论文部分内容阅读
目的:探究张应变条件下micro RNA-33(mi R-33)调控移植静脉内膜增生的机制,为缓解静脉移植内膜增生提供潜在治疗方法。方法:SD大鼠进行“套管法”自体静脉移植,Elastin-van Gesion染色观察内膜增生情况。使用FX4000细胞应力加载装置(Flexcell International)对静脉平滑肌细胞加载频率1.25 Hz、幅度10%的张应变以模拟静脉在动脉环境受到的张应变力学刺激。q RT-PCR检测mi R-33表达,Western blotting检测相关蛋白,Brd U增殖实验和CCK-8试剂盒检测细胞增殖。双萤光素酶报告基因验证mi R-33与靶基因的作用关系。骨形态发生蛋白3(BMP3)特异性si RNA干扰片段、重组蛋白以及mi R-33 inhibitor和mimics用于研究细胞功能和相关信号通路。在体局部注射mi R-33 agomir和antagomir来验证mi R-33在静脉移植内膜增生中的作用。结果:移植静脉出现明显内膜增生,mi R-33显著降低,而BMP3、p-Smad5和p-Smad2表达明显上升;牵拉条件下得到与移植静脉中相同的结果。双萤光素酶报告基因实验证明BMP3是mi R-33的靶基因。mi R-33 mimics抑制BMP3及下游信号分子p-Smad2、p-Smad5表达和细胞增殖;mi R-33 inhibitor或者BMP3重组蛋白得到类似结果。在体注射mi R-33agomir降低BMP3及下游信号分子表达,亦可缓解静脉移植内膜增生。结论:mi R-33-BMP3-Smad信号通路参与移植静脉平滑肌细胞增殖;mi R-33可以缓解静脉移植内膜增生过程,具有潜在临床应用前景。
OBJECTIVE: To explore the mechanism of micro RNA-33 (mi R-33) regulation of intimal hyperplasia of vein graft under tensile strain and to provide a potential therapeutic approach for relieving intimal hyperplasia of vein graft. Methods: Sprague - Dawley rats received autologous vein graft and endothelium proliferation was observed by Elastin - van Gesion staining. Vascular smooth muscle cells were loaded with tension strain of 1.25 Hz, amplitude 10%, using a FX4000 cell stress loading device (Flexcell International) to simulate tensile strain mechanical stimulation of the vein in the arterial environment. The expression of mi R-33 was detected by q RT-PCR, Western blotting analysis, BrdU proliferation assay and CCK-8 kit were used to detect cell proliferation. Dual Luciferase Reporter Validates the Role of mi R-33 in Targeting Genes. Bone morphogenetic protein 3 (BMP3) specific si RNA interference fragments, recombinant proteins, and mi R-33 inhibitor and mimics were used to study cellular functions and related signaling pathways. The effects of mi R-33 on vein graft intimal hyperplasia were verified by local injection of mi R-33 agomir and antagomir. RESULTS: Significant intimal hyperplasia was observed in the vein graft, while mi R-33 was significantly decreased, while the expressions of BMP3, p-Smad5 and p-Smad2 were significantly increased. The same results were obtained with the grafted veins. Dual luciferase reporter gene experiments show that BMP3 is mi R-33 target gene. mi R-33 mimics inhibited BMP3 and downstream signaling molecules p-Smad2, p-Smad5 expression and cell proliferation; mi R-33 inhibitor or BMP3 recombinant protein obtained similar results. In vivo injection of mi R-33agomir reduces the expression of BMP3 and downstream signaling molecules and may also attenuate graft-versus-host neointimal hyperplasia. Conclusion: The mi R-33-BMP3-Smad signaling pathway is involved in the proliferation of vein smooth muscle cells. Mi R-33 can alleviate the intimal hyperplasia of vein grafts and has potential clinical application.