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Objective To research the functional and structural change in TgAP-PswePS1 transgenic mice after intensive light exposure insult.Methods APPswe/PS1 transgenic mice at the age of 6 months old were grouped for experiments,the transgenic mice were replaced by light insult device for 6 months,while the control mice were kept in normal conditions.After 6 months light exposure,the eyes of control and experimental mice were examined with electroretinography (ERG).The retinal morphology change was investigated with H&E staining.All of the results were quantified and statistically analyzed.Results In the control group,the amplitudes of a and b wave in the rod response were (18.33 ±3.53) μV and (107.58 ± 14.72) μV,while (64.80 ±7.57) μV and (178.76 ± 14.47) μV for the amplitudes of a and b wave in the maximum response;After treated by 6 months of intensive light exposure,in experimental group mice,the amplitudes of a and b wave in the rod response were (17.92 ±4.89) μV and (21.83 ± 5.51) μV;While in the maximum response a striking decrease was detected with a wave (18.23 ±4.44) μV and b wave (24.50 ± 4.49) μV,by compared with control group,the difference were statistical significant (all P ＜ 0.05).Histopathological analysis found significant loss of outer nuclear layer,photoreceptor out segment,whereas controls remained little change in the retina.And the retinal thickness decreased significantly from (181.32 ± 13.47) μm in control group to (102.34 ±9.38) μm after light insults in experimental group,the difference was statistical significant (P =0.017).Conclusion Intensive light exposure can cause the retinal structural and functional disorder in the AP-Pswe/PS1 transgenic mouse.
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Objective To research the choroidal neovascularization (CNV)in TgAPPswePS1 transgenic mice after intensive light exposure injury.Methods Twenty TgAPPswe/PS1 transgenic mice at the age of 6 months were grouped for experiments.The treated groups of 12 mice were treated by a source of 10 000 lux cool full spectrum light for 6 months,12 hours per day;While the control groups of 8 mice were kept in normal conditions.The mice eyes of the experimental group and control group were examined with HE/Toluidin blue staining,the retinal structure was observed,and the number of CNV was counted.The expression of VEGF and Aβ were examined with immunofluorescence on the retinal pigment epithelial (RPE) flat mount.All of the results were quantified and statistically analyzed.Results After treated by 6 months of intensive light exposure in the experimental group,histopathological analysis has found significant loss of outer nuclear layer/photoreceptor out segment and outer plexiform layer as compared with the control group;At the same time,abnormal hypo-and hyper-pigmentation,vacuoles and disruption in the RPE layer,remarkable CNV were found in the experiment group by Toluidin blue staining,and the incidence of CNV was 18.75％.The VEGF expression domenstrated.a diffusive and deposition pattern along the neovessels which showed a significant increase of (6.59 ± 1.14) fold changes as compared with the control group.The difference was statistical significant (P ＜ 0.05).Then the Aβ deposits were positive expressed in the RPE layers after intensive light exposure treatment,and pathological deposition of Aβ in the RPE showed plaque like displayed by confocal Z-stack microscopy,and the drusenoid Aβ deposits were found alone with the neovessels on the RPE flat mount.The deposition of Aβ protein increased with (6.45 ± 2.93) fold changes as compared with the control group,and the difference was statistical significant.Conclusion CNV with degenerative changes in the outer retina can be induced by intensive light exposure in the APPswe/PS1 transgenic mouse.These results suggest that an Alzheimer's transgenic animal model might be an alternative animal model for CNV if combined with intensive light exposure.
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BrdU (5-Bromo-2'-deoxyuridine) is usually used to label the mitotic cells as well as to trace reagent in cell transplation. However, BrdU could also exert some side effect on cellular biological characteristics upon inappropriate use. To explore the appropriate concentration of BrdU for labelling retinal progenitor cells (RPCs), we co-cultured Embryonic day (E) 17. 5 RPCs with different concentrations of BrdU, which were 0.2, 1, 5 and 10 micromol/L, respectively. After 48 hours, the RPCs were proliferation- or differentiation-cultured. Immunofluorescence was used to detect the BrdU-positive ratio and differentiation potential. Cell count was used to evaluate proliferation ability, and lactate dehydrogenase (LDH) release assay was used to monitor cytotoxicity. The results showed that 0.2 micromol/ L BrdU could not label RPCs clearly, while BrdU of 1, 5 or 10 micromol/L could label the RPCs with similar ratios. 1 micromol/L BrdU displayed no obvious cytotoxicity and showed no obvious effect on the proliferation and differentiation ability. However, 5 micromol/L or 10 micromol/L BrdU could evidently inhibit RPCs proliferation, partly due to the cytotoxicity effect. Furthermore, 10 micromol/L BrdU could inhibit the differentiation of RPCs towards MAP2-positive nerve cells, but showed no influence on the differentiation of RPCs towards GFAP- and glutamine synthetase positive glial cells. This study suggested that 1 micromol/L BrdU could be an appropriate concentration for RPCs labelling and could efficiently label RPCs without obvious side effect.
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Animals , Mice , Bromodeoxyuridine , Chemistry , Cell Culture Techniques , Methods , Cell Differentiation , Cell Proliferation , Embryo, Mammalian , Immunohistochemistry , Retina , Cell Biology , Staining and Labeling , Stem Cells , Cell BiologyABSTRACT
ObjectiveTo investigate the role of p38 and ERK1/2 during rhesus monkeys mesenchymal stem cells differentiated into neuron-like cells.MethodsTo induce the neuronal phenotype,rhesus monkeys mesenchymal stem cells were maintained in sub-confluent cultures in serum-contain medium supplement with Sonic hedgehog.Western blot analysis the change of p38 and ERK1/2 during rhesus monkeys mesenchymal stem cells differentiated into neuron-like cells.Under transmission and scanning electron microscope,ultra-structure of the differentiated cell were observed.ResultsDuring BMSCs differentiated into neuron-like cells by SHH,Mitogen-activated protein kinases (MAPK) involved in their signal transduction,p38 was activated and ERK1/2 was inhibited.P38 inhibitor SB203580 increased induced differentiation time compared with normal induced cells,and inhibited neurite outgrowth.ConclusionActivation of p38 and inhibition of ERK was impacted on differentiation into neuron-like cells from rhesus monkeys mesenchymal stem cells induced by Sonic hedgehog,which may has potential application on neuroprotection of stem cells in Nervous system diseases
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BACKGROUND: Present studies have demonstrated that during neural development, differentiation of neural stem cells (NSCs) was affected by various regulatory factors from surrounding microenvironment. Sonic hedgehog (Shh) is a key induction signal during neural fetal development, and can be an effective inductor to regulate differentiation of neural cells. OBJECTIVE: To investigate the signal transduction pathway of SHH for differentiation of rhesus monkey bone marrow mesenchymal stem cells (BMSCs) into of neuron-like cells by sonic hedgehog factor. METHODS: Rhesus monkey BMSCs were isolated and cultured by conventional density gradient centrifugation. BMSCs in the induction group were treated with L-DMEM containing FGF2, B27 and fetal bovine serum for preinduction of 24 hours, and then with DMEM supplemented with 0.5 μmol/L retinoic acid or 400 μmol/L SHH for 8 days. Non-induced cells served as control group. Following labeling with neuron enolase, positive cells were screened by flow cytometry. RT-PCR and Western-blot were used to detect SHH- and retinoic acid-induced cell membrane receptor and intracellular signal protein changes. RESULTS AND CONCLUSION: SHH specific membrane receptor Ptc, retinoic acid specific receptor RARα, signal protein molecule ptch1 and Smad expressed in normal cells. Ptc expression upregulated in SHH-induced cells. High expression lasted for a long time with induction time, which was significantly stronger compared with the retinoic acid and control groups (P < 0.01). Intracellular ptch1 protein molecule expression showed similar tendency as this, but could not induce upregulation of RARα expression. During induction, retinoic acid-stimulated cells did not activate Ptc pathway. Four days following induction, RARα expression upregulated and lasted till 6 days, but there were no significant differences. No significant change in ptch1 expression was determined. SHH- and retinoic acid-induced cell Smad molecule expression upregulated, but no significant difference was determined. Results verified that SHH-induced scheme participated in cell induction and differentiation by persistently activating its specific receptors. However, there was no significant receptor pathway crossing between retinoic acid-induced and SHH-induced schemes.
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BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)differentiating into neural cells is an effective way of cell therapy of nervous system disease.However,the methods used nowadays still need to be improved.OBJECTIVE:To induce the differentiation of rhesus monkey BMSCs into neuron-like cells by using sonic hedgehog factor.METHODS:Rhesus monkey BMSCs differentiating into neuron-like cells was induced by typical retinoic acid and sonic hedgehog factor.Rhesus monkey BMSCs were isolated and cultured by density gradient centrifugation method.Cell growth was observed under an inverted phase contrast microscope and cell growth curve was determined using MTT assay.Flow cytometry was performed to characterize the phenotype of BMSCs,and immunohistochemistry was utilized to assess differentiated cells.Ultra-structure of the differentiated cells was observed by transmission and scanning electron microscopes.RESULTS AND CONCLUSION:Rhesus monkey BMSCs cultured in vitro were identified by flow cytometry,with high homogenicity.Following sonic hedgehog factor disposal for 7 days,differentiated cells were mainly positive for neurone specific enolase,neurofilament protein,Tau and glial fibrillary acidic protein(GFAP).Image statistical analysis found that in sonic hedgehog factor scheme,neural stem cells marker Neetin positive rate was significantly higher compared with the rstinoic acid scheme(P＜0.01).GFAP-positive rate was greater in the retinoic acid scheme than in the sonic hedgehog factor scheme(P＜0.05).Results indicated that sonic hedgehog factor scheme is an effective pathway of rhesus monkey BMSC differentiation into neuron-like cells.
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S. The migration of CNE-2Z cells was inhibited by chloride channel blockers (ATP, NPPB and tamoxifen), but the inhibitory effect of the blockers varied with cells at different stages. CONCLUSIONS: The migratory ability is associated with the cell cycle in CNE-2Z cells. Chloride channels play an important role in cell migration of CNE-2Z cells.
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AIM: The roles of Cl-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 ?mol/L arrested cells in G 1 phase (G 1 population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G 1 phase. The results suggest that activation of Cl-channels and RVD is necessary for facilitating cells to proceed to the S phase from G 1 phase and maintaining cell proliferation.