ABSTRACT
We emulated instances of open traumatic brain injuries (TBI) in a maritime disaster. New Zealand rabbit animal models were used to evaluate the pathophysiological changes in open TBI with and without the influence of artificial seawater. New Zealand rabbits were randomly divided into 3 groups. Control group consisted of only normal animals. Animals in TBI and TBI + Seawater groups underwent craniotomy with dura mater incised and brain tissue exposed to free-fall impact. Afterward, only TBI + Seawater group received on-site artificial seawater infusion. Brain water content (BWC) and permeability of blood-brain barrier (BBB) were assessed. Reactive oxygen species levels were measured. Western blotting and immunofluorescence were employed to detect: apoptosis-related factors Caspase-3, Bax and Bcl-2; angiogenesis-related factors CD31 and CD34; astrogliosis-related factor glial fibrillary acidic protein (GFAP); potential neuron injury indicator neuron-specific enolase (NSE). Hematoxylin & eosin, Masson-trichrome and Nissl stainings were performed for pathological observations. Comparing to Control group, TBI group manifested abnormal neuronal morphology; increased BWC; compromised BBB integrity; increased ROS, Bax, CD31, CD34, Caspase-3 and GFAP expressions; decreased Bcl-2 and NSE expression. Seawater immersion caused all changes, except BWC, to become more significant. Seawater immersion worsens the damage inflicted to brain tissue by open TBI. It aggravates hypoxia in brain tissue, upregulates ROS expression, increases neuron sensitivity to apoptosis-inducing factors, and promotes angiogenesis as well as astrogliosis.
Subject(s)
Brain Injuries, Traumatic/pathology , Seawater/adverse effects , Animals , Disease Models, Animal , Immersion , RabbitsABSTRACT
The purpose of the present study was to develop a novel transdermal drug-delivery system comprising a polyamidoamine dendrimer coupled with sonophoresis to enhance the permeation of diclofenac (DF) through the skin. The novel transdermal drug-delivery system was developed by using a statistical Plackett-Burman design. Hairless male Wistar rat skin was used for the DF-permeation study. Coupling media concentration, ultrasound-application time, duty cycle, distance from probe to skin, and a third-generation polyamidoamine-dendrimer concentration were selected as independent variables, while in vitro drug release was selected as a dependent variable. Independent variables were found to be statistically significant (P<0.05). DF gel without dendrimer and ultrasound treatment to skin (passive delivery, run 13) showed 56.69 µg/cm(2) cumulative drug permeated through the skin, while the DF-dendrimer gel without sonophoresis treatment (run 14) showed 257.3 µg/cm(2) cumulative drug permeated through the skin after 24 hours. However, when the same gel was applied to sonophoresis-treated skin, drastic permeation enhancement was observed. In the case of run 3, the cumulative drug that permeated through the skin was 935.21 µg/cm(2). It was concluded that dendrimer-coupled sonophoresis-mediated transdermal drug delivery system has the potential to enhance the permeation of DF through the skin.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Dendrimers , Diclofenac/administration & dosage , Drug Delivery Systems , Administration, Cutaneous , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Diclofenac/pharmacokinetics , Drug Liberation , Gels , Male , Permeability , Polyamines/chemistry , Rats , Rats, Wistar , Skin Absorption , Ultrasonography/methodsABSTRACT
OBJECTIVE: To investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR, respectively. The PCR products were inserted into plasmid pLXSN. The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE. The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method. RESULT: The interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively, and the production of A beta was reduced. CONCLUSION: BACE siRNA can inhibit the expression of BACE gene of mammalian cells.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , RNA Interference , RNA, Small Interfering/genetics , Amyloid Precursor Protein Secretases/genetics , Animals , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry , Mice , Microscopy, Fluorescence , NIH 3T3 Cells , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To test the effect of short interfering RNAs (siRNAs) of beta-site APP cleaving enzyme (BACE) on inhibiting the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR. The PCR products were inserted into the retrovirus plasmid pLXSN. The interfering vector was identified as pLXSN/ EGFP-U6-siBACE. The SK-N-SH cell line was produced, which can highly expressed BACE. The inhibitive effect of BACE siRNA on BACE expression was examined by fluoroscopy and immunohistochemistry tests. RESULTS: The interfering vector, pLXSN/EGFP-U6-siBACE, was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cell and reduced the production of Abeta. CONCLUSION: BACE siRNA inhibits the expression of BACE gene of mammalian, which has implications for RNA interference of Alzheimer's disease.
Subject(s)
Amyloid Precursor Protein Secretases/deficiency , Amyloid Precursor Protein Secretases/genetics , Genetic Engineering/methods , RNA, Small Interfering/genetics , Alzheimer Disease/genetics , Animals , Cell Line, Tumor , DNA, Recombinant/genetics , Gene Expression , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Humans , Mice , Molecular Sequence Data , RNA Interference , Retroviridae/genetics , Retroviridae/physiology , Viral LoadABSTRACT
OBJECTIVE: To investigate the tumor-suppression effect of PA combined with GM-CSF, TNF-alpha and IL-4 on cord blood mononuclear cells (CBMC). METHODS: The mononuclear cells were isolated from human umbilical cord blood and cultured with polyacttin A (PA), GM-CSF + TNF-alpha + IL-4 (GTI), and GTI + PA (GTIP) respectively. Six days later, surface antigen expression of the cultured cells, including CD1a and CD83, which were the specialized markers of dendritic cell (DC), were analyzed by immunohistochemistry technique. The CBMC were cultured with GTI for 24 h to enhance DC, then were added apoptotic/necrotic Hela/HepG2 tumor cells, and finally PA was co-cultured. The antitumor cytotoxicity of CBMC was measured by MTT assay. RESULTS: After the culture, CD1a and CD83 positive cell rates of the PA group inreased significantly, reaching (19.63 +/- 3.61)%, (9.28 +/- 4.31) % respectively, much higher than that of the control, but lower than that of the GTI group. The killing rate to the tumor cells of CBMC cultured with GTIP increased remarkably, much higher than the control, GTI and PA groups. After tumor antigens were added to the CBMC of GTIP group (GTIP + Tc), the killing rate increased. CONCLUSION: PA not only promotes the proliferation and maturation of cord blood derived DC, but also improves the tumor-suppression effect of CBMC cultured with GTI.
Subject(s)
Glycopeptides/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-4/pharmacology , Leukocytes, Mononuclear/immunology , Neoplasms/therapy , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, CD1/biosynthesis , Antigens, CD1/genetics , Cells, Cultured , Fetal Blood/cytology , HeLa Cells , Humans , Immunoglobulins/biosynthesis , Immunoglobulins/genetics , Immunotherapy , Leukocytes, Mononuclear/drug effects , Liver Neoplasms/pathology , Liver Neoplasms/therapy , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology , CD83 AntigenABSTRACT
AIM: To study the expression of fusion gene Abeta-HBcAg in E.coli and detect the immunogenicity of fusion protein Abeta-HBcAg. METHODS: The Abeta-HBcAg fusion gene was cut from recombinant plasmid pBV220/Abeta-HBcAg, and was inserted into the plasmid pYTA1 to construct recombinant plasmid pYTA/Abeta-HBcAg. The recombinant plasmid was transformed into E.coli DH5alpha, and expressed under IPTG induction. The expression of the fusion protein Abeta-HBcAg was detected by SDS-PAGE. The antigenicity of the fusion protein was detected by ELISA. BALB/c mice were immunized intraperitoneally with the fusion protein purified by salting out with saturate ammonium sulfate. The titers of anti- Abeta antibodies of the immunized mice was evaluated by ELISA. RESULTS: Fusion protein existed in supernatant of the bacteria lysate and its expression level was about 7% of the total bacteria protein. The fusion protein reacted with both Abeta and HBcAg. The highest titer of anti-Abeta antibodies could reach to 1:16 000 after immunization for 3 times. CONCLUSION: Recombinant gene Abeta-HBcAg can be expressed in E.coli DH5alpha. The expression protein exists in supernatant of the bacteria lysate and has good immunogenicity. This study lays the foundation for the experimental animal study of AD gene-engineering vaccine.
