转基因细胞片复合PLGA支架修复兔软骨缺损的研究

Repairing Larynx Cartilage Defects with Prefabricated PLAG Scaffold Compounded with BMSCs Cell Sheets Transfected by GDF5

细胞片技术是应用组织工程方法使培养细胞从培养表面分离而形成含有细胞外基质的一层完整片状结构,弥补了传统组织工程技术的不足,是获取种子细胞以及对种子细胞进行转移的一项新技术。为探讨体外生长分化因子-5(GDF5)基因转染修饰的BMSCs细胞片与GDF5转基因BMSCs负载的PLGA支架形成的共聚物修复兔甲状软骨缺损的效果,实验通过腺病毒转染GDF5基因至四代兔BMSCs,温度敏感性培养皿制备GDF5转基因细胞片并与负载有转染GDF5基因BMSCs的PLGA支架复合,移植至同种兔甲状软骨缺损处,分别于术后4、8周行大体观察和组织学检测其修复效果。实验分3组:(A)转基因细胞片包裹负载有转基因BMSCs的PLGA支架组;(B)负载有转基因BMSCs的PLGA支架组;(C)负载BMSCs的PLGA支架组。结果显示,体外成功收获了完整的GDF5转基因细胞片,Real time PCR检测到GDF5 mRNA的表达,行大体组织的II型胶原免疫组化和阿利新蓝染色显示:A组和B组均表达II型胶原和糖胺聚糖(GAG),但A组表达高于B组,有统计学意义(P<0.05)。由此可得,转基因细胞片包裹负载转基因BMSCs PLGA支架较传统转基因BMSCs负载PLGA支架方法具有更加优越的成软骨能力,能更有效地促进软骨缺损的修复。

Cell sheet technology is a new skill for cell harvesting and transferring in medicine tissue engineering, which makes the cultured cells separate from the dish surface to form a layer of complete lamellar structure containing extracellular matrix. It has circumvent these limitations, such as insufficient biological activity, low cell-attachment efficiency, which being in traditional tissue engineering. So a compound which was constituted with mesenchymal stem cell sheet transfected with GDF5 by adenovirus vector and PLGA scaffold prefabricated with GDF5 gene-transferred BMSCs was structured, explore the clinical application and make a preliminary assessment on the repairing effect. Isolated and cultured rabbit bone marrow mesenchymal stem cells in vitro, tansfected the passage 4 cells with exogenous recombinant growth differentiation factor 5 by adenovirus vector. Harvested the transgenic bone marrow mesenchymal stem cell sheet through temperature-responsive culture dish after culturing 7 - 14 days. Using real time PCR to detect the express of GDF5 in transgenic cell sheet. Preparing a prefabricated PLAG scaffold through injecting GDF5 gene-transferred BMSCs to PLGA scaffold, then co-culture the compound constituted with the cell sheet and prefabricated PLGA scaffold for 2 days. Transplant the compound to rabbit larynx cartilage defects and discuss the repair capacity after 4 or 8 weeks at the gross level as well as at the histological level. The experiment divided into three groups ： （A） the compound constituted with the transgenic cell sheet and prefabricated PLGA scaffold;（B） prefabricated PLGA scaffold;（C） PLGA scaffold loaded BMSCs. The Results showed the whole transgenic cell sheet have been harvested successfully, real time PCR can detect the express of GDF5 in transgenosis cell sheets. The results of immunohistochemistry and Alcian Blue staining showed the expression of collagen II and GAG in group A and group B, furthermore, there was more collagen II and GAG in group A than that in group B with statistical difference （ P 〈 0.05 ）. Thus, the compound constituted with the transgenosis BMSCs cell sheet and PLGA scaffold prefabricated with transgenosis BMSCs has the more superior chondrogenic capacity than traditional PLGA scaffold loaded with cells, it can promote the repair of cartilage effectively.