慢病毒载体caspase-3 RNA 干扰对染铝神经细胞凋亡的干预.docVIP

  慢病毒载体 caspase-3 RNA 干扰对染铝神 经细胞凋亡的干预# 张勤丽，徐丽，教霞，李娜，牛侨** 5 10 15 （山西医科大学公共卫生学院，太原 030001） 摘要：目的 通过体外实验来研究铝的神经毒性，探讨慢病毒载体 caspase-3 RNA 干扰对染 铝神经细胞凋亡的干预效果。方法 原代培养神经细胞，用 AlCl3·6H2O 染毒，选用病毒为 载体的 caspase-3 shRNA 试剂感染神经细胞，荧光标记感染成功的细胞计算神经细胞的感染 效率，荧光定量 PCR 技术检测干预后 caspase-3 基因的表达情况计算慢病毒载体的 caspase-3 shRNA 的抑制率。光镜、荧光染色观察神经细胞的形态学变化，并检测细胞活力；Annexin V-PI 双染法检测神经细胞感染后的凋亡坏死率。 结果 慢病毒载体的 caspase-3 RNA 干扰试 剂的感染效率大于 90%，抑制率为 66.47%。 用慢病毒载体的 caspase-3 shRNA 感染原代培 养神经细胞后可见染铝神经细胞活力显著上升(P0.01)；吖啶橙(AO)-溴化乙啶(EB)双荧光染 色法可以清楚地观察到染铝细胞的早期凋亡和晚期凋亡现象明显减少；Annexin V-PI 双染法 检测结果显示, 感染后染铝神经细胞凋亡率显著降低 (P0.01)。 结论 慢病毒载体的 caspase-3 shRNA 感染细胞后，可以显著抑制 caspase-3 基因的表达，神经细胞活力明显增 高，凋亡细胞的数量显著降低。 关键词：慢病毒载体；caspase-3；RNA 干扰；细胞凋亡 中图分类号：R994.6 20 Interference of caspase-3 shRNA With Lentiviral Vector on Neural Cells Apoptosis Induced by Aluminum ZHANG Qin-li, XU li, JIAO,xia, LI na, NIU qiao (Shanxi Medical University, Taiyuan 030001) 25 Abstract: Objective To study the neurotoxicity of aluminum (Al), and explore the intervention effect of caspase-3 shRNA on Al-induced apoptosis. Methods Primary cultured nerve cells were exposed to AlCl3·6H2O, caspase-3 shRNAs were infected in the cells. Fluorescent labeled shRNAs were used to label the successfully infected cells, and infectious rates were calculated. Interference rate of caspase-3 shRNAs on caspase-3 gene expression was calculated with 30 35 40 QRT-PCR method. Morphologic characteristics of caspase-3 shRNAs treated neural cells were observed under light microscope, and fluorescent microscope stained with AO-EB staining, the alternations of apoptosis rates were analyzed by cytometry. Results The infectious rate of caspase-3 shRNA was above 90%, the inhibition rate was 66.47%. Nuclei shrinkage or decreament of neurocytes volume were noticed in Al treated neural cells under light microscope， while the phenomenon decreased when treated with additional caspase-3 shRNA. Under fluorescence light, alive neurocytes showed a bright green color in the cell body