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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 mR NA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 mR NA和蛋白表达明显明显表达(P<0.05);caspase-8 m RNA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 m RNA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。
BACKGROUND: Caspase-8 is an important factor in the initiation of apoptosis. Diacetyl morphine can induce neuronal apoptosis, which is associated with diacetyl morphine-induced apoptosis of cerebellar granule neurons The relationship between death has not been reported. OBJECTIVE: To verify the apoptosis of cerebellar granule neurons induced by diacetylmorphine and the expression of caspase-8 gene in rat cerebellar granulosa cells, and to clarify the involvement of caspase-8 in neuronal apoptosis induced by diacetylmorphine. Methods: After cultured for 7 days, the cerebellar granule neurons of SD rats were cultured and cultured in vitro. After seven days, the morphological changes of diacetylmorphine (10, 40, 80, 100 and 120 mg / L) and C-terminal amino- Inhibitory effect of SP600125 on cerebellar granule neurons was observed in 24 hours in control group (0 mg / L diacetylmorphine), 80 mg / L diacetylmorphine group and diacetyl morphine + SP 600125 group. Hoechst 33258 staining Morphological changes, flow cytometry apoptosis rate, immunofluorescence, RT-PCR and Western Blotting detection of caspase-8 mR NA and protein expression. RESULTS AND CONCLUSION: 1 Apoptosis of neurons induced by different concentrations of diacetyl morphine was observed. Apoptotic bodies of translucent dark blue appeared apoptotic bodies. The nuclei showed shrinkage, cohesion and fragmentation. With the increase of concentration, the apoptosis rate Showing an upward trend (P <0.05). Compared with the control group, the caspase-8 mRNA and protein expression of caspase-8 mRNA were significantly increased (P <0.05) in the treatment of 80 mg / L diacetylmorphine; The expression of caspase-8 mRNA increased with the increase of <0.05). Caspase-8 mRNA and protein in diacetylmorphine + SP600125 group were significantly lower than those in 80 mg / L diacetylmorphine group (P <0.05). The results suggest that caspase-8 is involved in the apoptosis of cerebellar granule neurons induced by diacetylmorphine and is also an important candidate pro-apoptotic factor in the C-jun amino-terminal kinase signaling pathway.