【摘要】 本研究运用RNA干扰(RNAi)技术将内皮细胞MMP-2进行基因沉默,揭示MMP-2在内皮细胞增殖、迁移、侵袭、血管形成以及细胞周期等方面的作用。采用脂质体法将MMP-2小干扰RNA(siRNA)转化内皮细胞EAhy926,通过RT-PCR及流式细胞术分别在基因和蛋白水平验证转化的效率。应用MTT比色法测定经MMP-2 siRNA干扰不同时间EAhy926细胞的增殖能力;虎红染色测定光密度法观察干扰48小时后内皮细胞在两种趋化因子作用下的迁移及侵袭能力的变化;Matrigel胶三维培养法观察干扰48小时后内皮细胞血管形成能力的改变;流式细胞术及半定量RT-PCR法测定内皮细胞周期和相关基因的变化。结果表明:干扰因素施加后48小时MMP-2基因表达水平降低至谷底,较正常对照下降约82%;流式细胞术分析表明,干扰因素施加后60小时MMP-2蛋白表达水平降低至谷底,较对照下降约60%。MTT法检测显示干扰前后内皮细胞增殖无明显变化。内皮细胞经MMP-2 siRNA干扰48小时后在两种趋化因子作用下的迁移能力均受到抑制,且对COLⅣ的抑制作用强于Fn(Fn未干扰组0.581±0.012,干扰组0.261±0.002;COLⅣ未干扰组对干扰组为0.467±0.009vs0.110±0.010,p<0.01)。侵袭实验也有相似的结果(Fn未干扰组对干扰组为0.365±0.012vs0.101±0.002;COLⅣ未干扰组对干扰组为0.317±0.009vs0.102±0.010,p<0.01)。内皮细胞体外的血管形成能力在经siRNA干扰48小时后下降至正常的58.9%。内皮细胞经MMP-2siRNA干扰48小时及72小时后,有丝分裂后期细胞比例(G1)分别由对照组[(65.9±2.53)%;(63.2±1.89)%]上升至[(83.9±2.53)%,(89.2±1.24)%](p<0.01);DNA复制期(S)和有丝分裂前期(G2)期细胞比例分别由对照组[(32.7±1.91)%,(37.1±2.65)%]下降至[(18.1±1.49)%,(10.2±0.85)%](p<0.01)。半定量RT-PCR分析结果显示,内皮细胞Rb、cyclinD1以及PCNA基因表达量分别下降至未干扰者的35%,51%和22%。结论:MMP-2的表达与内皮细胞的增殖无明显相关性,但在内皮细胞迁移、侵袭和血管形成等方面发挥重要作用,同时还通过Rb、cyclinD1以及PCNA等基因参与内皮细胞周期的调控。更多还原

【Abstract】 The study was aimed to reveal the effects of matrix metalloproteinase-2(MMP-2)on cell proliferation,migration,invasion,angiogenesis and cell cycle.The small interfering RNA(siRNA)of MMP-2 transfected into endothelial cells EAhy926,the transformation efficiency at protein and gene levels was evaluated by using flow cytometry and RT-PCR respectively.MTT method was used to detect the proliferation ability of EAhy926.The migration and invasion abilities of EAhy926 induced by two kinds of factors,type Ⅳ collagen(COL Ⅳ)and fibronectin(Fn)were assayed with Rose Bengal dying method.The changes of angiogenesis were determined by three-dimension culture.The changes of cell cycle and related gene expression were assayed by using flow cytometry and RT-PCR respectively.The results indicated that the proliferation ability of EAhy926 had no obvious difference between interference or not.The migration ability of EAhy926 induced by two kinds of factors,type Ⅳ collagen(COL Ⅳ)and fibronectin(Fn)was inhibited after interfered by siRNA for 48 hours,and was stronger inhibition to COL Ⅳ than Fn(Fn concrol:0.581±0.012 vs 0.261±0.002;COL Ⅳ control:0.467±0.009 vs 0.110±0.010,p<0.01).The invasion test had the similar result as migration test.(Fn vs control:0.365±0.012 vs 0.101±0.002;COL Ⅳ vs control:0.317±0.009 vs 0.102±0.010,p<0.01).The angiogenesis ability of endothelial cells dropped to 58.9% of control after inteference with siRNA for 48 hours.In the cell cycle experiments,after RNAi for 48 hours and 72 hours,the cell ratio of G1 rose from [(65.9±2.53)%;(63.2±1.89)%] to [(83.9±2.53)%,(89.2±1.24)%]%(p<0.01);the cell ratio of S and G2 dropped from [(32.7±1.91)%,(37.1±2.65)%] to [(18.1±1.49)%,(10.2±0.85)%](p<0.01).The results analyzed by seml-qucmtitative RT-PCR showed that the expression levels of Rb,cyclin D1 PCNA genes dropped to 35%,51% and 22% of normal control respectively.It is concluded that the expression of MMP-2 does not distinctly correlate with the proliferation of endothelial cells,but plays a very important role in affecting endothelial cell migration,invasion and angiogenesis,and participates in regulating cell cycle through Rb,cyclinD1 and PCNA.更多还原