天津医药

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靶向P75 NTR的siRNA技术诱导U251胶质瘤细胞凋亡的实验研究

张泰1,亢建民2,蔡英3,4   

  1. 1. 天津医科大学在职研究生院，工作单位：天津市天和医院神经外科
    2. 天津市环湖医院
    3. 天津市环湖医院神经外科研究所
    4.
  • 收稿日期:2010-11-17 修回日期:2011-11-14 出版日期:2012-10-15 发布日期:2012-10-15
  • 通讯作者: 张泰

An experimental study of siRNA targeting P75NTR to induce apoptosis of U251 glioma cells

  • Received:2010-11-17 Revised:2011-11-14 Published:2012-10-15 Online:2012-10-15

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摘要： 目的：利用靶向P75神经营养因子受体（P75NTR）基因的siRNA技术诱导胶质瘤凋亡。方法：利用脂质体介导P75NTR-siRNA质粒转染U251胶质瘤细胞系，RT-PCR方法检测P75NTR的mRNA表达以确定基因沉默效果，绘制细胞生长曲线，原位细胞凋亡实验检测细胞凋亡情况，Western blot方法检测稳定转染后第3d时P75NTR、促凋亡基因Caspase-3和凋亡抑制基因Bcl-2的蛋白表达。结果：转染P75NTR-siRNA质粒后U251细胞P75NTR的mRNA表达水平显著下降（P<0.05），细胞增殖能力下降，凋亡细胞数量增加。与对照组和空载组比较，P75NTR与Bcl-2在RNAi组蛋白表达水平显著降低，Caspase-3则明显升高。结论：靶向P75NTR的siRNA技术可通过调控凋亡相关基因表达机制诱导胶质瘤细胞凋亡，有望成为一种新的治疗方法选择。

关键词: 胶质瘤, RNA干扰, 基因治疗, 凋亡

Abstract: Abstract Objective: To observe the therapeutic efficacy of siRNA silencing P75 neurotrophin receptor (P75NTR) to induce apoptosis of U251 glioma cells. Methods The siRNA fragments targeting P75NTR were transferred into glioma cell line U251. RT-PCR was used to explore the expression of P75NTR mRNA. Platelet counting method was employed to get cell growth curve, in situ apoptosis assay to detect cell apoptosis, and Western blot to proteins expression of P75NTR, pro-apoptotic gene caspase-3 and anti-apoptosis gene Bcl-2, after gene transfection 1,3,5 and 7d. Results The siRNA targeting P75NTR can decrease mRNA expression of P75NTR in U251 cell, decrease cell proliferation, and increase cell apoptosis. P75NTR and Bcl-2 protein level in RNAi group were lower than the other two groups. This trend was contrast with the caspase-3. Conclusion The gene silencing technique by siRNA targeting P75NTR can induce cell apoptosis through the mechanisms regulating the expression of apoptosis-related genes. It is expected to become a new choice for gene therapy of glioma.

Key words: glioma, RNA interfere, invasion, apoptosis