转化生长因子β1对组织工程瓣膜体外构建的作用

目的 研究转化生长因子β1（TGF-β1）对组织工程瓣膜（TEHV）体外构建的影响。方法 采用酶＋去污剂法制备去细胞瓣。脂质体介导pcDNA3．0／TGF-β1基因转染大鼠肌成纤维细胞,48h后G418筛选3周使TGF-β1基因稳定表达。去细胞瓣种植转基因细胞为实验组A,种植未转基因细胞且加TGF-β1（10 μg/L）为实验组B,种植未转基因细胞不加TGF-β1为对照组。2周后HE染色和扫描电镜观察,检测羟脯氨酸和DNA含量及瓣叶最大负荷力。结果 SABC法示转染48h和4周后肌成纤维细胞瞬时和稳定表达TGFβ1。形态学示实验组A、B细胞紧密联合且细胞外基质丰富。羟脯氨酸含量A组（5．83‰±0．67‰）和B组（5．02‰±0．40‰）,均高于对照组（4．34‰±0．47‰）;DNA含量A组（0．126‰±0．013‰）和B组（0．109‰±0．004‰）均高于对照组（0．089‰±0．011‰）;最大负荷力A组（13．4±1．0）N和B组（11．8±1．4）N均高于对照组（10．0±1．1）N增高。结论 TGF-β1能促进细胞生长和细胞外基质分泌,有利于提高TEHV力学性能。

Application of transforming growth factor-beta(1) on construction of tissue engineering heart valves: experimental in vitro

OBJECTIVE: To investigate the effects of transforming growth factor-beta1 (TGF-beta1) on construction of tissue engineering heart valves (TEHV). METHODS: Fresh porcine aortic valves were decellularized with trypsinase and detergent Triton X-100. Myofibroblasts were obtained from rat thoracic aorta, cultured, transfected with the vector contaonig TGF-beta1 gene-plasmid pcDNA3.0/TGF-beta1 mediated by lipofectamine 2,000 after 48 hours, screened by G418 for for 3 weeks. Decellularized valves were divided into 3 groups: Group A, seeded with the transfected myofibroblasts and cultured in medium without TGF-beta1, Group B, seeded with the transfected myofibroblasts and cultured in medium with TGF-beta1 10 ng/ml, and Group C, seeded with non-transfected myofibroblasts and cultured in medium without TGF-beta1. Hematoxylin-eosin staining and transmission electron microscopy were performed to observe the cell proliferation. DNA contents were measured. Hydroxyproline content was measured so as to indirectly test the collagen production. AGS-J mechanical testing instrument was used to test the mechanical properties of the strips of valves. RESULTS: Immunohistological investigation showed instant TGF-beta1 expression in the myofibroblasts 48 h after the transfection and stable TGF-beta1 expression 4 weeks later. Morphological examination showed that the myofibroblasts in Groups A and B were connected to one another closely with abundant extracellular matrix in the valves. The DNA contents of Groups A and B were (0.126 +/- 0.013) per thousand, and (0.109 +/- 0.004) per thousand, both significantly higher than that of Group (0.089 +/- 0.011) per thousand, both P < 0.011], with a significant difference between Groups A and B (P < 0.05). The hydroxyproline content of Groups A and B were (5.83 +/- 0.67) per thousand and (5.02 +/- 0.40) per thousand, both significantly higher than that of Group C (4.34 +/- 0.47) per thousand, both P < 0.05], with a significant difference between Groups A and B (P < 0.05). The maximum load of Groups A and B were (13.4 +/- 1.0) N and (11.7 +/- 1.4) N respectively, both significantly higher than that of Group C (10.0 +/- 1.1) N, both P < 0.05], with a significant difference between Groups A and B (P < 0.05). CONCLUSION: TGF-beta1 is an important and effective bioactive factor for cell proliferation and extracellular matrix growth of heart valve. It is of great value for constructing TEHV in vitro.