摘要
目的用RNA干扰(RNAi)技术抑制小鼠Fas基因表达,观察其对脑微血管内皮细胞bEnd.3增殖及FADD-FLIP-TRAF-NF-κB信号传导途径的影响,为后续FasL低剂量兴奋效应研究提供实验基础。方法设计合成靶向小鼠Fas基因的小干扰RNA(siRNA)片段,用脂质体包埋转染bEnd.3细胞;CCK-8法检测细胞增殖;RT-PCR检测bEnd.3细胞Fas mRNA的表达情况;western blot检测Fas、FADD、FLIP、TRAF蛋白表达水平。结果 CCK-8法检测显示,Fas-siRNA组细胞增殖水平与空白对照组比较,无统计学差异(t=1.805,P>0.05);RT-PCR结果表明,与空白组的Fas mRNA表达水平比较,Fas-228-、Fas-310-、Fas-427-、Fas-891-siRNA组mRNA表达水平降低,差异有统计学意义(F=123.127,P<0.05);western blot显示,Fas-siRNA组与空白对照组Fas蛋白表达水平比较,差异有统计学意义(t=12.101,P<0.01);Fas-siRNA组FADD、FLIP、TRAF蛋白相对表达水平与空白对照组比较,表达均下调(t=28.315、17.563、8.903,P<0.05)。结论 Fas-siRNA能有效封闭Fas基因的表达,抑制Fas下游信号FADD、FLIP、TRAF蛋白表达。
Objective To investigate the impact of reduced Fas expression by RNA interference on the proliferation of mice brain microvascular endothelial cell line bEnd. 3 and the signal transduction pathway of Fas-associated death domain-containing protein （FADD）, FADD-like IL-1 β-converting enzyme （FLIP）, tumor necrosis factor receptor-associated factor （TRAF） and nuclear factor KB （NF-KB）. Methods The siRNA fragment targeted to mice Fas gene was designed and synthesized, and transfected into bEnd. 3 ceils by Lipofection 2000. The cell proliferation was measured using cell counting kit-8 （CCK-8）. The expression levels of Fas mRNA and Fas protein were measured by real-time quantitative PCR and western blot respectively. The levels of FADD, FLIP and TRAF protein were measured by western blot simultaneously. Results CCK-8 assay demonstrated that no significantly difference of cell proliferation was found between Fas-siRNA group and blank control group （ t = 1. 805, P 〉 0.05 ）. Quantification analysis showed that Fas mRNA expression levels were markedly decreased in Fas-228-, Fas-310-, Fas-427- and Fas-891-siRNA group compared with the blank control group （ F = 123. 127, P 〈 0.05 ）. Western blot indicated that Fas-siRNA significantly reduced the expression of Fas protein （ t = 12. 101, P 〈 0.01 ）. The relative expression levels of FADD, FLIP and TRAF were significantly decreased as compared with blank group （ t = 28.315, 17. 563 and 8. 903, P 〈 0.05）. Conclusion Fas-siRNA can effectively block the expression of Fas gene in bEnd. 3 cell and decrease the protein levels of Fas downstream signal molecules FADD, FLIP and TRAF.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2013年第6期438-441,共4页
Chinese Journal of Clinical Laboratory Science
基金
国家自然科学基金(81171659)
江苏省自然科学基金(BE2010768)
