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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。
Objective To observe the effect of Astragalus injection on the expression of apoptosis-related gene Caspase-3 in hippocampal neurons of rats during hypoxia / hypoglycemia / reoxygenation. Methods Primary cultured rat hippocampal neurons for 8 days were randomly divided into normal control group, hypoxia / hypoglycemia / reoxygenation group, Astragalus injection solvent control group and Astragalus injection group. In addition to the normal control group were carried out 0.5h hypoxia-deficient glucose reoxygenation complex sugar. The numbers of Caspase-3 positive neurons were detected by immunohistochemical staining at 0h, 0.5h, 2h, 6h, 24h, 48h, 72h and 120h after reoxygenation in each group. The hippocampus was detected by in situ hybridization and RT-PCR Neurons caspase-3 mRNA expression, the experiment was repeated 6 times. Results The results of immunohistochemistry and in situ hybridization showed that the expression of Caspase-3 protein and caspase-3 mRNA in the hippocampus of the hypoxic-hypoglycemic / hypoglycemic groups was higher than that of the normal control group at all time points except 0h and 0.5h The percentage of neurons to the total number of neurons and mean absorbance increased significantly (P <0.05), reaching peak at 24h. Astragalus injection solvent control group indicators above the trend and the oxygen deficit / glucose / reoxygen glycosylation group (P> 0.05). Astragalus injection group except 0h and 0.5h, at each time point above the indicators than the oxygen-glucose deprivation / reoxygen glucose group significantly reduced (P <0.05). The results of RT-PCR showed that the average absorbance of caspase-3 mRNA in hippocampal neurons increased significantly (P <0.05) compared with the normal control group at all time points except 0h and 0.5h, The average absorbance value of reoxygenation complex 24h was the highest; compared with the oxygen-glucose deprivation / reoxygenation glucose group, the average absorbance value of caspase-3 mRNA in the astragalus injection solvent control group was not significantly different at 0h and 0.5h (P> 0.05), while the mean absorbance of caspase-3 mRNA in astragalus injection group was significantly lower at each time point (P <0.05). Conclusion Astragalus injection can inhibit the expression of caspase-3 in hippocampal neurons of hypoxia-deficient / hypoglycemic / reoxygenation complex, and thus inhibit the apoptosis of hippocampal neurons in hypoxic-hypoglycemic / reoxygenation rats.