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目的探讨星形胶质细胞与神经元共培养系统中,缺氧缺血后星形胶质细胞表达的整合素β8对神经元凋亡的影响。方法取新生1~3 d SD大鼠及孕16 d SD大鼠的胎鼠脑皮质,分别分离培养星形胶质细胞及神经元,免疫细胞化学染色鉴定细胞纯度。取缺氧缺血培养复氧后12 h、1 d、2 d星形胶质细胞(实验组)以及正常星形胶质细胞(对照组),RT-PCR检测细胞整合素β8表达变化。利用整合素β8小干扰RNA(small interfering RNA,siRNA)感染抑制星形胶质细胞整合素β8表达,实时荧光定量PCR检测其抑制效率。取正常神经元及星形胶质细胞共培养(共培养缺氧缺血组)、经整合素β8 siRNA感染后2 d的星形胶质细胞与正常神经元共培养(β8 RNA干扰组)以及正常神经元(单纯缺氧缺血组),行体外模拟缺氧缺血处理;复氧后1 d收集细胞,TUNEL染色法检测整合素β8对神经元凋亡的影响;以正常神经元作为对照组。结果免疫细胞化学染色鉴定示,星形胶质细胞胶质纤维酸性蛋白及神经元神经核阳性率均达90%以上。与对照组相比,实验组星形胶质细胞中整合素β8 mRNA表达在复氧后12 h即增加,1 d时达峰值,2 d时仍维持较高水平;各时间点实验组与对照组比较及实验组复氧后各时间点间比较,差异均有统计学意义(P<0.05);整合素β8 siRNA感染后,实验组整合素β8 mRNA表达明显降低,感染后2 d降至最低值(P<0.05)。TUNEL检测示,与对照组相比,单纯缺氧缺血组、共培养缺氧缺血组及β8 RNA干扰组神经元凋亡明显增多(P<0.05);共培养缺氧缺血组及β8 RNA干扰组细胞凋亡指数较单纯缺氧缺血组明显降低,共培养缺氧缺血组低于β8 RNA干扰组,差异均有统计学意义(P<0.05)。结论缺氧缺血后,星形胶质细胞中整合素β8表达增高,神经元凋亡率下降,对受损神经元具有明显保护作用。
Objective To investigate the effect of integrin β8 expressed by astrocytes on the apoptosis of neurons after astrocytes co-cultured with astrocytes and neurons. Methods The astrocytes and neurons were isolated and cultured from 1 to 3 d neonatal SD rats and 16 d SD pregnant rats. Immunocytochemical staining was used to identify the cell purity. Hypoxia and ischemia reoxygenation 12 h after reperfusion, 1 d, 2 d astrocytes (experimental group) and normal astrocytes (control group), RT-PCR detection of cell integrin β8 expression. Infection with small interfering RNA (siRNA) of integrinβ8 inhibited the expression of integrinβ8 in astrocytes, and the inhibition efficiency was detected by real-time fluorescence quantitative PCR. Co-culture of normal neurons and astrocytes (co-cultured with hypoxic-ischemic group), astrocytes co-cultured with normal neurons (β8 RNA interference group) 2 days after infection with integrin β8 siRNA and The normal neurons (hypoxia-ischemia group) were randomly divided into hypoxia-ischemia group and hypoxia-ischemia group. The cells were harvested at 1 day after reoxygenation and the neuronal apoptosis was detected by TUNEL staining. The normal neurons were used as control group. Results Immunocytochemical staining showed that the positive rates of astroglial glial fibrillary acidic protein and neuronal neurons reached more than 90%. Compared with the control group, the expression of integrinβ8 mRNA in astrocytes in experimental group increased 12 h after reoxygenation and peaked at 1 d, and remained high at 2 d; at each time point, (P <0.05). After integrin β8 siRNA infection, the expression of integrin β8 mRNA in the experimental group was significantly decreased, and the lowest in the experimental group 2 d after the reoxygenation Value (P <0.05). TUNEL assay showed that compared with the control group, the apoptosis of neurons in hypoxic-ischemic and co-cultured hypoxic-ischemic and β8-RNAi groups was significantly increased compared with the control group (P <0.05). The co-cultured hypoxic-ischemic group and β8 The apoptosis index of RNA interference group was significantly lower than that of hypoxia - ischemia group, and the difference was statistically significant (P <0.05). Conclusion After hypoxia and ischemia, the expression of integrin β8 is increased in astrocytes, and the apoptosis rate of neurons is decreased, which has a significant protective effect on damaged neurons.