增强型绿色荧光蛋白标记兔间充质干细胞体外复合小肠黏膜下层的生长特性

目的利用绿色荧光蛋白（EGFP）标记兔来源的骨髓间充质干细胞（BMSC）,观察其作为种子细胞复合SIS基质体外培养的生长特性,为复合物回植体内时间提供参考。方法通过物理和化学方法制备小肠黏膜下层（SIS）;取新西兰大白兔分离BMSC,体外培养增殖至P3代,行转染增强型EGFP,转染前后根据增殖速度,绘制生长曲线。然后将转染后的第3代间BMSC,以30×106的浓度接种于1 cm×1 cm大小的SIS支架材料上培养。观察细胞转染后的生长特性,检测复合后的细胞总数,荧光显微镜观察复合后细胞形态。结果 BMSC经转染后生长增殖速度未受明显影响。细胞DNA检测显示复合物培养第7天培养结果显示细胞增殖状态最佳差异有统计学意义（P〈0.05）。荧光显微镜观察显示复合物培养7天细胞增殖良好差异有统计学意义（P〈0.05）,第10天后呈现老化状态。结论增强型的EGFP转染不影响间充质干细胞传代以及增殖。间充质干细胞-SIS基质复合物体外培养7 d回植较为合适。

Growth characteristics of rabbit BMSCs labelled with EGFP on SIS in vitro

Objective To observe the growth characteristics of rabbit bone marrow- derived mesenchymal stem cells （BMSC） with enhanced green fluorescent protein （EGFP） which were used as seeding cells for small intestinal submucosa （SIS） in vitro. Methods The SIS was prepared using physical and chemical methods. BMSC were isolated from New Zealand rabbits and transfected with EGFP by virus. The characteristics of BMSC before and after transfection were observed. Labeled BMSC were seeded at the concentration of 30 × 10^6 on the 1 cm × 1 cm SIS. The proliferation and morphology of EGFP labeled BMSC were observed with fluorescent microscope after 3, 7, and 10 days＇ co-culture. Results Transfection of BMSC with EGFP virus did not affect the proliferation and morphology of BMSC. DNA measurement of these cells was showed that labelled BMSC proliferated well on SIS at day 7; however these cells showed senescence at day 10. Conclusion The EGFP has no effect on BMSC proliferation. The BMSC-SIS should be used for transplantation at about day 7 after co-culture.