转化生长因子β_1对组织工程瓣膜体外构建的作用

Application of transforming growth factor -B_1 on construction of tissue engineering heart valves:experimental in vitro

目的研究转化生长因子β_1(TGF-β_1)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-β_1基因转染大鼠肌成纤维细胞,48 h 后 G418筛选3周使 TGF-β_1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-β_1(10μg/L)为实验组 B,种植未转基因细胞不加 TGF-β_1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48 h 和4周后肌成纤维细胞瞬时和稳定表达 TGF-β_1。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-β_1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。

Objective To investigate the effects of transforming growth factor-β1 （TGF-β1） on construction of tissue engineering heart valves （TEHV）. Methods Fresh porcine aortic valves were deeellularized with trypsinaso and detergent Triton X-100. Myofibroblasts were obtained from rat thoracic aorta, cultured, transfected with the vector contaonig TGF-β1 gene plesmid pcDNA3.0/TGF-β1 inedited by lipofectamine 2000 after 48 hours, screened by G418 for for 3 weeks. Decellularized valves were divided into 3 groups： Group A, seeded with the transfected myofibroblests and cultured in medium without TGF-β1, Group B, seeded with the transfected myofibroblests and cultured in medium with TGF-β1 10 ng/ ml, and Group C, seeded with non-transfected myofibroblests and cultured in medium without TGF-β1. Hemataxylin-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 lmmunohistological investigation showed instant TGF-β1 expression in the myofibroblasts 48 h after the transfection and stable TGF-IM 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）‰, and （0. 109±0.004） ‰, both signitlcantly higher than that of Group [（0.089±0.011） ‰, 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 ） %0 and （5.02 ±0. 40） ‰, both significantly higher than that of Group C [ （4. 34 ±0. 47） ‰, 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-β1 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 vitax