【摘要】 目的观察黄芪注射液对缺氧缺糖/复氧复糖大鼠海马神经元凋亡相关基因Caspase-3表达的影响。方法取原代培养8d的大鼠海马神经元,随机分为正常对照组、缺氧缺糖/复氧复糖组、黄芪注射液溶剂对照组和黄芪注射液组。除正常对照组外均进行缺氧缺糖0.5h再复氧复糖。各组于复氧复糖后0h、0.5h、2h、6h、24h、48h、72h和120h采用免疫组织化学染色法检测Caspase-3阳性神经元数目,采用原位杂交和RT-PCR方法检测海马神经元caspase-3 mRNA的表达,实验重复6次。结果免疫组织化学和原位杂交结果显示,与正常对照组相比,除0h和0.5h之外,缺氧缺糖/复氧复糖组各时间点海马Caspase-3蛋白、caspase-3 mRNA阳性神经元数目占神经元总数的百分率及平均吸光度值均明显增加(P<0.05),于24h达到高峰。黄芪注射液溶剂对照组以上指标的变化趋势与缺氧缺糖/复氧复糖组一致(P>0.05)。黄芪注射液组除0h和0.5h之外,各时间点以上指标比缺氧缺糖/复氧复糖组显著减少(P<0.05)。RT-PCR结果显示,除0h和0.5h外,各时间点缺氧缺糖/复氧复糖组海马神经元caspase-3 mRNA的平均吸光度值均较正常对照组明显增加(P<0.05),复氧复糖24h平均吸光度值最高;与缺氧缺糖/复氧复糖组相比,除0h和0.5h外,黄芪注射液溶剂对照组各时间点caspase-3 mRNA的平均吸光度值无明显变化(P>0.05),而黄芪注射液组在各个时间点caspase-3 mRNA的平均吸光度值明显降低(P<0.05)。结论黄芪注射液可抑制缺氧缺糖/复氧复糖大鼠海马神经元凋亡相关基因caspase-3的表达,从而抑制缺氧缺糖/复氧复糖大鼠海马神经元的凋亡。更多还原

【Abstract】 Objective To investigate the effect of astragalus injection on the expression of caspase-3 interrelated by apoptosis after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats.Methods Divided the hippocampal neurons cultured for eight days into four groups: normal control group(normal),hypoxia/hypoglycemia and reoxygenation group(model),astragalus injection solution group(solution) and astragalus injection group(astragalus).All groups were treated with reoxygenation after deprived of oxygen and glucose for 30 minutes except normal group.Immunohistochemistry was used to measure the number of Caspase-3 positive neurons,in situ hubridization and RT-PCR were used respectively to measure the expression of caspase-3 mRNA after hypoxia/ hypoglycemia and reoxygenation 0,0.5,2,6,24,48,72,120 hours.The experiment was repeated 6 times.Results The results of immunohistochemistry were similar to that in in situ hybridization.It could be observed in normal control group that hippocampal neuronal nucleolus were integrated,cellular tuber was distinct and cytokinesis were dyed by yellow a lot;It could be observed in hypoxia/ hypoglycemia and reoxygenation group that hippocampal neuronal nucleolus were crimple,cellular tuber was shrinked,largenumbers of cytokinesis were dyed by yellow and there were yellow granules.Compared with normal control group,the numbers,the percentage and the average absorbance(AA) of Caspase-3 protein and caspase-3 mRNA positive neuronal cells every time points increased obviously in the hypoxia/ hypoglycemia and reoxygenation group except 0 hour and 0.5 hours(P < 0.05),and peaked at 24 hours.The change of neuronal in configuration,numbers,percentage and the average absorbance of caspase-3 mRNA positive neuronal cells in astragalus injection solution group were accorded with hypoxia/ hypoglycemia and reoxygenation group(P > 0.05).Compared with hypoxia/hypoglycemia and reoxygenation group,the numbers,the percentage and the average absorbance of caspase-3 mRNA were less obviously in the astragalus injection group except 0 hour and 0.5 hours(P < 0.05).RT-PCR result showed that compared with normal control group,the average absorbance of expression of caspase-3 mRNA in hippocampal neurons of rats every time points increased obviously in hypoxia/hypoglycemia and reoxygenation group except 0 hour and 0.5 hours(P < 0.05),and peaked at the 24th hour;Compared with hypoxia/hypoglycemia and reoxygenation group,the average absorbance of expression of caspase-3 mRNA every time points in astragalus injection solution group had no obvious change,however the average absorbance of expression of caspase-3 mRNA in hippocampal neurons of rats every time points decreased obviously in the astragalus injection group except 0 hour and 0.5 hours(P < 0.05).Conclusion Astragalus injection could inhibit the expression of caspase-3 after hypoxia/hypoglycemia and reoxygenation,accordingly inhibited hippocampal neuronal apoptosis.更多还原