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目的探讨神经酰胺对孕期酒精暴露所诱导的小鼠神经细胞凋亡的影响。方法建立神经鞘磷脂合成酶2基因敲除(SMS2-/-)小鼠和野生型(WT)小鼠的孕期酒精暴露模型,将出生后的不同基因型仔鼠(共360只)分为对照组和酒精组。用酶学法检测生后0 d(P0)仔鼠血清神经鞘磷脂(SM)含量,利用免疫荧光染色法观察对照组与模型组各年龄点仔鼠齿状回苔藓细胞凋亡数量的变化,免疫印迹法检测P7、P14仔鼠海马组织Caspase-8、Caspase-3激活蛋白的相对表达量。结果酒精暴露后仔鼠血清SM水平降低,且具有剂量依赖性(F=41.08,P<0.05);SMS2-/-仔鼠血清SM水平低于同组WT仔鼠(F=53.34,P<0.01)。酒精诱导WT和SMS2-/-仔鼠苔藓细胞凋亡(F=15.61,P<0.05),有剂量依赖性和长时程效应,与同年龄、相同处理条件的WT仔鼠相比,SMS2-/-仔鼠苔藓细胞凋亡数量较多(F=11.72,P<0.05)。Western blotting检测结果与免疫荧光结果一致。结论 SMS2基因缺失使血清SM水平降低,可引起神经酰胺在体内蓄积;神经酰胺促进酒精暴露诱导神经细胞凋亡的过程;孕期酒精暴露主要通过死亡受体途径诱导神经细胞凋亡的发生。
Objective To investigate the effect of ceramide on neuronal apoptosis induced by alcohol exposure during pregnancy. Methods The gestational alcohol exposure model of sphingomyelinase synthetase 2 gene knockout (SMS2 - / -) mice and wild type (WT) mice was established. The different genotypes of offspring born after birth were divided into control Group and alcohol group. Serum sphingomyelin (SM) levels were detected by enzyme-linked immunosorbent assay (ELISA) at 0 d postnatal day. The changes of apoptotic numbers in the germinalbladder moss cells of the control and model groups were observed by immunofluorescence staining. Western blotting was used to detect the relative expression of Caspase-8 and Caspase-3 activation proteins in hippocampus of P7 and P14 offspring. Results The level of serum SM was decreased in a dose-dependent manner (F = 41.08, P <0.05). The level of serum SM in SMS2 - / - offspring was lower than that in WT group (F = 53.34, P <0.01 ). Alcohol induced apoptosis of moss cells in WT and SMS2 - / - offspring mice (F = 15.61, P <0.05), and there was a dose-dependent and long-term effect. Compared with WT mice of the same age and same treatment conditions, / - pups moss cell apoptosis more (F = 11.72, P <0.05). Western blotting results are consistent with immunofluorescence results. Conclusions The deletion of SMS2 gene can decrease the level of serum SM and lead to the accumulation of ceramide in vivo. Ceramide can promote the apoptosis of nerve cells induced by alcohol exposure. The alcohol exposure during pregnancy induces the apoptosis of nerve cells mainly through the death receptor pathway.