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Objective To understand and compare the proportion of neural stem cells (NSCs) in the whole brain and cerebral cortex of mice at different embryonic days, and provide quantitative data for the later optimization of NSCs isolation and culture.Methods The whole brains (at embryonic 12.5, 14, 16 and 18 days) and cerebral cortex (at embryonic14, 16 and 18 days) were isolated and digested into single cell suspension, and were adherently cultured for 3-4 h.Immunofluorescence staining of Nestin, a NSCs specific marker, was used to statistically analyze the proportion of NSCs in each group.Expression of Nestin mRNA in the cerebral cortex of mice at E12.5, E14, E16, and E18 was detected by real-time fluorescence quantitative PCR.Results The result of immunofluorescence assay showed that there were Nestin-positive cells in the whole brain and cerebral cortex of mice at different embryonic days.In the whole brain,the proportion of NSCs was highest at E12.5 (53.42±1.57%) and lowest at E18(25.96±1.31%), and the proportions at E14 and E16 were placed in the middle among the groups.In the cerebral cortex, the highest proportion of NSCs was at E14 (33.65±0.29%), and the lowest at E18(25.29±0.28%), and the middle at E16 (26.82±0.30%).The result of real-time PCR showed that when the mRNA expression of Nestin in the cerebral cortex was set to 1, the relative mRNA expression of Nestin was 0.83±0.04 at E14, 0.77±0.05 at E16, and 0.44 ±0.05 at E18.Thus, the mRNA expression level of Nestin in the mouse cerebral cortex was gradually decreasing with the increase of embryonic days.Conclusions During the brain development, the proportion of NSCs is gradually decreasing in the whole brain and cerebral cortex of mice with the increase of embryonic days.
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BACKGROUND:At present, mouse embryonic neural stem cells (NSCs) culture has been skillfully operated by many labs, but there are differences existing about which part are dissociated to get NSCs. Embryonic 14 days (E14) mouse brain tissues are widely used for culturing NSCs, but there are less studies about the detailed percentage and difference of NSCs separated from different brain tissues. OBJECTIVE: To test the proportion and difference of NSCs and neurons percentage from E14 mouse whole brain, cortex and forebrain, providing quantized data for optimizing the isolation of high-purity NSCs. METHODS:E14 C57BL/6 mouse whole brain, cortex and forebrain tissues were separated and dissociated into single cells that were adherently cultured for 3.0-4.0 hours and labeled by DAPI. Then the cells were immunostained with NSCs specific marker, Nestin, and neuron specific marker, Tuj1, to identify NSCs and neurons percentage by calculating Nestin+/DAPI and Tuj1+/DAPI. In addition, real-time PCR assay was used to test Nestin and Tuj1 mRNA expression in the E14 mouse whole brain, cortex and forebrain. RESULTS AND CONCLUSION: (1) Immunocytochemical results showed that there were a large amount of Nestin+ and Tuj1+ cells in the whole brain, cortex and forebrain of E14 mice. NSCs percentage in the forebrain was obviously higher than that in the whole brain (P 0.05); the Tuj1 mRNA expression in the forebrain was significantly lower than that in the whole brain (P < 0.05) and in the cortex (P < 0.05). These findings indicated that the forebrain had the most NSCs and the least neurons compared with the whole brain and the cortex. In summary, E14 mouse forebrain has the highest percentage of NSCs compared with the whole brain and cortex, which is a better source to obtain NSCs for the following cell culture experiments.
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<p><b>OBJECTIVE</b>To construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product.</p><p><b>METHODS</b>The coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2. The positive recombinants were transformed into E.coli BL21 cells, and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein.</p><p><b>RESULTS</b>A gene fragment 300 bp in length was amplified. Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2. The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE. Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein.</p><p><b>CONCLUSION</b>The constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein, which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.</p>
Subject(s)
Animals , Humans , Mice , Epitopes , Allergy and Immunology , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Mice, Inbred C57BL , Peptides , Genetics , Metabolism , Recombinant Proteins , Genetics , Allergy and Immunology , Vaccines, DNA , Allergy and Immunology , tau Proteins , Genetics , Allergy and ImmunologyABSTRACT
Objective To detect the frequency of micronucleated polychromatic erythrocytes(fMNPCE) in mice bone marrow after irradiated with X-ray by flow cytometer(FCM) to study whether fMNPCE detecting method could become a rapid biodosimeter for early radiation damage. Methods BALB/c mice were randomly assigned to receive the X-ray irradiation at dose of 0.5,3.0,6.0 Gy(n=4 for each irradiation dose),and the bone marrow was collected in 6,12,24 h after irradiation.Another 4 mice were intraperitoneally injected with cyclophosphamide twice as positive control,of which the bone marrow was collected in 42 h after intoxication began and 4 treated with spurious radiation as normal control.Results fMNPCE in mice bone marrow increased remarkably in 24 h after exposure to 0.5 Gy X-ray(P
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Objective To study the effects of the cytotoxicity, cell cycle and time dependent apoptosis of Jurkat T lymphoma cells induced by Tripterygium Hypoglaucum (Levl) Hutch (THH) alkaloids so ad to explore the mechanisms of the apoptosis induced by the THH alkaloids. Methods Cell vitality and cell proliferation were measured by Typan blue staining. Cell cycle and the time dependent apoptosis were determined by DNA staining, TUNEL labeling and flow cytometry. Results THH alkaloids could effectively inhibit cell proliferation of Jurkat cells and induce the G 1 arrest and could induce apoptosis in G 2/S phase first and then G 1 phase. Conclusion THH alkaloids can inhibit DNA synthesis, cell proliferation and can also induce apoptosis of Jurkat T lymphoma cells in all phases.
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Objective To investigate the effects of Tripterygium Hypoglaucum (Levl) Hutch alkaloids on the nuclear DNA strand breaks and DNA fragmantation in Jurkat T lymphoma cells to understand the mechanisms of the apoptosis. Methods After Jurkat cells were induced by the alkaloids, DNA stand breaks were labeled by TUNEL assay and DNA fragmentation was analyzed by DNA content analysis. Flow cytometry was performed to determine these phenomena. Results THH alkaloids could effectively induce DNA stand breaks and DNA fragmentation. Conclusion There are great changes in nuclear DNA in the apoptosis of Jurkat T lymphoma cells induced by THH alkaloids.
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Objective To explore the roles of [Ca 2+ ]i in the apoptosis of Jurkat cells induced by Tripterygium Hypoglaucum (Levl) Hutch alkaloids. Methods After Jurkat cells were stained with Indo 1 AM and PI, changes of [Ca 2+ ]i were assessed by flow cytometry at the single cell level. Results THH alkaloids could induce no changes of [Ca 2+ ] inside cytoplasm. Conclusion THH alkaloids induce the apoptosis of Jurkat cells via other pathways rather than via the [Ca 2+ ] pathway.
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Objective To explore the acute toxicity and LD 50 of Tripterygium Hypoglaucum (Levl) Hutch(THH) solution to provide information for safe clinical application. Methods After oral administration of THH solution in mice, the mortality and the physiological and pathological changes were observed. Results The LD 50 (95% confidence limit) of THH in male and female mice was 79 g/kg(69～89 g/kg) and 100 g/kg (90～112 g/kg), respectively. No marked pathological change of the organs was found. Conclusion According to the standard of grading of acute toxicity, THH solution belongs to the moderate class. Therefore, it is safe in clinical practice and has a wide application.
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Objective To study the availability of escharectomy in shock phase and its role in preventing complications.Methods To make an analysis between the escharectomies and skin grafting of 79 cases in or beyond shock phase in the incidence of sepsis,visceral complications,MODS,mortality,healing time and the expenditure.Result The cases of the early operations revealed much better consequences than those performed beyond shock phase.Conclusion The escharectomy in shock phase proves to be available and significant in reducing post burn complications.