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Objective: To discuss the protective effect of velvet antler polypeptides (VAP) combined with Schwann cells (SCs) modified by glial cell line derived neurotrophic factor (GDNF) gene on the apoptosis of spinal cord neurons induced by beta-amyloid 25-35 (Aβ25-35). Methods: The spinal cord cells of fetal mice were prepared and the spinal cord neurons in logarithmic growth phase were taken; the apoptosis of spinal cord neurons was induced by Aβ25-35. The spinal cord neurons were divided into normal cell group (the normal spinal cord neurons), induced apoptosis group (the apoptotic spinal cord neurons induced by Aβ25-35, SCs group (the apoptotic spinal cord neurons induced by Aβ25-35 + SCs), GDNF group (the apoptotic spinal cord neurons induced by Aβ25-35 + GDNF), SCs + GDNF group (the apoptotic spinal cord neurons induced by Aβ25-35 + GDNF-transfected SCs) and VAP combination group (the apoptotic spinal cord neurons induced by Aβ25-35 + VAP combined with GDNF-transfected SCs). Flow cytometry was used to detect the apoptotic rates of spinal cord neurons in various groups; immunohistochemical staining was used to detect the number of caspase-3 positive cells in the spinal cord neurons in various groups. Results; After suspension inoculation of fetal spinal cord neurons, most of them were round at the initial stage. The flow cytometry results showed that compared with induced apoptosis group, the apoptotic rates of spinal cord neurons in SCs, GDNF, SCs + GDNF, and VAP combination groups were decreased (P<0.05). Compared with SCs + GDNF group, the apoptotic rate of spinal cord neurons in VAP combination group was decreased (P<0. 05). The immunohistochemistry results showed that the expression of caspase-3 in spinal cord neurons in various groups could be found. There were no significant differences in the number of caspase-3 positive cells and cell staining between SCs group, GDNF group and SCs + GDNF group; but the number of caspase-3 positive cells in SCs group, GDNF group and SCs + GDNF group were significantly higher than that in induced apoptosis group (P<0. 05). Conclusion: VAP combined with GDNF-transfected SCs has the protective effect on the apoptosis of spinal cord cells by reducing the expression of caspase-3 in spinal cord neurons.
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@#Objective To study the differentiation and proliferation ability of the spinal neural stem cells (NSCs) at different gestational ages in fetal rats. Methods Sprague-Dawley fetal rats were divided into group A (12 days of pregnancy), group B (14 days of pregnancy) and group C (16 days of pregnancy). NSCs were separated with enzyme-assisted microdissection. The diameter and numbers of NSCs balls were measured at different time. The cell growth curve was drawn with CCK8 colorimeter. NSCs were identified with BrdU/Nestin immunohistochemical staining. They were induced with 10% fetal bovine serum for 10 days, and the expression of β-tubulinⅢ and glial fibrillary acidic protein was detected with immunocytochemistry. Results There were cells expressed BrdU, Nestin, β-tubulinⅢ and GFAP in all the group. The most cells (22.74±0.79%) expressed β-tubulinⅢ in the group B, but no significant difference between group B and group C. The cell vitality on the 5th day of third-generation neural stem cells was the most in group B. Conclusion For enzyme-assisted microdissection, it may obtain more neurons to isolate the neural stem cells from 14 days of pregnancy pregnant rats.
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Objective:To investigate spinal cord neurons fos,heat shock protein 70(HSP70)change and influence of Danshen Injection after the rats’limb ischemia reperfusion.Methods:Through temporarily interdict the rat’s one side iliac artery and femoral artery to establish the model of the limb ischemia reperfusion injury.Using the immunohistochemical method of ABC to observe the expressions about neurons fos and HSP70 positive neurons and the character about their distributions,as well as their change after being intervened by Danshen Injection n.Results:The rats’spinal cord neurons fos and HSP70 expressions increased in four hours later the limb ischemia(P
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Objective To establish a model of injury to primarily cultured spinal cord neurons,mimicking the neuronal injury after complete transactional spinal cord trauma,for the sake of exploring changes in expression of an immediately-early gene,c-jun,in central nervous system injury. Methods Spinal cords were removed form fetal Wistar rats at the 14th gestation day and the neurons were cultured for 10 to 12 days.Then,mechanical injury was applied to the neurons by making regular scores on the culture disk under direct vision with the aid of a self-made standard template.Morphology of the injured neurons and changes in expression of c-Jun protein were observed before and at different intervals after injury. Results c-Jun expression was noted in neuronal nuclei 10 min after injury and its peak appeared at 2 hrs.Besides,the density of positive neurons bore evidently an inverse proportion with their distance from the scores.Conclusion\ Positive expression in injury neurons show that c-jun gene enters the nucleoli of injured neurons and takes the role of “the third messenger” at early time after neuronal injury.
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Objective In this experiment,we explored the effects of rAAV\|hGDNF on protecting spinal neurons From death. Methods rAAV\|hGDNF particals were produced by recombinant virus technolgy,and infected the culture spinal neurons which were exposed to glutamate.We counted the mortality rate and detected the expression of NOS mRNA by RT\|PCR. Results In the group transfering rAAV\|hGDNF the death rate was inhibited(50%?0^02,control 59^25%?0^023, P
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In order to study the expression and the feasibility of scaled production of neuropeptide in the routine expression system such as E.coli with the pituitary adenylate cyclase activating polypep tide(PACAP) as an example, the following experiments were carried out. First, on the basis of the reported amino acid sequence of PACAP, DNA sequence of PACAP w as deduced and six partially complementary oligonucleotide fragments were design ed. The coding region of PACAP was obtained by renaturing the DNA fragments and ligation and identified by DNA sequencing. The coding region of PACAP was cloned into plasmid pGEX-4T-3 and transformed into E.coli BL21(DE3 ). An expression strain BLPACAP was selected. SDS-PAGE analysis revealed that t he GST-PACAP fusion protein was highly expressed and accumulated to about 30% o f the total bacterial proteins. By affinity chromatography, up to 90% GST-PACAP was purified by one step from bacterial lysate. The purified protein could prom ote neurite outgrowth of PC12 cells and the survival of spinal cord neurons.