CAJ | 학술논문

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72 h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-siRNA 72 h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细胞凋亡.
목적 초보탐토파향감성성섬유세포생장인자(bFGF)적소분자간우RNA(siRNA)유도효질류U251세포조망적작용궤제.방법 장U251세포분위대조조、공재조화실험조,공재조화실험조안감염복수(MOI)=100분별진행공재체선병독(Ad-null)화함유bFGF소분자간우RNA적중조선병독(Ad-bFGF-siRNA)전염,전염72 h후통과Western blot방법검측상관단백적표체,병응용류식세포의화격광공취초현미경검측선립체막전위적변화,다조간균수비교채용단인소방차분석( One-way ANOVA).결과 실험조세포경전염Ad-bFGF-siRNA 72 h후,Western blot결과현시,여대조조화공재조상비bFGF단백표체명현강저,동시Cytochrome C、Caspase-3급Bax조망상관단백적표체증강,Bcl-xl화Bcl-2단백적표체강저；경류식세포의검측,선립체막전위고적세포소점적비례실험조위74.4％±4.7％여대조조적92.1％±2.5％、공재조적90.9％±1.8％상비명현하강,차이유통계학의의(F=28.805,P＜0.05)；격광공취초현미경적결과현시실험조적홍색형광여록색형광적비치위0.83±0.12여대조조적1.36±0.40화공재조적1.32±0.35상비명현하강,차이구유통계학의의(F=7.920,P＜0.05).결론 파향bFGF적siRNA가능통과선립체도경유도효질류U251세포조망.
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 (ΔΨm) 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 mitochondrial membrane potential of cells by flow cytometry,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.