Angiogenic gene-modified muscle cells for enhancement of tissue formation
Angiogenic gene-modified muscle cells for enhancement of tissue formation
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DOI：
10.1089/ten.2005.11.1034
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发表时间：
2005-07-01
影响因子：
--
通讯作者：
Soker, S
中科院分区：
文献类型：
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作者：
De Coppi, P;Delo, D;Soker, S
血管不足的工程化肌肉组织可能会导致纤维化和肌肉功能的丧失。在这项研究中，我们将成肌细胞的分离和基因工程与组织移植相结合，试图创造出血管良好的肌肉组织。从成年Lewis大鼠肌纤维的单个外植体中获得成肌细胞，并将其分别导入血管内皮生长因子(VEGF)和绿色荧光蛋白(GFP)双顺反子表达载体或无功能的血管内皮生长因子-碱性磷酸酶(AP)融合蛋白表达载体。用Western印迹分析、ELISA法和荧光显微镜分别检测成肌细胞中血管内皮生长因子和绿色荧光蛋白的表达，结果表明重组蛋白在成肌细胞中得到了成功表达。将转基因细胞悬浮在I型胶原中，注射到裸鼠皮下。对回收的工程化肌肉组织进行RT-PCR免疫染色和荧光分析，结果显示表达了血管内皮生长因子和绿色荧光蛋白。植入后1周、3周和4周的肌肉组织免疫组织化学分析证实了肌肉表型。与非功能性血管内皮生长因子转基因细胞相比，功能性血管内皮生长因子转基因细胞的新生血管形成和肌肉组织质量显着增加。综上所述，这项研究表明，当使用表达血管内皮生长因子的肌肉细胞时，体内工程化肌肉组织的体积增加。
Engineering muscle tissue with inadequate vascularity may lead to fibrosis and loss of muscle function. In this study we combined the isolation and genetic engineering of myoblasts with tissue transplantation in an attempt to create well- vascularized muscle tissue. Myoblasts were obtained from a single explant of adult Lewis rat myofibers and transfected with a bicistronic plasmid encoding vascular endothelial growth factor ( VEGF) and green fluorescent protein ( GFP) or with a plasmid encoding a nonfunctional VEGF - alkaline phosphatase ( AP) fusion protein. VEGF expression and GFP expression in vitro were, respectively, assessed by Western blot analysis ELISA and fluorescence microscopy, showing that the myoblasts were successfully expressing the recombinant proteins. The transfected cells were suspended in collagen type I and injected subcutaneously into nude mice. Analysis of the retrieved engineered muscle tissues by RT- PCR immunostaining and fluorescence showed expression of VEGF and GFP proteins. Immunohistochemical analysis of the muscle tissues 1, 3, and 4 weeks after implantation confirmed the muscle phenotype. Neovascularization and muscle tissue mass significantly increased with functional VEGF- transfected cells compared with nonfunctional VEGF- transfected cells. In conclusion, this study demonstrates that in vivo engineered muscle tissues improve their volumes when VEGF- expressing muscle cells are used.