摘要
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
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第23期1622-1626,共5页
National Medical Journal of China
基金
国家自然科学基金(C30371414
C30571839)
关键词
心脏瓣膜
人工
转化生长因子Β
转染
Heart valve prosthesis
Transforming growth factor beta
Transfection