目的:观察体外培养星形胶质细胞损伤后S100β的表达变化,进一步了解脑损伤修复的分子机制,以期为法医病理学鉴定提供更多的依据。方法:取出生后24h内SD大鼠大脑皮质进行星形胶质细胞体外分离培养和纯化。随机分为对照组和损伤后30min、1h、3h、6h、12h、24h、3d、7d组,应用ABC法检测不同时间段S100β表达的差异。结果:体外培养星形胶质细胞的纯度达95％以上。对照组可见少量S100β蛋白表达;损伤后30min,S100β开始增加,3~6h达高峰,随后下降,24h时低于对照组水平,此后略有上升,7d时恢复至对照组水平。结论: 机械性划痕损伤后S100β总体表达呈单峰,而在体动物实验结果表明大鼠大脑损伤后S100β总体表达成双峰趋势,但均具有时序性变化规律,说明损伤后S100β表达可为脑损伤时间的推断依据之一;体外培养细胞损伤模型对组织细胞损伤的分子机制研究具有优越性。
Objective To investigate the division, prol iferation and differentiation abil ities of nestin+/GFAP+cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs). Methods Twelvemale SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group inwhich the spinal cord injury model was establ ished by aneurysm cl ip compression method, and control group in which no processing was conducted. At 5 days after model ing, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cellsuspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was appl ied to induce differentiation. Immunohistochemistry detection and flow cytometry were appl ied to observe and analyze the type of cells and their capabil ity of division, prol iferation and differentiation. Results At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 ± 0.71 and 1.12 ± 0.38, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Concerning cell cycle, the proportion of S-phase cell and prol iferation index of the model group (15.49% ± 3.04%, 15.88% ± 2.56%) were obviously higher than those of the control group (5.84% ± 0.28%, 6.47% ± 0.61%), indicating there were significant differences between two groups (P lt; 0.01). In the model group, primary cells gradually formed threedimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multi ple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of β-tubul in III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ ol igodendrocyte, β-tubul in II+ neuron and GalC+ cell body and protruding were observed. Conclusion Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the abil ity of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
ObjectiveTo illustrate the role of epidermal growth factor (EGF) secreted by astrocytes in the process of tacrolimus (FK506) in promoting neurite outgrowth. MethodsThe spinal cord astrocytes and neuronal cells were isolated respectively from 2-day-old Sprague Dawley (SD) rats and 15-day SD pregnant rats, and cultured in vitro and identified by immunofluorescence staining. The spinal cord astrocytes were cultured with 20 μmol/L FK506 medium in the experimental group, and with FK506 free medium in the control group. The supernatant was collected after 24 hours for preparing conditioned medium, and astrocytes were collected. EGF proteins in the conditioned medium were detected with ELISA, and EGF gene expressions of astrocytes were detected with real-time quantitative PCR (RT-qPCR). The spinal cord neurons were cultured respectively with conditioned medium from the experimental group (FK506-CM) and the control group (C-CM) in group A and group B, also with neutralized C-CM and neutralized FK506-CM with anti-EGF neutralizing antibodies in group C and group D. Both the total neurite length and the longest neurite length were measured and compared among groups. ResultsBoth astrocytes and neurons were confirmed by immunofluorescence staining. The EGF content of experimental group (0.241±0.044) was significantly higher than that of the control group (0.166±0.014) (t=3.93, P=0.01); EGF gene expression of the experimental group (1.12±0.25) was significantly higher than that of the control group (0.46±0.11) (t=5.78, P=0.00). The neurite length measurement displayed that the total neurite length and the longest neurite length of groups C and D were significantly shorter than those of groups A and B (P<0.05). Both the total and longest neurite length of group A were significantly longer than those of group B (P<0.05), but no significant difference was shown between groups C and D (P>0.05). ConclusionThe EGF secreted by spinal cord astrocytes can promote the neurite outgrowth. So spinal cord astrocytes can be used as an important intermediary target of FK506 to promote the recovery of neurological function.
Developmental and epileptic encephalopathy (DEE) is a genetic neurological disease affecting 0.27–0.54 per 1000 newborns, with a strong genetic association. Currently, the majority of known pathogenic genes in genetic DEE can be classified into six functional categories: ion channels, organelles and cell membranes, growth and development, synaptic function, neurotransmitters and receptors, DNA and RNA regulation, and signal transduction pathways. Emerging evidence suggests that inflammatory regulation may play a critical role in genetic DEE pathogenesis. Specifically, astrocyte and microglial activation contributes to neuroinflammation in genetic DEE, while pro-inflammatory cytokines disrupt neuron-glia interactions, exacerbating epileptic seizures and neuronal damage. Targeting the source mechanism of neuroinflammation in genetic DEE, such as the activation of astrocytes and microglia, and intervening from the source, is expected to be a new target for the treatment of genetic DEE.
ObjectiveTo observe the effect of integrin β8 on the neuronal apoptosis after hypoxia ischemia (HI) in astrocyte/neuron co-culture system. MethodsAstrocytes and neurons were cultured in vitro from cerebral cortex of the P1-3 days Sprague Dawley rats and E16 days fetal rats, respectively. Immunocytochemistry staining was used to identify the purity of cells. Integrin β8 mRNA expression was qualified in the astrocytes at 12 hours, 1 day, and 2 days after HI and reoxygenation (experimental group) and in normal astrocytes (control group) by RT-PCR. Integrin β8 small interering RNA (siRNA) system was established to specifically block astrocyte β8 expression, the efficiency of integrin β8 inhibition was detected by real-time fluorescent PCR. The astrocytes and neurons were co-cultured to established the astrocyte/neuron co-culture system. The neuronal apoptosis was detected with TUNEL in the normal neurons/astrocytes group (co-cultured HI group), the astrocytes infected by integrin β8 siRNA for 2 days/normal neurons group (β8 RNA interference group), and normal neurons in vitro with HI treatment group (HI group) at 1 day after HI and reoxygenation. The normal neurons without treatment as control (control group). ResultsGlial fibrillary acidic protein and neuronal nuclei staining suggested a purity of more than 90% in cultured cells. HI resulted in an increase of integrin β8 mRNA expression at 12 hours after reoxygenation in astrocytes, which peaked at 1 day after reoxygenation, then slowly decreased and remained higher at 2 days, showing significant differences between control group and experimental group and among different time points in experimental group (P<0.05). RNA interference efficiency was most significant at 2 days after astrocytes infected with integrin β8 siRNA (P<0.05). The neuronal apoptosis was significantly increased in HI group, co-cultured HI group, and β8 RNA interference group when compared with control group (P<0.05). But neuronal apoptosis index (AI) was significantly decreased in co-cultured HI group and β8 RNA interference group when compared with HI group (P<0.05). The significant difference of AI was found between co-cultured HI group and β8 RNA interference group (P<0.05). ConclusionIntegrin β8 expression can be induced with hypoxic-ischemic brain damage, leading to decreased AI of neurons and obvious protective effect.
Neuropathic pain has been redefined by NeuPSIG as “pain arising as a direct consequence of a lesion or disease affecting the somatosensory syste”. However, pharmacological management for neuropathic pain is not effective, which is correlated with the uncertainty of pathogenesis. For a long time, neuron had been considered acting a major role in the development of neuropathic pain. In recent years, a majority of studies revealed that glia cell also involved in the occurrence and development of neuropathic pain, and neuron-glia interaction is one of the key mechanism of neuropathic pain, including complex signaling pathways as purinergic signaling. This review focuses on recent advances on the role of purinergic receptors in neuropathic pain.
OBJECTIVE: To study the nerve growth factor (NGF) expression and the influence of IL-1 alpha or IL-1 beta on NGF secretion in newborn rat astrocytes. METHODS: Astrocytes obtained from the brain cortex of newborn rats were cultured and purified, and they were divided into three groups, experimental, control and blank groups. IL-1 alpha or IL-1 beta were added into the experimental group with 25, 50 and 100 U/ml, each group was cultured for 24, 48 or 72 hours, and then the NGF contents in cultured astrocytes suspension media were measured by a two-cite enzymelinked immunoserbent assay (ELISA). RESULTS: Astrocytes could secret NGF by themselves and each concentration of IL-1 alpha or IL-1 beta media at any testing time could enhance NGF secreting in newborn rat astrocytes in certain degrees. The effects of IL-1 beta were ber than IL-1 alpha, the best effect in the unit time was observed in IL-1 beta with 50 U/ml for 24 hours. CONCLUSION: Astrocytes can express NGF, and IL-1 alpha or IL-1 beta can enhance the NGF expression in newborn rat astrocytes.