小脑颗粒神经元细胞凋亡中caspase-8基因变化与二乙酰吗啡的影响

背景：半胱-天冬氨酸蛋白酶8(Caspase-8)是细胞凋亡过程起始的重要因子，二乙酰吗啡可致神经元细胞凋亡，其与二乙酰吗啡致小脑颗粒神经元细胞凋亡之间关系尚未见报道。 目的：验证二乙酰吗啡诱导小脑颗粒神经元细胞凋亡与caspase-8基因表达情况，明确caspase-8参与二乙酰吗啡致神经元凋亡过程。 方法：取出生7 d SD大鼠小脑颗粒神经元细胞进行体外培养，第7天后，以不同质量浓度的二乙酰吗啡(10，40，80，100，120 mg/L)和C-jun氨基末端激酶通路特异性抑制剂SP600125作用小脑颗粒神经元细胞24 h，并分对照组(0 mg/L二乙酰吗啡)，80 mg/L二乙酰吗啡组，二乙酰吗啡+SP 600125组；采用Hoechst 33258 荧光染色观察细胞形态学改变，流式细胞仪测细胞凋亡率，免疫荧光、RT-PCR及Western Blotting检测caspase-8 mRNA和蛋白表达情况。 结果与结论：①施加不同质量浓度二乙酰吗啡致神经元细胞凋亡，凋亡细胞出现透亮深蓝色的凋亡小体，细胞核呈现固缩、凝聚及断裂，随给药浓度增加细胞凋亡率呈现升高趋势(P < 0.05)。②与对照组相比，在80 mg/L二乙酰吗啡干预时caspase-8 mRNA和蛋白表达明显明显表达(P < 0.05)；caspase-8 mRNA随给药浓度增加呈现升高趋势(P < 0.05)，二乙酰吗啡+SP600125组中caspase-8 mRNA和蛋白较80 mg/L二乙酰吗啡组明显下调(P < 0.05)。结果提示caspase-8参与二乙酰吗啡致小脑颗粒神经元细胞凋亡过程，同时也是C-jun氨基末端激酶信号通路中的重要候选促凋亡因子。 中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：

Effects of diacetylmorphine on caspase-8 gene in apoptosis of cerebellar granule neurons

BACKGROUND: Caspase-8 plays an important role in starting the process of cell apoptosis, and diacetylmorphine can induce neuronal apoptosis. The relationship between the apoptosis of cerebellar granule neurons cells induced by diacetylmorphine and caspase-8 has not been reported. OBJECTIVE:To investigate the effect of caspase-8 on diacetylmorphine-induced neuronal apoptosis. METHODS: Cerebellar granule neurons from Sprague-Dawley rats aged 7 days were cultured in vitro. At 7 days, the cells were cultured with different dosage of diacetylmorphine (10, 40, 80, 100, 120 mg/L) and SP600125 for 24 hours, and were divided into control group, 80 mg/L diacetylmorphine group, diacetylmorphine+SP600125 group. Cell morphology was observed by Hoechst 33258 fluorescent staining, and cell apoptosis rate was measured by flow cytometry. Immunofluorescence staining, RT-PCR and western blot assay were used to detect caspase-8 mRNA and protein expression. RESULTS AND CONCLUSION:(1) After different dosage of diacetylmorphine was used to induce neuronal apoptosis, dark blue translucent apoptotic bodies were found in apoptotic cells, appearing with nucleus condensation, cohesion and fracture, and the apoptosis rate presented an increasing trend with increasing of drug dosage (P < 0.05). (2) Compared with the control group, caspase-8 mRNA and protein expression was remarkable under the intervention of 80 mg/L diacetylmorphine (P < 0.05); caspase-8 mRNA expression exhibited an increasing trend with increasing dosage of diacetylmorphine (P < 0.05), while caspase-8 mRNA and protein expression in the diacetylmorphine+SP600125 group was significantly lower than that in the 80 mg/L diacetylmorphine group (P < 0.05). These findings indicate that caspase-8 is involved in the process of diacetylmorphine-induced neuronal apoptosis, and meanwhile, it is also an important candidate of pro-apoptotic factors in the c-jun N-terminal kinase signaling pathway.