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Objective To investigate the expressions of IgG,Gab2 and PTEN in human gliomas and analyze their relationship with patient prognosis. Methods Immunohistochemistry was performed to detect the expressions of IgG,Gab2 and PTEN in 55 cases of gliomas and 20 cases of normal brain tissues. The mRNA expression of IgG in glioma tissues was detected by in situ hybridization. Results IgG protein and IgG mRNA were expressed in glioma tissues. IgG,Gab2,PTEN expression rate in normal brain tissues was significantly different to that in glioma-tissues(5.0%vs. 69.0%,5.0%vs. 52.7%,and 85.0%vs. 25.5%,P<0.05). Expression of IgG was positively related with expression of Gab2 in glioma(r = 0.3124,P < 0.05),IgG and PTEN expression were negatively related with each other(r=-0.422,P<0.05). Conclusions IgG and Gab2 are highly expressed in glioma. The expression of PTEN was downregulated in glioma. IgG,Gab2 and PTEN might be involved in the development of glioma.
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Objective To explore the role of homocysteine(Hcy)on angiogenesis at peri infarct region after focal cere-bral ischemia in rats, to elucidate inhibitory factors of angiogenesis, and to establish a clinic foundation for clinical brain functional recovery. Methods Spragur-Dawley (SD) male rats (n=36) were randomly divided into three groups with 12 rats in each group including Sham Operation (SO) group, Middle cerebral artery occlusion (MCAO) group and MCAO+Hcy group. The rats in Sham and MCAO groups were intra-peritoneally injected with 5 mL/(kg·d) saline and rats in MCAO+Hcy group were injected with 2%5 mL/(kg·d) Hey solution from the same route. MCAO was introduced by intraluminal filament meth-od after 7 d Hcy intervention. Rat brains were harvested on the 7th day after MCAO. BrdU(50 mg/kg, as a marker of cell pro-liferation)was intraperitoneally injected three days before the rats were killed. High performance liquid chromatography (HPLC)was used to measure serum Hcy concentration in rats. Brain infarction size was observed by TTC staining. Immuno-fluorescence staining was used to detect the number of BrdU+/laminin+cells at the thalamus of infarction side. Results Se-rum Hcy concentration significantly higher in MCAO+Hcy group than in SO and MCAO groups(P<0.05). Brain damage increased and the number of BrdU+/laminin+cells decreased in MCAO+Hcy group compared with those of MCAO group (P<0.05). Conclusion Increased Hcy concentration in rats lead to more severe damage of cerebral infarction as well as to inhibit the angiogenesis at surrounding ischemia area.
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Objective To explore the effects of homocysteine (Hcy) on neural stem cell (NSC) proliferation and the mRNA expression level of Notch1 and Hes1 related Notch signaling pathway from neonatal rats in vitro. Methods NSCs from neonatal rats were cultured by serum-free culture method in vitro. Cells were divided into four groups: control group (Hcy-C), low dose Hcy (Hcy-L) group, middle dose Hcy (Hcy-M) group and high dose Hcy (Hcy-H) group. NSCs were iden- tified by immunofluorescent staining using the antibodies against Nestin, β-tubulin Ⅲ and GFAP. The proliferation ability of NSCs was detected by MTT. The mRNA expressions of Notch1 and Hes1 were detected by Real-time PCR. Results In the serum free suspension medium, neurospheres that consisted of a great number of nestin-positive cells were found. β-tu- bulin Ⅲ positive neurons and GFAP positive astrocytes were detected by immunofluorescence staining on the 6 th day of cell induction. MTT assay showed that the cell viability was significantly lower in three Hcy treatment groups than those of con- trol group (P ＜ 0.05). And the effect of concentration-dependent was observed. The results of RT-PCR showed that mRNA expression of Hes1 was significantly lower in three Hcy treatment groups than that in control group (P ＜ 0.05). The mRNA ex- pression of Notch1 was significantly lower in Hcy-H group than that of other three groups (P ＜ 0.05). The mRNA expression of Notch1 was significantly lower in Hcy-M group than that of Hcy-L group and control group (P ＜ 0.05). Conclusion Hcy could inhibit the proliferation of NSCs by down-regulating mRNA expression levels of Notch1 and Hes1 genes related to Notch signal pathway.
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BACKGROUND: Previous studies have verified that under normal culture of neural stem cells (NSCs), folic acid can accelerate proliferation of NSCs by phosphorylation of mitogen activated protein kinase path activation ERK1/2. OBJECTIVE: To investigate the effect of folic acid on NSC extracellular signal regulatory protein kinase pERK1/2 under hypoxic condition. METHODS: NSCs from Neonatal rats were cultured in vitro by serum-free culture method, and incubated in a flask at 1×10~8/L. Except normal control group, self-made hypoxia equipment was used in the hypoxia model, folic acid deficiency and folic acid supplemented groups at day 3. At 37 ℃, hypoxia culture was conducted in the thermostat for 6 hours. The contents of folic acid were 4 mg/L, 4 mg/L, 0.65 mg/L, 8 mg/L in the four groups. Cells following 6 days were collected to count the density using trypan blue. RT-PCR was utilized to detect pERK1/2 mRNA expression. Western blot assay was employed to determine pERK1/2 protein expression. RESULTS AND CONCLUSION: Compared with the normal control group, the proliferation of NSCs and the expression of ERK1/2mRNA and pERK1/2 protein were decreased significantly in the hypoxia model group. Compared with the hypoxia model group, the proliferation of NSCs and the expression of ERK1/2mRNA and pERK1/2 protein were increased in the folic acid supplemented group, whereas decreased in the folic acid deficiency group. There were significant differences among groups (P < 0.001). Above-described results verified that folic acid supplementation can activate ERK1/2 phosphorylatin and accelerate proliferation of NSCs under hypoxia condition.
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Objective To study the effects of folate(Fol) on proliferation,apoptosis and the antioxidative capacity of the NSCs under hypoxia in vitro.Method The NSCs taken from infant rats brain were cultured by serum-free medium in vitro and divided into four group which were normal control group(NC),hypoxia group(Hyp),Hpy+Fol-D(folate deficient)group and Hpy+Fol group.Except NC group,the other groups were cultured in hypoxia condition for 6 h.The proliferation of NSCs after hypoxia damage was detected by MTT test.The NSCs were collected after being cultured 6 d and then the activity of SOD and GSH-PX was examined.The expression of activated caspase-3 was detected by Western blot.Results Compared with NC group,the proliferation of NSCs and the activity of SOD and GSH-PX decreased significantly after hypoxia,while the expression of activated caspase-3 increased0 The proliferation of NSCs and the activity of SOD and GSH-PX in Hyp group were significantly higher than Hyp+Fol-D group but much lower than Hpy+Fol group(P
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Objective To investigate the effects of folic acid on apoptosis of neural cells after focal cerebral ischemia injury in rats and the mechanisms.Method Thirty two adult male Sprague-Dawley(SD) rats were randomly divided into sham operation group(SO),middle cerebral artery occlusion group(MCAO),MCAO+ low dose folic acid group(MCAO+FA-L) and MCAO+ high dose folic acid group(MCAO+FA-H).The model of middle cerebral artery occlusion(MCAO) was set up by using intraluminal filament method.The rats were sacrificed at D7 day after cerebral ischemia.The apoptotic rate of neural cells was examined by TUNEL test,and the expression of pERK1/2 protein was detected by Western blotting method,The MDA content and serum SOD and GSH-Px activities in rats were measured before and 28d after folic acid treatment and 7th day after ischemia.Results Compared with ischemia group,the apoptotic rate of neural cells and MDA content in both folic acid supplemented groups were decreased significantly(P
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Objective To explore the effect of folic acid on neural stem cells(NSCs) proliferation from fetal rats in vitro.Method NSCs were isolated and cultured by microdissection,mechanical blowing and serum-free suspension culture,and identified by immunofluorescent staining using antibody against nestin.BrdU(5’bromo-2’deoxyuridine) was used to mark dividing neural stem cells.Cultured NSCs were divided into four groups:control group,low,high dose group(liquid media with added 4,40 mg/L folic acid),and deficiency group(liquid media with added 0.4 mg/L methotrexate,MTX).Monotetrazolium(MTT) and double-label immunofluorescence technique detected NSCs proliferation under the condition of folic acid.Results In the serum-free suspension medium,neurospheres that consisted of a great number of nestin-positive cells could be obtained.The proliferative ability of NSCs were observed by BrdU labeling methods.MTT assay and double-label immunofluorescence for nestin+BrdU showed that the growth tendency was increased with folate concentration in the medium.Compared with control group,NSCs growth rate of folate group was significantly increased in vitro.Conclusion The culture of NSCs isolated from fetal rats possesses the abilities of proliferation and self-regeneration.Folic acid may stimulate proliferation of NSCs efficiently.