三羟异黄酮对丙烯酰胺致大鼠小脑颗粒神经元凋亡的影响

目的探讨不同浓度三羟异黄酮(GEN)对丙烯酰胺(ACR)诱导大鼠小脑颗粒神经元(CGNs)凋亡的影响。方法取新生5-7 d SD大鼠小脑皮质细胞培养,采用神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经元,将培养8 d的神经元进行随机分组,正常对照组、ACR染毒组(浓度为10 mmol/L)、GEN预处理组Ⅰ、Ⅱ、Ⅲ、Ⅳ(浓度为10、25、50、100μmol/L的GEN预先处理细胞12 h,再予ACR作用24 h)。甲基噻唑基四唑(MTT)法检测神经元活性;相差显微镜及Hoechst33342染色分别观察细胞及其核形态学变化;原位细胞凋亡检测法(TUNEL)观察神经元凋亡细胞数。结果 10μmol/L组及25μmol/L组GEN可拮抗ACR所致的大鼠CGNs凋亡,提高神经元活性,降低TUNEL阳性细胞数,减少ACR所致的神经元胞体皱缩、细胞核固缩等特征;而50μmol/L组及100μmol/L组对ACR引起的神经元凋亡没有保护作用。结论一定浓度范围的三羟异黄酮可以保护丙烯酰胺所致的大鼠CGNs凋亡。

EFFECT OF GENISTEIN ON RAT CEREBELLAR GRANULE NEURON APOPTOSIS INDUCED BY ACRYLAMIDE

Objective To investigate the effects of genistein(GEN) on apoptosis of rat cerebellar granule neurons induced by acrylamide(ACR).Method Rat cerebellar granule neurons were prepared from the cerebellar cortex cells of 5-7 d-old Spragule-Dawley rat pups.The neurons were identified by neuron-specific enolase immunofluorescence technic.The 8 d cultured cells were divided randomly into control group,ACR model group,GEN pretreatment group Ⅰ,Ⅱ,Ⅲ,Ⅳ in which the cerebellar granule neurons were pretreated with 10,25,50,100 μmol/L GEN for 12 h;and the culture medium was cleared,then changed to fresh DMEM/F-12 solution with the above mentioned GEN and 10 mmol/L ACR added for 24 h.The neuronal viability was measured by metylthiazdyltetrazolium(MTT);and their nuclei were observed by phase-contrast and Hochest33342 staining separately.Neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling(TUNEL).Results GEN at the dose of 10,25 μmol/L significantly blocked apoptosis of primary cultured cerebellar granule neurons induced by ACR.The diminished neuronal body,chromatin concentration and the number of TUNEL positive cells induced by ACR were markedly weakened.The protective effect of GEN on apoptotic cells was not observed in 50,100 μmol/L groups.Conclusions Genistein at appropriate concentration is found to protect against apoptosis induced by ACR in primary cultured cerebellar granule neurons.