人骨髓基质干细胞作为心血管瓣膜组织工程种子细胞的实验研究

Experimental Study on Human Bone Marrow Stromal Cells as the Cell Source for Tissue Engineering of Heart Valve

目的探讨人骨髓基质干细胞(human bone marrow stromal cells,hMSC)作为心脏瓣膜组织工程种子细胞的可行性。方法取胸外科手术患者肋骨骨髓,Ficoll离心法获取单个核细胞,体外培养扩增,以流式细胞仪检测hMSC的表面抗原。将自然分化的hMSC接种于去细胞猪心瓣膜上2周,免疫组化检测细胞分化结果;扫描电镜和透射电镜观察细胞种植情况;生化检测种植细胞DNA含量和细胞外基质,并和隐静脉来源成纤维细胞构建的组织工程瓣膜作对比。结果hMSC在自然条件下能够分化表现成纤维细胞特性,Vimentin和-αSMA染色阳性;电镜观察活性成肌纤维细胞在瓣膜表面生长均匀平滑;瓣膜组织DNA和胶原含量显著高于去细胞瓣组,与成纤维细胞对照组无明显差异。结论hMSC能够在自然条件下先向成纤维细胞分化,并在去细胞猪心瓣膜上增殖生长,分泌胶原,可作为心血管瓣膜组织工程的种子细胞。

Objective To investigate the feasibility of using human bone marrow stromal cells （hMSC） as an alternative cell source for tissue engineering of heart valve. Methods hMSC were isolated from bone marrow and expanded in culture. The cultured hMSC were characterized by flow eytometry and immunohistoehemistry and seeded onto deeellularized porcine aortic valves. After 14 days of culture, the cell-seeded deeellularized heart valves were analyzed by histological, immunohistoehemieal, transmission （TEM） and scanning eleetorn microscopy （SEM） examinations as well as biochemical assays （DNA and collagen assay） for the tissue formation, extraeellular matrix protein production and cells proliferation. Results hMSC showed myofibroblast-like morphology characterized by positive staining for α-smooth muscle aetin （α-SMA） and vimentin. After 14 days of culture, the TEM and SEM showed a new confluent and viable myofibroblasts covering the surface deeellularized valves. The hMSCs-engineered heart valves demonstrated significantly higher DNA and collagen content than deeellularized valve. There were no statistical differences in DNA and collagen contents between the hMSC-seeded and saphenous vein myofi brobalsts-seeded valves. Conclusion Isolated hMSC demonstrated similar characteristics of vascular-derived myofibroblast that could proliferate and synthesize extraeelluar protein when cultured on the deeellularized porcine aortic valve, hMSC represent an alternative, promising, easy-accessible, autologous cell source for tissue engineering of cardiovascular structures and the invasive surgical procedure for harvesting vascular myofibroblasts can be spared.