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目的:探讨脑源性神经营养因子(brain derived neurotrophic factor,BDNF)在PC12细胞凋亡中的作用。方法:设计并合成针对BDNF mRNA序列的小片段干扰RNA(siRNA),利用lipofectamine 2000将siRNA转染入PC12细胞中或给与6-OHDA损伤,给与/不给予BDNF蛋白保护,采用定量PCR和免疫荧光法检测BDNF mRNA和蛋白表达水平;采用上清液乳酸脱氢酶(LDH)释放量测定和流式细胞仪法检测siRNA对细胞凋亡的影响。结果:转染siRNA的细胞的BDNF mRNA的表达量比正常组细胞减少73%,而转染作为对照的scrambled siRNA的细胞的BDNF mRNA的表达没有明显变化。BDNF RNA干扰与6-OHDA神经毒性一样可诱导PC12细胞的LDH释放和细胞凋亡。给予BDNF蛋白保护后细胞毒性减轻。结论:BDNF基因下调可以导致PC12细胞的凋亡,BDNF蛋白对PC12细胞有保护作用,为进一步进行动物体内研究奠定了基础。
Objective: To investigate the role of brain derived neurotrophic factor (BDNF) in the apoptosis of PC12 cells. Methods: Small interfering RNA (siRNA) targeting BDNF mRNA sequence was designed and synthesized. Lipofectamine 2000 was used to transfect siRNA into PC12 cells or to injure 6-OHDA with and without BDNF protein protection. Quantitative PCR and The expression of BDNF mRNA and protein was detected by immunofluorescence. The effect of siRNA on apoptosis was detected by the determination of LDH release and the flow cytometry. Results: BDNF mRNA expression was decreased by 73% in cells transfected with siRNA compared to normal cells, whereas BDNF mRNA expression in cells transfected with scrambled siRNA did not change significantly. BDNF RNA interference, like 6-OHDA neurotoxicity, induces LDH release and apoptosis in PC12 cells. Cytotoxicity was reduced after BDNF protein protection. CONCLUSION: Down-regulation of BDNF gene can lead to apoptosis of PC12 cells. BDNF protein has a protective effect on PC12 cells, which lays the foundation for the further study in vivo.