血管成形术后外膜细胞表型转化和迁移的实验研究

Phenotype change and migration of adventitial fibroblasts during postangioplasty

目的观察和分析血管成形术后血管外膜成纤维细胞表型转化和外膜细胞向血管内膜的迁移。方法改良导丝法损伤24只大鼠颈总动脉(CCA),制作血管再狭窄模型;采用5-溴,2尿苷嘧啶(BrDU)标记增殖和迁移的成纤维细胞,以免疫组化,BrDU单染结合α-肌动蛋白(α-actin)复染,光镜、扫描电镜和图像分析仪观察和分析损伤后3、7、14和28d,血管外、中和内膜上与BrDU结合的成纤维细胞的动态分布。结果1.免疫组化染色:BrDU结合的外膜成纤维细胞在术后第3天外膜上分布较多,至第7天达到峰值并表达α-actin,成纤维细胞发生表型转化成为成肌纤维细胞,细胞外基质(ECM)沉积;第14天,外膜上阳性细胞数下降,中膜和内膜上数量显著上升,内膜增厚,管腔狭窄;第28天,外膜、中膜和内膜阳性细胞数量回归到基线,但内膜ECM沉积较多,内膜仍增厚,管腔狭窄。不同时间点,血管三层结构内阳性细胞数比较均有显著差异(P<0.05)。2.电镜观察:血管成形术后,成纤维细胞胞质饱满,粗面内质网发达,表面分泌颗粒丰富,合成大量微丝束,转化为成肌纤维细胞;第7和14天,成肌纤维细胞形成宽大的伪足,分别伸向血管外弹力板窗孔和内弹力板窗孔,细胞呈腔内方向迁移趋势。结论血管成形术后血管外膜成纤维细胞发生表型转化,合成分泌α-actin;外膜成纤维细胞转化为成肌纤维细胞,向血管内膜迁移、增殖,成为新生内膜的细胞成分;外膜细胞和血管再狭窄有关。

Objective To verify fibroblasts translocation from adventitia into neointima by labeling adventitia cells with bromodcoxyuridine （BrDU）after angioplasty, and to explore the relationship of adventitial fibroblast with restenosis. Methods Vascular restenosis model was created by injured intima of common carotid artery （CCA）of mouse with gnidewire, adventitial fibroblasts were labeled with BrDU, and dynamic distribution of myofibroblasts in adventitia, media and neoitima was observed at different times（3 d, 7 d, 14 d and 28 d）by means of single/doable-label immunohistochemistry, light microscope, electronic microscope and image analysis system. Results l.Immunohistochemistry： More adventitial fibroblasts combined with BrDU could be found in adventitia on the 3rd day of postangioplasty, and the number of this kind of cells reached the peak on 7 th day, and at the same time fibroblasts changed their phenotypes and became myofibroblasts, which produced α-actin and extracellular matrix （ECM）. On 14th day, the number of the positive cells decreased in adventitia, increased in media and neointima associated with intima thickening; on 28th day, while the number of fibroblasts labeled by BrDU returned to the basic-line in adventitia, media and intima, nevertheless, intima thickening and vascular stenosis and intimal ELM precipitation were still present. There were significant differences in the number of fibroblasts labeled with BrDU located in three layers of artery（P 〈 0.05）. 2. Electronic microscope： After angioplasty, the plasm of fibroblasts became rich, mitochondrious and increase of Golgi apparatus ; and the amount of rough endoplasmic reticulums rose with more secretory granules,together with a great amount of collagen synthesized forming the microfilaments ; on days of 7th and 14th, the wide pseudopodia of myofibroblasts could be found extending into the windows on the external elastic lamina （ELL）and the internal elastic lamina （ILL）; and showing the tendency of cellular migration. Conclusions Adventitial fibroblasts change their phenotype in postangioplasty and become myofibroblasts, which can synthesize α-actin, and translocate into the neointima. The adventitial fibroblasts are correlated with the postangioplasty restenosis. （J Intervent Radiol, 2006, 15： 424-429）