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目的探讨第10染色体同源丢失性磷酸酶张力蛋白基因(phosphatase and tensin homolog deleted on chromo-some ten,PTEN)对大鼠原代海马神经元牵张损伤后AMPA受体GluR2亚单位定位表达的调节作用。方法选用新生SD大鼠培养原代海马神经元,建立神经元牵张损伤模型,利用RNAi慢病毒载体敲减神经元中PTEN基因的表达。采用Tunel染色评估神经元牵张各时相点的损伤情况。采用Western blot检测牵张各时相点AMPA受体GluR2亚单位在细胞中的表达定位变化。结果慢病毒介导的RNAi能有效下调PTEN基因的表达。PTEN基因表达下调后,0、2、6h细胞凋亡比例无明显差异,但24h细胞凋亡比例由(68.7%±5.1%)下降至(39.3%±3.5%),48h细胞凋亡比例由(76.7%±5.3%)下降至(52.0%±4.3%)。神经元细胞膜中的AMPA受体GluR2亚单位含量在牵张损伤6、24、48h明显降低。下调PTEN基因的表达后,神经元细胞膜中的AMPA受体GluR2亚单位含量在牵张损伤6h无明显降低,牵张损伤24、48h后降低。细胞损伤6、24、48h时,下调PTEN基因的表达能够明显上调神经元细胞膜中的AMPA受体GluR2亚单位含量。结论海马神经元受到机械性牵张损伤后,下调PTEN的表达能够有效地阻止AMPA受体GluR2亚单位在细胞膜上定位的降低,降低凋亡细胞比例。PTEN对AMAP受体的调节作用可能为预防和治疗脑创伤提供一条新途径。
OBJECTIVE: To investigate the regulation of the localization of GluR2 subunit of AMPA receptor by stretch of chromosome 10 (PTEN) on the primary cultured hippocampal neurons in rats effect. Methods Primary cultured hippocampal neurons were cultured in neonatal SD rats to establish a model of neuronal stretch injury. The expression of PTEN gene in neurons was knocked down by RNAi lentiviral vector. Tunel staining was used to assess the injury of neuronal stretch at each time point. Western blot was used to detect the expression and localization of AMPR receptor GluR2 subunits in cells at different time points. Results Lentivirus-mediated RNAi effectively down-regulated PTEN gene expression. After PTEN gene expression was down-regulated, there was no significant difference in the proportion of apoptotic cells at 0, 2 and 6 h, but the percentage of apoptotic cells decreased from (68.7% ± 5.1%) to (39.3% ± 3.5% 76.7% ± 5.3%) to (52.0% ± 4.3%). Neuronal cell membrane AMPA receptor GluR2 subunit content in the stretch injury 6,24,48 h significantly reduced. After down-regulating the expression of PTEN gene, the content of GluR2 subunit of AMPA receptor in neuronal cell membrane was not significantly decreased 6 h after stretch injury, and decreased 24 and 48 h after stretch injury. At 6, 24, and 48 h after cell injury, the down-regulation of PTEN gene expression significantly increased the GluR2 subunit content of AMPA receptor in neuronal cell membrane. Conclusion The down-regulation of PTEN expression in hippocampal neurons after mechanical stretch injury can effectively prevent the decrease of AMPA receptor GluR2 subunit localization on the cell membrane and decrease the percentage of apoptotic cells. The regulatory effect of PTEN on AMAP receptors may provide a new way to prevent and treat brain trauma.