【摘要】 目的探讨BMP-2基因修饰的复层乳牙牙髓干细胞膜片的体外构建方法及基本生物学特性。方法分离培养脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHEDs),并通过转染BMP-2基因进一步提高SHEDs的成骨能力,随后以SHEDs为种子细胞构建复层细胞膜片。利用倒置显微镜对细胞转染效率及成骨能力进行观察;通过HE染色和Masson染色对细胞膜片的结构和胶原分布进行检测;以免疫组织化学染色检测BMP-2蛋白的表达情况。结果体外分离培养了SHEDs,并证实其具有多向分化潜能。采用富含维生素C的培养基连续培养10 d左右即可获得白色膜样结构,该细胞膜片具有较好的弹性,叠加两层单层膜片继续培养24 h可构建具有良好韧性和机械性能的复层膜片。HE染色及Masson染色观察发现SHEDs膜片由多层细胞及丰富的细胞外基质组成。免疫组织化学染色示SHEDs膜片可表达BMP-2蛋白。结论 BMP-2基因修饰的SHEDs体外采用富含维生素C的培养基连续培养可构建具有良好机械性能的复层细胞膜片并持续表达BMP-2蛋白,该膜片将为牙周骨缺损修复提供一种新思路。更多还原

【Abstract】 Objective To investigate the construction method and biological characteristics of BMP-2 gene modified bilayer human exfoliated deciduous dental pulp stem cell sheet. Methods Stem cells from human exfoliated deciduous teeth( SHEDs) were isolated and cultured,and the osteogenic ability of SHEDs was further improved by transfection of BMP-2 gene and then the bilayer cell sheet was constructed with SHEDs as seed cells. The transfection efficiency and osteogenic ability of cells were observed by microscope. The structure and collagen distribution of the cell sheet were detected by HE staining and masson staining. The expression of BMP-2 protein was detected by immunohistochemical staining. Results SHEDs were isolated and cultured in vitro and confirmed to have multi-directional differentiation potential. The white membrane-like structure was obtained by continuous culture of SHEDs in a medium containing vitamin C for about 10 days. The cell sheet had a good elasticity,and a bilayer cell sheet with good toughness and mechanical properties were constructed by superimposing two monolayer cell sheets for 24 h. HE staining and masson staining showed that SHEDs sheet was composed of multi-layer cells and rich extracellular matrix. Immunohistochemical staining showed that BMP-2 protein was expressed in SHEDs sheet. Conclusion BMP-2 gene modified SHEDs can be continuously cultured with a medium rich in vitamin C to construct a bilayer cell membrane with good mechanical properties and continuously express BMP-2 protein in vitro,which will provide a new idea for the repair of periodontal bone defects.更多还原