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目的:观察银杏内酯(ginkgolides,Gin)、刺五加皂甙(acanthopannxsenticosussaponins,ASS)对脊髓运动神经元缺血缺氧有无保护作用,并探讨其可能的机制。方法:无菌下取出孕15dSD大鼠的胚鼠,分离胚鼠脊髓运动神经元进行原代分离培养,免疫组化鉴定后建立缺血缺氧诱导的神经元凋亡模型。取运动神经元培养在24孔板中,分为对照组(无缺氧损伤)、缺氧诱导组、Gin保护组、ASS保护组、神经胶质细胞源性神经营养因子(glialcellderivedneurotrophicfactor,GDNF)保护组,每组10孔。其中保护组分别于缺氧前24h每孔加入Gin37.5μg/ml,ASS50μg/ml,GDNF0.1μg/ml(每孔800μl),缺氧后继续培养3d。倒置显微镜及电镜下观察细胞形态学变化,MTT法测定神经元细胞活性,检测细胞外液LDH释放量,提取细胞匀浆液,Western印渍法检测运动神经元HIF-1α的表达。结果:神经元的细胞活性Gin组(0.25±0.059)、ASS组(0.21±0.028)和GDNF组(0.20±0.026)均比缺氧诱导组(0.15±0.012)高(P<0.01),且Gin组效果优于ASS组(P<0.05),GDNF作用与ASS相仿(P>0.05);LDH释放量Gin组(22.8±1.645)、ASS组(28.6±1.309)和GDNF组(26.5±0.885)均比缺氧诱导组(40.7±1.885)低(P<0.01);神经元HIF-1α的相对表达量缺氧诱导组(0.72±0.027)比对照组(0.16±0.003)高(P<0.01),Gin组(1.28±0.019)、ASS组(1.15±0.016)和GDNF组(1.12±0.016)均高于缺氧诱导组及对照组(P<0.01)。结论:Gin、ASS和GDNF均可提高体外缺血缺氧损伤的脊髓运动神经元的细胞活性,对细胞的缺血缺氧损伤有明显的保护作用,Gin的保护作用强于ASS,ASS的保护作用与GDNF相仿;其保护作用的发挥,可能与增强细胞膜的稳定性及提高细胞HIF-1α的表达有关。
OBJECTIVE: To observe whether ginkgolides (Gin) and acanthopannx senticosusaponins (ASS) have no protective effects on ischemic and hypoxic neurons in spinal motor and discuss its possible mechanism. METHODS: Embryonic rats were harvested from gestational 15d SD rats and the spinal cord motoneurons were isolated and cultured. The model of neuronal apoptosis induced by ischemia and hypoxia was established after immunohistochemical identification. The motoneurons were cultured in 24-well plates and divided into control group (no hypoxic injury), hypoxia induction group, Gin protection group, ASS protection group, and glial cell derived neurotrophic factor (GDNF) protection. Groups, 10 holes per group. In the protective group, Gin 37.5 μg/ml, ASS 50 μg/ml, and GDNF 0.1 μg/ml (800 μl per well) were added to each well 24 h before hypoxia, and the culture was continued for 3 days after hypoxia. The morphological changes of the cells were observed under inverted microscope and electron microscope. The activity of neuronal cells was measured by MTT assay. The release of extracellular fluid LDH was measured. The cell homogenate was extracted and the expression of HIF-1α in motor neurons was detected by Western blotting. RESULTS: The cellular activities of neurons in the Gin group (0.25±0.059), the ASS group (0.21±0.028), and the GDNF group (0.20±0.026) were higher than those in the hypoxia-induced group (0.15±0.012) (P<0.01), and Gin The effect of group was better than that of ASS group (P<0.05). The effect of GDNF was similar to that of ASS (P>0.05). The release of LDH in Gin group (22.8±1.645), ASS group (28.6±1.309) and GDNF group (26.5±0.885) were both significant. It was lower than that of hypoxia-induced group (40.7±1.885) (P<0.01). The relative expression of neuronal HIF-1α was significantly higher in the hypoxia-induced group (0.72±0.027) than in the control group (0.16±0.003) (P<0.01). The Gin group (1.28±0.019), ASS group (1.15±0.016) and GDNF group (1.12±0.016) were higher than the hypoxia induction group and the control group (P<0.01). Conclusion: Gin, ASS and GDNF can increase the cell activity of spinal motoneurons injured by ischemia and hypoxia in vitro, and have obvious protective effects on ischemic and hypoxic injury of cells. The protective effect of Gin is stronger than that of ASS and ASS. The effect is similar to that of GDNF; its protective effect may be related to the enhancement of cell membrane stability and increase of HIF-1α expression.