转染OPN-002-siRNA对大鼠颈动脉球囊损伤后内膜增生和OPN、TGF-β1、PCNA表达的影响

Effects of OPN-002-siRNA Transfection on Intimal Hyperplasia and OPN,TGF-β1,PCNA Expression after Carotid Balloon Injury in Rat

目的观察经动脉外膜转染OPN-002-siRNA对大鼠颈动脉球囊损伤后内膜增生和骨桥蛋白(OPN)、转化生长因子β1(TGF-β1)及增殖细胞核抗原(PCNA)表达的影响。方法通过前期细胞实验筛选出最敏感的OPN-002-siRNA序列作为动物实验转染基因。72只Wistar大鼠,随机分为假手术组、球囊损伤组、OPN-SCR-siRNA转染组和OPN-002-siRNA转染组。用免疫荧光、HE染色、real-time RT-PCR和Western blot观察大鼠颈动脉球囊损伤后内膜增生情况和OPN、TGF-β1及PCNA的表达变化,以及OPN-002-siRNA对它们的影响。结果 (1)球囊损伤术后3天未见明显的新生内膜,7天内膜开始增厚,14天时内膜增厚显著。(2)OPN、TGF-β1 mRNA和蛋白水平于术后3、7、14天时持续升高,而PCNA mRNA和蛋白水平均于术后3天开始升高,7天达高峰,14天时下降。(3)与球囊损伤组、OPN-SCR-siRNA转染组比较,OPN-002-siRNA转染组在各个时间点新生内膜厚度明显减轻(P<0.001),OPN、TGF-β1、PCNA mRNA和蛋白表达显著减少(P<0.001)。结论 OPN-002-siRNA可能通过特异性抑制OPN、TGF-β1、PCNA的表达,从而减轻血管损伤后的内膜增生。

Aim To investigate the effects of OPN-002-siRNA transfection on intimal hyperplasia and osteopontin( OPN),transforming growth factor-β1( TGF-β1),proliferating cell nuclear antigen( PCNA) expression after carotid balloon injury in rat. Methods Through preliminary experiment,OPN mRNA in cultured vascular smooth muscle cells was tested by real-time reverse transcription polymerase chain reaction( real-time RT-PCR),and OPN-002-siRNA was determined as the most sensitive sequence and used as transfected siRNA in the subsequent animal experiments. Seventy-two rats were randomly divided into four groups: sham group,balloon injury group,OPN-scramble-siRNA( OPN-SCRsiRNA) group and OPN-002-siRNA group. Changes of intimal hyperplasia and OPN,TGF-β1,PCNA expressions were detected by immunofluorescence,hematoxylin-eosin( HE) staining,real-time RT-PCR and Western blot,and also the effect of OPN-002-siRNA was studied on them. Results( 1) There was no apparent neointima on the 3rd day after balloon injury. Intima began to thicken on the 7th day after injury,and intimal thickening was significant on the 14 th day.( 2) The expression of OPN,TGF-β1 mRNA and protein started to increase on the 3rd day and persisted until the14 th day. The expression of PCNA mRNA and protein started to increase on the 3rd day,peaked on the 7th day,decreased on the 14 th day.( 3) Compared with balloon injury group and OPN-SCR-siRNA group,the neointima thickness decreased significantly on each time point in OPN-002-siRNA group( P < 0. 001),and both mRNA and protein expression of OPN,TGF-β1 and PCNA reduced significantly on each time point in OPN-002-siRNA group( P < 0. 001). Conclusion OPN-002-siRNA can inhibit intima hyperplasia after artery injury by decreasing the expression of OPN,TGF-β1 and PCNA.