应用GFP基因转染技术示踪组织工程技术构建角膜基质

Tissue engineering of corneal stroma with GFP marked cells

目的 应用基因转染技术将绿色荧光蛋白 (GFP)标记角膜基质细胞 ,以此为种子细胞构建角膜基质并对构建过程进行示踪。方法 构建携带EGFP基因的重组逆转录病毒载体PLNCX2 EGFP ,转染兔角膜基质细胞 ,然后将该细胞接种于PGA ,形成细胞 -生物材料复合物 ,移植于母兔角膜基质板层内。术后 8周 ,组织学切片 ,HE染色 ,荧光显微镜下对绿色荧光蛋白 (GFP)进行示踪观察。结果 8周后 ,组织工程化角膜组织形成 ,组织学切片显示PGA完全降解 ,新生组织形成 ,组织排列规整 ,荧光显微镜下见新生组织呈绿色 ,提示EGFP表达。结论 组织工程角膜基质的组织学结构与角膜基质相类似。新生组织表达GFP 。

ObjectiveTo reconstruct corneal stroma with cultured cells and trace the cultured cells by Green Fluorescence Protein (GFP) gene transfection.MethodsCorneal stromal cells were isolated from newborn rabbit cornea and were cultured and expanded in vitro. At the time of reaching confluency, corneal stromal cells were mixed with Polyglicolic Acid (PGA) to form a cell scaffold construct. At 1 week after culture, the constructs were implanted into mother rabbit corneal stroma and portions of corneal stromal cells were transfected with GFP gene to mark the seed cells by retroviral vector. Tissues were harvested at 8 weeks for histologic evaluation.ResultsThe engineered tissue proceeded to become transparent over an 8 week period. Stroma engineered in cornea revealed the histology of lamellar relatively similar to that of natural stroma. Additionally, a green colored stroma was observed when engineered with GFP labeled cells under fluorescence light microscope, suggesting that the stroma was constituted by implanted cells. ConclusionsThe results demonstrate that stroma could be engineered in recipient cornea and the composed cells originate from cultured cells.