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
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, typeⅣ 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 EAliy926 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 E（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 sernl-qucmtitative RT-PCR showed that the expression levels of Rb, cyclin D1 PCNA
出处
《中国实验血液学杂志》
CAS
CSCD
2008年第2期381-386,共6页
Journal of Experimental Hematology
基金
苏州大学医学发展基金项目(编号EE122522)
