Li Jinhui, Chen Dapeng, Tang Jun, Wu Jinlin, Qu Yi, Mu Dezhi
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2014 Mar;28(3):366-70.
To observe the effect of integrin beta8 on the neuronal apoptosis after hypoxia ischemia (HI) in astrocyte/neuron co-culture system.
Astrocytes 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 beta8 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 beta8 small interering RNA (siRNA) system was established to specifically block astrocyte beta8 expression, the efficiency of integrin beta8 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 beta8 siRNA for 2 days/normal neurons group (beta8 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).
Glial fibrillary acidic protein and neuronal nuclei staining suggested a purity of more than 90% in cultured cells. HI resulted in an increase of integrin beta8 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 beta8 siRNA (P < 0.05). The neuronal apoptosis was significantly increased in HI group, co-cultured HI group, and beta8 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 beta8 RNA interference group when compared with HI group (P < 0.05). The significant difference of AI was found between co-cultured HI group and beta8 RNA interference group (P < 0.05).
Integrin beta8 expression can be induced with hypoxic-ischemic brain damage, leading to decreased AI of neurons and obvious protective effect.
观察整合素β8在星形胶质细胞/神经元共培养体系中对缺氧缺血（HI）后神经元凋亡的影响。
分别从出生1 - 3天的Sprague Dawley大鼠和胚胎16天的胎鼠大脑皮质体外培养星形胶质细胞和神经元。采用免疫细胞化学染色鉴定细胞纯度。通过逆转录聚合酶链反应（RT-PCR）检测HI及复氧后12小时、1天和2天（实验组）星形胶质细胞以及正常星形胶质细胞（对照组）中整合素β8 mRNA的表达。建立整合素β8小干扰RNA（siRNA）系统特异性阻断星形胶质细胞β8表达，通过实时荧光定量PCR检测整合素β8抑制效率。将星形胶质细胞和神经元共培养建立星形胶质细胞/神经元共培养体系。在HI及复氧后1天，采用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测正常神经元/星形胶质细胞组（共培养HI组）、整合素β8 siRNA感染2天的星形胶质细胞/正常神经元组（β8 RNA干扰组）以及体外HI处理的正常神经元组（HI组）中的神经元凋亡情况。未处理的正常神经元作为对照（对照组）。
胶质纤维酸性蛋白和神经元核染色显示培养细胞纯度超过90%。HI导致复氧后12小时星形胶质细胞中整合素β8 mRNA表达增加，在复氧后1天达到峰值，随后缓慢下降，但在2天时仍高于对照组，对照组与实验组之间以及实验组不同时间点之间差异均有统计学意义（P < 0.05）。整合素β8 siRNA感染星形胶质细胞2天后RNA干扰效率最为显著（P < 0.05）。与对照组相比，HI组、共培养HI组和β8 RNA干扰组神经元凋亡均显著增加（P < 0.05）。但与HI组相比，共培养HI组和β8 RNA干扰组神经元凋亡指数（AI）显著降低（P < 0.05）。共培养HI组和β8 RNA干扰组之间AI差异有统计学意义（P < 0.05）。
整合素β8表达可被缺氧缺血性脑损伤诱导升高，导致神经元AI降低，具有明显的保护作用。
