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目的:研究哈巴苷对缺氧缺糖(OGD)诱导下海马神经细胞内游离Ca2+浓度的影响。方法:原代培养新生24 h内SD乳鼠海马神经细胞,采用神经元特异性烯醇化酶(NSE)免疫组织化学染色鉴定并检测其纯度;哈巴苷(0.5、1、5、10、25、50、100、150、200、250、500、1 000μmol/L)预处理48 h后,OGD 4 h,另设正常对照组及模型组,采用MTT法检测各组海马神经细胞活力,筛选哈巴苷有效浓度范围;将SD乳鼠海马神经细胞随机分为正常对照组、模型组、哈巴苷各剂量(50、75、100、125μmol/L)组,采用Fluo-3/AM负载的海马神经细胞,应用荧光倒置显微镜及荧光分光光度计观测哈巴苷各剂量对OGD海马神经细胞内游离Ca2+浓度的影响。结果:原代SD乳鼠海马神经细胞培养至第7天,经神经元特异性烯醇化酶(NSE)免疫组织化学染色鉴定为海马神经细胞,并检测其神经细胞纯度为85%~90%;MTT结果显示:与模型组比较,哈巴苷10、25、50、100μmol/L能显著增强海马神经细胞的活力(P<0.05或P<0.01),确定哈巴苷预处理48 h的有效浓度范围为10~100μmol/L;荧光倒置显微镜下观察,正常对照组海马神经细胞Ca2+荧光强度弱,模型组海马神经细胞Ca2+荧光强度强;与模型组比较,哈巴苷各剂量预处理48 h,均能降低OGD诱导的海马神经细胞Ca2+的荧光强度,其中75μmol/L组荧光强度降低最为明显。荧光分光光度法测定显示:与正常对照组比较,模型组海马神经细胞内游离Ca2+浓度显著升高(P<0.01);与模型组比较,哈巴苷75μmol/L能显著降低OGD诱导下海马神经细胞内游离Ca2+的浓度(P<0.05)。结论:哈巴苷预保护48 h能降低OGD诱导的海马神经细胞内游离Ca2+的浓度,以75μmol/L剂量效果最好,其保护作用与降低细胞内游离Ca2+浓度有关。
Objective: To study the effects of hababoside on intracellular free Ca2 + concentration in hippocampal neurons induced by oxygen-glucose deprivation (OGD). Methods: Primary cultured hippocampal neurons of SD neonatal rats within 24 h were identified by neuron-specific enolase (NSE) immunohistochemical staining and their purity was detected. Habaside (0.5, 1, 5, 10, 25, 50, 100, 150, 200, 250, 500 and 1 000 μmol / L) for 48 h, OGD 4 h, another set of normal control group and model group, MTT assay of hippocampal neuronal cell viability, The hippocampal neurons of SD suckling mice were randomly divided into normal control group, model group and Habasidone (50, 75, 100, 125μmol / L) groups. The hippocampal neurons loaded with Fluo-3 / Fluorescence microscopy and fluorescence spectrophotometer were used to observe the effects of various doses of hababoside on intracellular free Ca2 + concentration in OGD hippocampal neurons. RESULTS: Primary cultured SD rat neonatal rat hippocampal neurons were cultured on the 7th day. Neurons were identified as hippocampal neurons by neuron - specific enolase (NSE) immunohistochemical staining. The purity of neurons was 85% ~ 90%. MTT results showed that compared with the model group, 10, 25, 50, 100 and 100μmol / L of hababine significantly increased the viability of hippocampal neurons (P <0.05 or P <0.01), and the effective concentration range of pretreatment for 48 h 10 ~ 100μmol / L. Fluorescence inverted microscope showed that Ca2 + fluorescence intensity of hippocampus neurons in normal control group was weak, Ca2 + fluorescence intensity of hippocampal neurons in model group was stronger than that in model group, OGD-induced Ca2 + fluorescence intensity of hippocampal neurons, of which 75μmol / L group, the most obvious decrease in fluorescence intensity. Fluorescence spectrophotometry showed that compared with the normal control group, the intracellular free Ca2 + concentration in hippocampal neurons increased significantly in the model group (P <0.01). Compared with the model group, 75|Ìmol / L of habacin significantly reduced the hippocampal neurons Intracellular free Ca2 + concentration (P <0.05). CONCLUSION: Pretreatment with habobin for 48 h reduced the intracellular free Ca2 + concentration induced by OGD in hippocampal neurons. The protective effect was best at 75 μmol / L, which was related to the decrease of intracellular free Ca2 + concentration.