique of immunohistochemistry and in situ hybridization.

RESULTLight microscopy showed media thickness of pulmonary arterioles was much higher in LH3 group than that of NC group, and, vessel cavity turned more straiter in LH3 group than that of NC group. Howerer, the damage of pulmonary arterioles in LHP3 group was much slighter than that of LH3 group. The levels of plasma ET-1 and lung homogenates Hyr and MDA were much higher in rats of LH3 group than those of NC group (P < 0.01), and lower in LHP3 group than LH3 groups (P < 0.01). The activities of SOD in lung homogenates were significantly lowered in hypoxic and hypercapnic groups compared with control group (P < 0.01), but higher in LHP3 group than that of LH3 group. Plasma NO content of group LH was lower than that of group NC (P < 0.01), it was highter in group LHP3 than that of group LH3 (P < 0.01). Expression of collagen I and collagen I mRNA in pulmonary arterioles were significantly higher in rats of LH groups than those of NC group (P < 0.01), and they were lower in rats of LHP3 group than those of LH3 group (P < 0. 01). Expression of collagen III and collagen III mRNA were not significant difference among three groups.

CONCLUSIONPuerarin could improve pulmonary vascular remodeling in rats with pulmonary hypertension by inhibiting the deposition of collagen.

Animals ; Hypercapnia ; complications ; Hypertension, Pulmonary ; drug therapy ; Hypoxia ; complications ; Isoflavones ; pharmacology ; Male ; Oxidative Stress ; drug effects ; Pulmonary Artery ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo culture olfactory ensheathing cells (OECs) of rats in vitro and to investigate its morphology, mitosis and immunocytochemistry, and to explore if the OECs could be a new donation for transplantation.

METHODSOECs were harvested from olfactory mucosa of Sprague Dawley rats based on the differing rates of attachment of the various cell types, followed by glial fibrillary acidic protein (GFAP), nerve growth factor (NGF), anti-low affinity receptor for NGF (NGFRp75), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and S-100 immunocytochemistry. The morphological changes and mitosis were observed under a phase contrast microscope at different culture time.

RESULTSThree morphologically distinct types of cells, bipolar, multipolar and flat morphology were present in the primary culture of adult rat olfactory mucosa. Mitosis was characterized by a retraction of all processes, forming a sphere that divided into spherical daughter cells, the daughter cells sent out their processes. The OECs were immunoreactive for GFAP, NGFRp75, S-100, NGF, BDNF and NT-3.

CONCLUSIONSThe OECs from nasal olfactory mucosa cultivated in the medium with fetal bovine serum could survive, divide, differentiate, and express the neurotrophin. It may become an accessible source for autologous grafting in spinal cord injury.

Animals ; Cells, Cultured ; Disease Models, Animal ; Immunohistochemistry ; Male ; Mitosis ; Olfactory Mucosa ; cytology ; transplantation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo construct eukaryotic expression vectors for HA-tagged receptor for advanced glycation end products (RAGE) mutants.

METHODSSite-directed mutagenesis was applied to wild-type RAGE gene cloned in the pcDNA3 vector with HA tag to obtain the mutants pcDNA3-HA-RAGE(S391A), pcDNA3-HA-RAGE(S399A), pcDNA3-HA-RAGE(S400A), and pcDNA3-HA-RAGE(T401A). After identification by sequencing, the mutants were transfected into HEK293 cells, and the expression of these mutants were detected by Western blotting using anti-HA antibody.

RESULTSThe HA-tagged RAGE mutants constructed were verified successfully by sequencing, and highly expressed in HEK293 cells.

CONCLUSIONThe success in constructing HA-tagged RAGE mutants, which are highly expressed in eukaryotic cells, may facilitate the functional study of RAGE in cell signal transduction.

Cell Line ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; biosynthesis ; genetics