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BACKGROUND:It is reported that tailless-like protein (TLX) plays critical roles in the regulation of early developmental processes in vertebrates, and it plays a key role in stem cells proliferation and differentiation into neurons. OBJECTIVE: To construct recombinant plasmid pEGFPN1-TLX and study the transfection into dermal multipotential stem cells. DESIGN, TIME AND SETTING: Cytogene experiment was performed at the Department of Pathogen Biology, School of Basic Medical Science, the Third Military Medical University of Chinese PLA from March to December 2007. MATERIALS: An adult SD was obtained from the Experimental Animal Center of the Third Military Medical University of Chinese PLA; dermal moltipotential stem cells (DMSCs) were cultured by the Institute of Combined Injury of the Third Military Medical University of Chinese PLA; pEGFPN1 and DH5α was gifted by professor Xu.METHODS: Total RNA was extracted from rat brain tissue to amplify TLX-coded cDNA sequence using RT-PCR. T/A was cloned on pMD18-T vector and determined using BamHI and Hindlll. The products were positive recombinant plasmid pMD18-T-TLX segments, which were sub-cloned in pEGFPN1 to construct recombinant plasmid pEGFPN1-TLX. Finally, pEGFPN1-TLX was transfected into DMSCs.MAIN OUTCOME MEASURES: The fluorescence protein expression was observed under fluorescence microscope at 24 hours after transfection; TLX mRNA expression was detected using RT-PCR; neuronal differentiation was observed using immunohistochemical staining.RESULTS: TLX full length cDNA was successfully cloned into pEGFPN1, and pEGFPN1-TLX was successfully constructed by means of sequence analysis and enzyme cutting identification. As compared with non-transfected DMSCs, pEGFPN1-TLX transfected DMSCs were observed after 10 days, formed resistant clones after 15 days, and shown a green fluorescent protein expression. However, non-transfected DMSCs died at day 10. RT-PCR indicated that pEGFPN1-TLX transfected DMSCs could express TLX mRNA. At day 3 after induction, NF200 positive cells were increased, but glial fibrillary acidic protein positive cells were decreased after induction of pEGFPN1-TLX transfected DMSCs.CONCLUSION: TLX was successfully constructed and transfected into DMSCs. After transfection, neuronal differentiation of DMSCs was enhanced, and the differentiation to gliocytes was inhibited.
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Objective: To identify the mutation points of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis(ALS) family with a unique phenotype,and to compare the value of single strand conformation polymorphism(SSCP) and denaturing high performance liquid chromatography(DHPLC). Methods: Five exons of SOD1 gene were amplified by PCR. The difference of these products were analyzed by PCR-SSCP and DHPLC.DNA sequencing was used to examine the mutation. Results: ①Mutations were found in exons 2 and 5 in several family members.DNA sequencing revealed that a base pair insertion occurred in the codon area of exon 2 and in the non-codon area of exon 5.②The results of DHPLC tests proved double peaks in one member with ALS symptoms(Ⅲ1),which indicated the possibility of mutation in SOD1 exon 4.DNA sequencing revealed that there was a heterozygote,with a mutation of GAA to GGA in exon 4 in the member with double peak. Conclusion: ①The mutations in exons 2,4,5 were proved.Insertion of exon 2 may be responsible for the disease of the ALS family in Chongqing.②Compared with PCR-SSCP,DHPLC technique has been proven to be a rapid and reliable method for screening mutation site in large samples.
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Objective To observe the effect of ecdysterone(EDS)on the level of VEGF protein in the brain,angiogenesis and neurologic function after focal cerebral ischemia in rats.Methods Rat with focal cerebral ischemia were established by occluding their middle cerebral artery.The established rats(n=36)were randomly and equally divided into EDS treatment group and ischemia group.EDS(20 mg?kg-1?d-1 for 7 d)was intraperitoneally injected into the rats of EDS treatment group 2 h after operation,and the animal of ischemia group received an intraperitoneal injection of the same solvent as in EDS group.Another 6 rats served as normal control.Rats were sacrificed in 7,14 and 21 d after operation,and the VEGF protein level and microvessel density(MVD)was detected with immunohistochemical methods and analyzed quantitatively with image system.Effect of EDS on neurologic recovery following brain ischemia were assessed using the neurologic severity scores(NSS).Results VEGF expression was not seen in normal control,and was higher in ischemia group than in the EDS treatment group at day 7 and 14,but the significant difference was only observed at day 7(P
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Objective To identify the point mutation of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis(ALS) family and observe the value of denaturing high performance liquid chromatography(DHPLC). Methods DHPLC and DNA sequencing were used to examine SOD1 gene of the ALS family which had not been found mutation by PCR-SSCP. Results DHPLC tests proved double peaks in one member(Ⅲ_1), Which indicated the possibility of mutation in SOD1 exon 4. DNA sequencing revealed that there was a heterozygote,with mutation of GAA to GGA in exon 4, and with a substitution of glutacid by glycine. Conclusion As compared with PCR-SSCP, DHPLC technique has proved to be a rapid and reliable method for screening mutation site in large samples.
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Objective To explore the relationship of the changes of memory behavior and the of changes of the parameter of synapse structure in the brain cortex and hippocampus of chronic cerebral hypoperfusion rats Methods Chronic cerebral hypoperfusion rat model was established by ligating bilateral common carotid arteries of the old rats (over 12 months) The rats were divided into 3 groups, normal, 2 months ischemia, and 4 months ischemia groups The memory behavior changes were observed with a computerized shuttle training case The ultrastucture of synapse were observed with electron microscopy for the number density, length of activity cord, area of synapses disk, surface density of the synapse, and the results were analyzed with stereology and image analyses The relationship between the behavior and the ultrastructure were studied Results Active avoidance response (AAR) and passive avoidance response (PAR) were decreased in the 2 months ischemia group and the 4 months ischemia group The number density (Nv) of the synapses in the brain cortex and hippocampus were reduced in the 2 months ischemia group and the 4 months ischemia group The length of activity cord (L), the area of synapses disk (S), the area density (Sv) in the hippocampus decreased in the 2 and 4 months ischemia group, and those in the brain cortex did not change in the 2 groups Conclusion The decrease of the number density (Nv) and the length of activity cord (L) of synapses in the chronic cerebral hypoperfusion is related to the memory behavior changes
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Objective To study the effects of estradiol benzoate on the morphology and number of neurons in cerebral frontal cortex and hippocampal CA1 area of 2VO OVX rats Methods Eighteen female Wistar rats were randomly divided into 3 groups, that is, sham operation, 2VO OVX (bilateral common carotid artery occlusion and ovariectomy)+ sesame oil, and 2VO OVX+estrogen replacement (named as S, O and E groups respectively) The changes of morphology and number of neurons in frontal cortex and hippocampal CA1 were studied with light and electron microscopy Results The numbers and shape of neurons in frontal cortex and CA1 were well maintained in E group, better than in O group but worse than in S group Significant differences were found in neuron number among 3 groups ( P
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Objective To study the pattern of activities of microglia, the patholgical changes of chronically hypoperfused brain of rats and the interrelationship between them. Methods Model of chronically hypoperfused brain was established in rat by the ligation of both common carotid arteries, and the histopathological changes of brain were observed with light microscope. The activity of microglia of the brain were also observed with immunohistochemistry method and the number of microglia was measured with image analyzer. Results Extensive activation of microglia was observed after chronic cerebral hypoperfusion and the activation was increased with the elapse of time of hypoperfusion. There were obvious pathological change in the brain after the chronic cerebral hypoperfusion, such as myelinic degeneration and formation of glial nodule in white matter. After treated with cyclosporin A, the number of microglia was obviously reduced, but the pathological change was evidently decreased. Conclusion The activation of microglia resulted from chronic cerebral hypoperfusion is relates to the pathological changes of the brain. However, cyclosporin A can decrease the pathological change and inhibit the activation of microglia.
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Objective To observe the ultrastructural changes of the infarcted region after transplantation of mesenchymal stem cells (MSCs) into the infarcted rats. Methods Model of middle cerebral artery occlusion (MCAo) in Wistar rats was established. The isolated and cultured MSCs in vitro were injected into the infarcted region. The ultrastructural changes of the infracted region were observed at 4 w after transplantation by electron microscopy. Results Some immature cells surrounding the neurons were found to survive in the infracted region. Massive free ribosomes were observed in the neural cytoplasm in the infarcted region in the rats receiving MSCs transplantation, but neurons with nuclear pyknosis and neurophagia were observed in the infarcted region of rats in the control group. Conclusion MSCs transplantation might improve neuron repair in cerebral ischemic injury in rats.
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Objective To observe the morphology of embryonic stem cell-derived neurons after transplanted into A?-injured rat hippocampus. Methods Neural precursor cells (NPCs) were generated from mouse embryonic stem cells (ESCs) expressing EGFP with modified serum-free methods and then transplanted into the hippocampus of A?1-40-injuried rats. The morphology, neurotransmitter phenotypes and receptors of EGFP-positive donor cells were observed with immunofluorescene methods. Results The engrafted NPCs survived and differentiated into glutamatergic and GABAergic neurons and expressed both glutamatergic and GABAergic neurotransmitter receptors. Synaptophysin-positive dots were found surrounding somata and dendrites of transplanted neurons, suggesting the presence of presynaptic terminals adjacent to their membranes. Conclusion NPCs derived from ESCs can differentiate into excitatory and inhibitory neurons after grafted into Alzheimer's disease model rats, and maybe form synapse with the host neurons.