P53凋亡刺激蛋白2在gp120诱导的神经细胞凋亡中的作用

Investigation of apoptosis-stimulating protein 2 of P53 in gp120 induced neuronal apoptosis

目的研究P53凋亡刺激蛋白2(ASPP2)在人类免疫缺陷病毒外膜糖蛋白(gp120)诱导的神经细胞凋亡中的作用。方法体外培养小鼠大脑皮质神经细胞,给予gp120处理,免疫荧光技术检测P53和ASPP2在细胞内的表达与分布,蛋白印迹法检测活性caspase-3表达情况,原位末端转移酶标记技术(TUNEL)检测神经细胞凋亡。分析gp120对神经细胞内P53、ASPP2表达影响,微小RNA干扰技术反向验证ASPP2的作用。结果 gp120处理前,神经细胞内无P53表达,ASPP2表达在细胞浆中;gp120处理后,神经细胞出现P53表达并定位于细胞核,部分ASPP2由细胞浆移位到细胞核,与P53定位于同一部位。gp120处理前,caspase-3无活性,神经细胞凋亡率为5%,gp120处理后caspase-3被激活,神经细胞凋亡率为50%,微小RNA干扰ASPP2表达后,神经细胞凋亡率由50%降低到15%。结论 ASPP2参与了gp120诱导的经P53介导的神经细胞凋亡。

Objective To study the role of apoptosis-stimulating protein 2 of P53 （ASPP2） in envelope glycoprotein of human immunodeficiency virus （gp120） induced neuronal apoptosis. Methods Mouse cortical neurons cultured in vitro were treated with gp120. Then, P53, ASPP2 were detected by immunofluorescence, caspase-3 expression was analyzed by western blot, neuronal apoptosis was detected by TUNEL assay. To verify the role of ASPP2 in gp120 induced neuronal apoptosis,microRNA interference was used. Results P53 could not be detected and ASPP2 was detected in the cytoplasm within the control group. But, if the neurons were treated with gp120, P53 expression was enhanced and located in the nucleus, ASPP2 content shifted from the cytoplasm to the nucleus and located in the same position of P53. Caspase-3 was not activated and the neuronal apoptosis ratio was 5 % within control group. However,caspase-3 was activated into cleavage and the neuronal apoptosis ratio was elevated to 50% in gp120 treated group. When ASPP2 expression was reduced by ASPP2-microRNA lentivirus, the neuronal apoptosis ratio was reduced from 50% to 15%. Conclusion It is implied that ASPP2 plays an important role in the P53-mediated gp120- induced neuronal apoptosis.