靶向碱性成纤维生长因子的小分子干扰RNA诱导胶质瘤U251细胞凋亡机制的初步探讨

A preliminary study about the apoptostic mechanism of RNA targeting basic fibroblast growth factor in glioma U251 cells

目的初步探讨靶向碱性成纤维细胞生长因子（bFGF）的小分子干扰RNA（siRNA）诱导胶质瘤U251细胞凋亡的作用机制。方法将U251细胞分为对照组、空载组和实验组，空载组和实验组按感染复数（MOI）=100分别进行空载体腺病毒（Ad—null）和含有bFGF小分子干扰RNA的重组腺病毒（Ad-bFGF-siRNA）转染，转染72h后通过Western blot方法检测相关蛋白的表达，并应用流式细胞仪和激光共聚焦显微镜检测线粒体膜电位的变化，多组间均数比较采用单因素方差分析（One—way ANOVA）。结果实验组细胞经转染Ad—bFGF—siRNA72h后，Western blot结果显示，与对照组和空载组相比bFGF蛋白表达明显降低，同时Cytochrome C、Caspase-3及Bax凋亡相关蛋白的表达增强，Bcl—xl和Bcl-2蛋白的表达降低；经流式细胞仪检测，线粒体膜电位高的细胞所占的比例实验组为74．4％±4．7％与对照组的92．1％±2．5％、空载组的90．9％±1．8％相比明显下降，差异有统计学意义（F=28．805，P〈0．05）；激光共聚焦显微镜的结果显示实验组的红色荧光与绿色荧光的比值为0．83±0．12与对照组的1．36±0．40和空载组的1．32±0．35相比明显下降，差异具有统计学意义（F=7．920，P〈0．05）。结论靶向bFGF的siRNA可能通过线粒体途径诱导胶质瘤U251细胞非淋菌性尿道凋亡。

Objective To preliminarily investigate the mechanism of small interfering RNA （siRNA） induced apoptosis in glioma U251 cells by silencing basic fibroblast growth factor （bFGF）. Methods U251 cells were divided into the normal control group, the mock group and experiment group, the mock and experiment group were transfected with mock vector （Ad-null） and the recombinant adenovirus carrying bFGF-siRNA （ Ad-bFGF-siRNA ） respectively at a multiplicity of infection （ MOI ） of 100. After 72 hours, the expression of related proteins was revealed by the method of Western blot. Mitochondrial transmembrane potential （△ψm） was measured with flow cytometry and confocal microscopy, Groups were compared using single factor analysis of variance （One-way ANOVA）. Results After U251 cells were transfected with bFGF-siRNA, the results of Western blot showed that after 72 hours of transfection the bFGF protein in the experiment group decreased obviously, meanwhile Cytochrome C, Caspase-3 and Bax showed increased expression while in the Bcl-xl and Bcl-2 proteins decreased expression. The proportion of high mitoehondrial membrane potential of cells by flow eytometry, the experimental group was 74.4% ± 4. 7% decreased significantly compared with the control group 92.1% ± 2. 5%, the mock group 90. 9% ± 1.8%（ F = 28. 805, P 〈 0. 05 ） ; laser scanning confocal microscopy results showed that the red fluorescence and green fluorescence ratio of the experimental group was 0. 83 ± 0. 12 decreased significantly compared with 1.36 ± 0. 40 of the control group and 1.32 ± 0. 35 of the mock group （ F = 7. 920, P 〈 0. 05 ）. Conclusion siRNA targeting bFGF induced U251 cell apoptosis may be achieved through the mitochondrial pathway.