骨髓单个核细胞构建细胞化生物组织工程血管支架的研究

An Experimental Research on Construction of Tissue Engineering Blood Vessel Scaffold with Cells from Bone Marrow Mononuclear Cells

目的研究鼠骨髓来源平滑肌祖细胞(SPCs)和内皮祖细胞(EPCs)在细胞外基质支架上的生长特性,为生物组织工程血管寻找更新的种子细胞和支架。方法将鼠骨髓来源单个核细胞(BMCs)分别用不同的培养基进行诱导分化,于培养10 d后用免疫荧光双标技术(CD34/VWF)鉴定EPCs,用-αSMA/CD14鉴定SPC。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作用下,构建毯状细胞外基质(ECM)支架,在支架表面种植诱导培养骨髓来源的第2代SPCs和EPCs。用扫描电镜和透射电镜观察和分析的支架表面结构以及细胞在支架上生长情况。结果单个核细胞经EBM-2培养基诱导,培养至10 d双荧光染色结果表明,培养贴壁细胞中约90%的EPCs呈VWF/CD34双阳性;经bFGF的M-199培养基诱导阳性率逐渐增加,培养至10 d双荧光染色结果表明,培养贴壁细胞中SPCs呈-αSMA/CD14双标阳性100%。将诱导培养的第2代SPCs,种植在支架上,4 d时倒置显微镜示SPCs在支架表面细胞融合呈现典型的"峰"、"谷"样形态,当EPCs被种植10 d时扫描电镜显示在支架表面形成一个完整的较平整的细胞平面。透射电镜发现在多层平滑肌祖细胞表面有单层内皮祖细胞,呈三维立体结构,具有天然血管的内膜和中膜,更接近天然血管的结构。结论BMCs将是组织工程血管最理想的细胞来源,细胞外基质支架能显著促进SPCs和EPCs粘附、增殖和分化,可作为SPCs和EPCs生长的载体,可作为一种新颖的合成人造血管的生物组织工程支架。

Objective To explore the characteristics of smooth muscle progenitor cells （SPCs） and endothelial progenitor cells （EPCs） on the extracellular matrix （ECM） scaffolds and to find a novel seeded cells and bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels. Methods Induced, proliferated and differentiated in different culture media from rat bone mononuclear cells （BMCs）. The EPCs and SPCs were identified by immunofluorescent staining （CD34/VWF dipl-mark）and （α-SMA/CD14 dipl-mark） at 10 d. SPCs and EPCs of the second filial generation from mononuclear cells isolated and cultured and induced from rat bone marrow were grown on the scaffold, that comprises three major ECM proteins （i. e. fibrinogen, fibronectin and laminin） which can be easily molded into mat-like form and gelled by the addition of fresh plasma. the surface structure of the scaffold and growth state of SPC_,s and EPCs on the scaffold were observed and analysed by scanning electron microscope （SEM） and transmission electron microscope （TEM）. Results BMCs were induced in EBM-2 culture medium, EPCs percentage of the cells that were dipl-positive for CD34/VWF was 90% by immunofluorescent staining at 10 d. BMCs were induced in M-199 culture medium with bFGF, and SPCs percentage of the cells that were dipl-positive for α-SMA/CD14 was 100 %. Second filial generation SPCs that induced were planted on the ECM-scaffold, SPCs under inverted/fluorescent microscope present typical ＂peak＂＂valley＂ at 4 d; After EPCs were planted for 10 d, a level cell surface on scaffolds was achieved under SEM. And multiplayer SPCs were found on the surface of monolayer EPCs under TEM, which had three-diamensional stereochemical structure with endomembrane and tunica media and got close to natural blood vessel. Conclusion BMCs will be most ideal cells source for Tissue engineering Blood Vessel. ECM scaffolds can promote adhesion, proliferation and differentiation on SPCs and EPCs. ECM scaffolds can be used as the matrix for SPCs and EPCs and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.