平滑肌祖细胞在新型细胞外基质支架上的生长特性

Characteristics of smooth muscle progenitor cells growing on novel extracellular matrix scaffolds

背景:血管壁细胞包括内皮细胞和平滑肌细胞,平滑肌细胞合成分泌的胶原蛋白、弹力蛋白和蛋白聚糖等细胞外基质是提供血管塑形及张力的最主要成分,平滑肌细胞在细胞外基质支架上生长可能更接近体内生长环境特性。目的:观察鼠骨髓来源平滑肌祖细胞在毯状细胞外基质支架上的生长特性。设计、时间及地点:细胞水平对比观察实验,于2007-07/2008-07在江苏大学临床检验实验室完成。材料:将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝coagulation作用下,构建毯状细胞外基质支架。方法:实验组诱导培养骨髓来源的第2代平滑肌祖细胞种植在毯状细胞外基质支架表面。对照组将骨髓来源的第2代平滑肌祖细胞接种到无菌圆盖玻片上。主要观察指标:于培养1,4,7,10,14,21d用电镜观察支架表面结构及平滑肌祖细胞生长情况;WesternBlotting法、Real-TimePCR法分别检测α-SMA蛋白及mRNA表达变化。结果:实验组平滑肌祖细胞贴壁率、增殖数明显高于对照组(P<0.01);电镜显示细胞在支架表面形成较平整的平面,如典型"平滑肌"形状;实验组平滑肌祖细胞为多层、三维立体、更接近天然血管的结构;实验组α-SMA蛋白及基因表达明显高于对照组(P<0.05)。结论:细胞外基质支架能显著促进平滑肌祖细胞黏附、增殖和分化,可作为一种新颖的合成人造血管的生物组织工程支架。

BACKGROUND： The cells in vessel wall contain endothelial cells and smooth muscle cells. The extracellular matrix （ECM）, such as collagen protein, elastin, as well as proteoglycan, which synthesis and secreted by smooth muscle cells, are the essential component for remodeling and tension of vessels. In addition, the growth of smooth muscle cells on ECM scaffolds is more approximate to growth environment in vivo. OBJECTIVE： To explore the characteristics of smooth muscle progenitor cells （SPCs） on the ECM scaffolds. DESIGN, TIME AND SETTING： The contrast observation at cell level was performed at the Clinical Examination Laboratory of Jiangsu University from July 2007 to July 2008. MATERIALS： Fibrinogen, laminin and fibronectin were mixed with certain proportion to construct mat-like ECM-scaffold under coagulation of rats＇ blood plasma. METHODS： Second generation of SPCs were grown on the ECM scaffolds as the experiment group, and SPCs inoculated on the cover glass were served as the control group. MAIN OUTCOME MEASURES： The surface structure of the scaffolds and growth state of SPCs on the scaffolds were observed electron microscope at the days 1,4, 7, 10, 14, and 21; Meantime, the expression changes of a-SMA and mRNA was .detected by Western blotting and real-time PCR technique. RESULTS： The ratios of cell adhesion and proliferation on scaffolds were higher in the experimental group than that of the control group （P 〈 0.01 ）. Furthermore, electron microscopy showed that morphology of SPCs on scaffolds was more plat just as typical smooth muscle cells with multiplayer, 3 D stereochemical structure, which was close to structure of natural vessels. Western Blotting assays revealed that the expression of a-SMA protein and gene was greater than the control group （P 〈 0.05）. CONCLUSION： ECM scaffolds can promote adhesion, proliferation and differentiation of SPCs, which can be used as the matrix for SPCs and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.