向内皮细胞分化的骨髓间充质干细胞体外三维构建组织工程心脏瓣膜的实验研究

Three-diamensional Construction of Tissue Engineering Heart Valve in vitro with Sheep Bone Marrow Mesenchymal Stem Cells Induced to Differentiate into Eendothelial Cells

目的以绵羊骨髓间充质干细胞在体外向内皮细胞诱导分化(MSC-ECs)作为种子细胞,去细胞猪主动脉瓣为支架,体外三维构建组织工程心脏瓣膜(TEHV)。方法羊骨髓穿刺液,以Percoll梯度分离液离心法获取骨髓间充质干细胞(MSCs),在内皮细胞诱导分化液中体外培养、扩增,鉴定;以酶消化法制备去细胞猪主动脉瓣叶支架,将体外培养扩增的诱导分化后的MSCs种植于支架上,体外三维培养,构建组织工程心脏瓣膜,研究细胞功能及组织学结构。结果MSC-ECs为梭形的成纤维细胞状形态,呈网格状排列;α-SMA、Vimentin、CD-31及Ⅷ因子细胞免疫染色阳性,培养上清液NO及ET-1分别为(165.6±8.9)μmol/L及(50.6±5.6)ng/L,其中NO值明显高于未诱导组(P<0.05),猪去细胞瓣膜支架去细胞完全,弹力纤维及胶原纤维保持完整;诱导分化的MSCs在支架上生长良好,形成完整的细胞层。结论MSCs在体外向内皮细胞诱导分化后已初步具有内皮细胞功能,在去细胞猪主动脉瓣膜支架上生长良好,可以在体外初步构建TEHV。

Objective To develop tissue engineering heart valves （TEHV） in vitro by seeding sheep bone marrow mesenchymal stem cells induced to differentiate into endothelial cells （MSC-ECs） on acellular porcine aortic valve matrices. Methods Sheep MSCs-ECs were obtained by bone marrow punc- tures （iliac crest）, isolated by Percoll gradient centrifugation, cultured and expanded by VEGF, bFGF and ECGF to differentiate into endothelial cells in vitro and characterized by immunohistochemistry. The secretion levels of nitric oxide （NO） and endothelin I（ET-1） of the MSC-ECs were measured; Decellularized porcine aortic valve matrices were created by 0.05 %Trypsin＋0.02 %EDTA digestion procedure; TEHVs were constructed by seeding the sheep MSC-ECs on the acellular porcine aortic valve matrices in vitro and the histological structures were studied by light microscopy （LM） and scanning electron microscopy （SEM）. Results Sheep MSC-ECs were spindle-shaped with both positive staining of α-SMA, Vimentin and CD-31, vWF, and the levels of NO and ET-1 were （165.6±8.9）μmol/L and （50.6±5.6）ng/L, respectively. The level of NO in MSC-ECs was significantly higher than that in MSCs. Porcine cells could not be detected by H-E staining after decellularization, with elastic fibers and collagenou fibers well- preserved. LM and SEM demonstrated a confluent seeding MSC-ECs in the surface of TEHV. Conclusion In vitro sheep MSC-ECs could function as vascular endothelial cells, which may be a potential cell source for TEHV. It is feasible to construct TEHVs with sheep MSC-ECs on porcine acellular scaffolds.