BDNF RNA干扰在多巴胺能PC12细胞凋亡中的作用研究

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。

Effect of BDNF RNA interference on Doparminergic PC12 cell apoptosis

Objective： To approach the exact role of brain derived neurotrophic factor （BDNF） in Doparminergic PC12 cell apoptosis. Methods： Specific small double-stranded interfering RNA （siRNA） against BDNF mRNA was designed, synthesized and transfected into PC12 cell line using lipofectamine 2000 with or without BDNF protein protection. Real time PCR and immunocytochemistry assay were used to check the expression of BDNF mRNA and protein; lactate dehydrogenase release and flow cytometry assay were used to detect the effect of siRNA on cell apoptosis. Results： BDNF mRNA reduced more than 70% in the siRNA transfected cells as compared with the control group, but not in the scrambled siRNA. BDNF RNAi induced lactate dehydrogenase release and cell apoptosis of PC 12 cell coincide with 6-Hydroxydopamine （6-OHDA） neurotoxicity. Cell apoptosis was alleviated when post-incubated with BDNF protein. Conclusion： The present study showed that downregulation of BDNF could induce PC 12 cell apoptosis but BDNF protein had protective effect on PC12 cells.