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Objective To study the expression of special AT-rich sequence-binding protein 1(SATB1) gene and its relationship with SLC22A18 protein expression in astrocytoma.Methods Fifty-six patients with astrocytomas (12 with grade Ⅰ,13 with grade Ⅱ,15 with grade Ⅲ,and 16 with grade Ⅳ),performed surgical excision in our hospitals from September 2006 to June 2010 and from September 2003 to June 2006,were chosen in our study; another 10 brain tissues from patients performed decompression operation resulting from cerebral hernia were selected as the controls.RT-PCR and Western blotting were used to detect the mRNA and protein expressions of SATB1.The SLC22A18 protein expression was detected by immunohistochemical assay.The relations between SLC22A18expressions and SA TB1 levels,and these two and the degree of malignancy were analyzed.Results RT-PCR and Western blotting revealed that positive mRNA and protein expressions were noted in 35patients with astrocytomas; the mRNA and protein expression rate and value of SATB1 in the astrocytoma tissues were significantly different among different grades of tumors (P＜0.05); the higher the malignancy grade,the higher mRNA and protein expression rate and value ofSA TB1; the protein expression value of SA TB1 had a positive correlation with the malignancy grade of tumors (r=0.987,P=0.000).And a few expressions of SA TB1 mRNA and protein were found in the tissues of controls.Immunohistochemical assay indicated that positive protein expression of SLC22A18 was noted in 19 astrocytoma tissues,and the protein expression rate of SLC22A18 in the astrocytoma tissues was significantly different among different grades of tumors (P＜0.05); the higher the malignancy grade,the lower expression of SLC22A18.And the protein expression of SLC22A18 was found in all the tissues of controls.The SATB1 expression rate in the tissues with negative SLC22A18 expression (81.1％) was significantly higher than that in the tissues with positive SLC22A18 expression (26.3％,P＜0.05).Conclusion SATB1 expresses in the astrocytoma tissues,indicating that it may play an important role in the pathogenesis of astrocytoma;up-regulation of SATB1 expression and dysfunction of SLC22A18 may play synergetic roles in the process of carcinogenesis of astrocytoma.
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Objective To investigate the relationship between aberrant methylation of SLC22A18 gene promoter and SLC22A18 expression in human glioma. Methods Thirty patients with glioma and 10 patients with craniocerebral injury performed decompression were chosen in our study;their tissue samples were prepared. Methylation-specific PCR (MSP) was used to detect the methylation status of SLC22A18 gene promoter;and Western blotting and RT-PCR were employed to measure the protein and mRAN expressions of SLC22A18 in these tissue samples. U251 cells were cultured in vitro with demethylating agent 5-aza-2-deoxycytidine (experimental group, 2μmol/L) and common medium (control group), resepectively;the re-expression of SLC22A18 in U251 cells was measured by Western blotting and cell growth suppression induced by 5-aza-2-deoxycytidine was also observed 3, 5 and 7 d after the culture. Results The methlylation of SLC22A18 gene promoter existed in glioma tissues of 15 patients (50%) but that did not exist in the tissues of patients with craniocerebral injury. The protein and mRAN expressions of SLC22A18 in the tissue samples of these 15 patients were significantly decreased as compared with those in patients with craniocerebral injury (P<0.05);cell counting of U251 cells in the experimental group on the 5th and 7th d of culture was significantly decreased as compared with that of those in the control group (P<0.05). On the 7ht d of culture, Western blotting indicated that the protein b expression level of SLC22A18 in the experimental group was obviously higher than that in the control group. Conclusion The aberrant methylation of SLC22A18 gene promoter plays a key role in down-regulating SLC22A18 expression, and demethylation agents can restore the SLC22A18 expression and suppress the growth of U251 cells.
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Objective To investigate the effect ofstromal cell derived factor-1 (SDF-1) on the regulation of neural stem cells (NSCs) migration.Methods NSCs were obtained from the cerebral cortex of embryonic rats and cultured in serum-free medium,and their stem cell properties were assessed by means of induced differentiation in vitro into neurons and astrocytes.After in vitro cell culture,the purity of NSCs and the co-expression rate of CXCR4/nestin were detected by flow cytometry.Blind-well chambers were employed to detect the chemotactic effects of SDF-1 by counting the cells which had crossed a 8 μm pore membrane when confronted with varying concentrations of SDF-1 (0,1,10,50,100,500 and 1000 ng/mL),and the distribution of cells migrated out of the same neurosphere was overviewed by μ-slides in the persistent concentration gradient of SDF-1.Results Neurospheres were formed by persistent proliferation of NSCs, which were capable of differentiating into neurons (β-tubulin+) and astrocytes (GFAP+) in media without mitogens,and flow cytometry analyses showed that most of the cultured cells expressed nestin and the co-expression rate of CXCR4/nestin was nearly 80%.SDF-1 showed great chemotaxis to NSCs,and the amount of cells having migrated through the membrane in 500 ng/ml SDF-1 group was higher than that in other groups (P<0.05).When the cells were confronted with a linear concentration gradient (from 500 to 0 ng/mL),which was generated by diffusion and stable for at least 48 h,the cells migrated out ofa neruosphere could distribute irregularly with more cells locating in the region of higher concentration of SDF-1 and longer migration distance away from the center of the neurosphere than the opposite.Conclusion SDF-1 binding to its specific receptor CXCR4 was capable of inducing NSCs migrating directionally to the source of SDF-1.
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Objective To explore the expression of human brain-derived neurotrophic factor (hBDNF) and green fluorescent protein (GFP) in hBDNF-GFP gene-transfected rat neural stem cells (NSCs) and the changes in the biological characteristics of the transfected cells. Methods NSCs were transfected with a lentiviral vector carrying hBDNF and GFP genes (hBDNF-GFP-NSCs) or GFP gene only (GFP-NSCs), with normal NSCs as the control. The expression levels of hBDNF mRNA and hBDNF protein in all the 3 groups were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to detect hBDNF level in the cell culture medium before and after hBDNF-GFP gene transfection. Dorsal root ganglion (DRG) neurons and NSCs were cultured with the supernatants of the transfected NSCs and normal NSCs, and the growth status of the DRG neurons was observed and the proportion of NSCs differentiating into neurons determined. Results Compared with GFP-NSCs and normal NSCs, hBDNF-GFP-NSCs showed obvious hBDNF overexpression at both rnRNA and protein levels 7 days after the transfection, hBDNF content in the supematant of hBDNF-GFP-NSCs culture increased significantly with time and peaked 5 days after the transfecfion (P<0.05). Four days after culture in hBDNF-GFP-NSCs supernatant, the DRG neurons and adherent NSCs extended cells processes, and the ratio of the NSCs differentiating into neurons was higher in cells cultured in hBDNF-GFP-NSCs supematant than in those culture in GFP-NSCs and normal NSCs supematants. Conclusion Lentivitus can be used as the vector for hBDNF and GFP gene transfection into NSCs, and hBDNF-GFP gene-transfected NSCs maintain the basic characteristics of NSCs and are capable of stable expression and secretion of hBDNF and GFP.
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Objective To investigate the effects of different medium and rat age on optic nerve tissue culture of rats.Methods Theoptic nerves from newborn rats(4dpostbirth)or adult rats(3-month old)were cultured on the rat-tailed collagen slide,pely-L-Lycine(PEL)slide,and Biocoat culture inserts,respectively.Their growth status was dynamically observed under a phase contrast microscope every day.The adherence rate of explant was recorded 48 h after culture.The maximum migration distance Was measured by an image analysis system on the 5th day after culture.The activity of lactic dehydrogenase (LDH)in the tissue culture medium Was measured dynamically.Morphological observance Was carried out by routine HE staining and the ultrastmcture of the tissue explants Were observed by a transmission electronmicroscope. Results The tissue adherence rate was higher in the Bioeoat insert group than in the rat-tailed collagen slide group or PLL slide group.The magnum migration distance of the tissue explants cultured in the Bioeoat insert group was longer than that in the rat-tailed collagen slide group or the PLL slidegroup.The maximum migration distance of the newborn rats Was longer than that of the adult rats under same culture condition(P<0.05).The LDH activity in the tissue culture medium began to descend 3 d after culture.The LDH activity in the adult rat group increased again on the 9th day since culturewhile it remained low level in the newborn rat group even on 12th day since culture.The cell processes showed up from the edge of explants and neuralgia cell migration was observed at the early stage,especially in newborn rats.The optic nerve structure gradually died out with the increase of culture time.The survival timeofopticnerve explant from newborn rats was longer than that of adult rats. Conclusion The optic nerve tissue can be cultured for a long time under suitable culture conditions.
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Recently,the progress in employing transplanting stem cells to cure injured retina is very fast and has been continuously yielding exciting results.Various sources are used in the studies,including retina-derived cells such as M?ller cells and ciliary body cells,and non-retina-derived cells such as embryonic stem cells and brain-derived stem cells.This review briefly discusses the recent progress of these studies.
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<p><b>OBJECTIVE</b>To investigate the effects of magnesium sulfate on traumatic brain edema and explore its possible mechanism.</p><p><b>METHODS</b>Forty-eight Sprague-Dawley (SD) rats were randomly divided into three groups: Control, Trauma and Treatment groups. In Treatment group, magnesium sulfate was intraperitoneally administered immediately after the induction of brain trauma. At 24 h after trauma, total tissue water content and Na(+), K(+), Ca(2+), Mg(2+) contents were measured. Permeability of blood-brain barrier (BBB) was assessed quantitatively by Evans Blue (EB) dye technique. The pathological changes were also studied.</p><p><b>RESULTS</b>Water, Na(+), Ca(2+) and EB contents in Treatment group were significantly lower than those in Trauma group (P<0.05). Results of light microscopy and electron microscopy confirmed that magnesium sulfate can attenuate traumatic brain injury and relieve BBB injury.</p><p><b>CONCLUSIONS</b>Treatment with MgSO4 in the early stage can attenuate traumatic brain edema and prevent BBB injury.</p>