siRNA干扰人脑源性神经营养因子表达对U251细胞凋亡的作用机制研究

Studies on the mechanism of U251 cell apoptosis by interfering BDNF expression with siRNA

目的应用特异性脑源性神经营养因子-小干扰核糖核酸(brain derived neurotrophic factor-small interfering RNA,BDNFsiRNA)下调U251细胞中BDNF表达,观察对胶质瘤细胞凋亡的影响,并初步探讨其相关机制。方法将经体外培养的人胶质瘤细胞系U251细胞随机分为3组,其中A组为对照组,B组为non-silencing-siRNA组,C组为BDNF-siRNA组。通过Western blot法检测细胞BDNF、AKt、p AKt蛋白的表达,ELISA检测各组BDNF的分泌水平,流式细胞仪法检测各组U251细胞凋亡情况。结果 Western blot结果显示:与A组和B组相比,C组BDNF、p AKt蛋白表达显著降低(P<0.05)。ELISA结果表明C组U251细胞培养上清中BDNF含量为(70.20±3.05)pg/m L,与A相比有所降低,差异有统计学意义(P<0.05)。流式细胞仪检测结果可见C组细胞的凋亡率为(19.62±2.50)%,显著高于A组[(1.63±0.61)%]和B组[(1.78±0.94)%],差异均有统计学意义(P<0.05)。结论体外培养U251细胞时,采用特异性BDNF-siRNA能够显著降低BDNF、p AKt表达,同时,干扰BDNF表达能促进U251细胞的凋亡,推测与Trk B-Akt通路密切相关。

Objective To discuss the effect of siRNA specificly for BDNF on U251 cell apoptosis and its related mechanism. Methods Human gliomas cell line U251 were cultured in vitro and divided into three groups,group A as control group,group B as non-silencing-siRNA group,group C as the BDNF-siRNA group. Expression of BDNF,AKt and p AKt in three groups was examined by Western blot and BDNF secretion level was evaluated by ELISA. Cell apoptosis in each graup were examined by flow cytometry instrument method. Results Western blot results showed that compared with group A and group B,protein expression of BDNF,p AKt in group C was decreased. ELISA results showed that BDNF concentration in the supernatant of group C was( 70. 20 ± 3. 05) pg / m L,which lower than group A,the difference was statistically significant( P < 0. 05). The cell apoptosis rate of group C was( 19. 62 ± 2. 50) %,which was higher than group A( 1. 63 ± 0. 61) % and group B( 1. 78 ± 0. 94) %,the difference were statistically significant( P< 0. 05). Conclusion When cultured in vitro,specific BDNF-siRNA can significantly reduce the expression of BDNF,p AKt,meanwhile,when interference expression of BDNF can promote the apoptosis of U251 cell,it may closely related to the Trk B-Akt pathway.