杆状病毒转导的小鼠羊水干细胞保持成骨分化潜能的实验研究_医学论文.docVIP

杆状病毒转导的小鼠羊水干细胞保持成骨分化潜能的实验研究_医学论文 杆状病毒转导的小鼠羊水干细胞保持成骨分化潜能的实验研究_医学论文 【摘要】 目的 探讨带有绿色荧光蛋白基因的重组杆状病毒修饰后的小鼠羊水干细胞(mouse amniotic fluid stem cells, mAFSs)是否仍保持成骨分化潜能。方法 体外原代培养小鼠羊水干细胞，并进行成脂成骨诱导分化，鉴定多向分化潜能；体外构建并扩增重组杆状病毒（Baculovirus-CMV-GFP, Bv-GFP）；Bv-GFP体外转导小鼠羊水干细胞，流式细胞仪检测其转导效率；经Bv-GFP基因修饰后的小鼠羊水干细胞进行成骨诱导分化。结果 小鼠羊水干细胞体外具有良好的增殖能力，3代后的细胞呈现较均一的梭形、漩涡状生长，且小鼠羊水干细胞体外具有成脂成骨分化潜能。体外成功构建重组杆状病毒，经Sf9细胞扩增后，极度稀释法测定其滴度可达109v.g/ml；Bv-GFP体外可较有效转导小鼠羊水干细胞，其转导效率为52.85％，且经Bv-GFP基因修饰后的小鼠羊水干细胞仍保持成骨分化潜能。结论 小鼠羊水来源干细胞是一种理想的干细胞，杆状病毒是一种新型病毒基因载体，体外能较有效转导小鼠羊水干细胞且不改变其成骨分化潜能。经重组杆状病毒基因修饰的羊水干细胞会为今后的骨组织工程提供一种新的治疗模式。 【关键词】 杆状病毒 羊水细胞 基因修饰 成骨分化 【Abstract】 Objective To explore whether the mouse amniotic fluid stem cells(mAFSs) maintain the potential of osteogenesis differentiation after gene modification by green fluorescent protein(GFP) labeled baculovirus. Methods mAFSs were primary cultured in vitro and were induced for osteogenic and adipogenic differentiation, the multi-directional differentiation was identified. Baculovirus-CMV-GFP(Bv-GFP) was constructed and amplified, then it was transfected to mAFSs, the transfection efficiency was tested by flow cytometer osteogenic differentiation was induced on the transfected stem cells. Results The mAFSs had good proliferation capacity, cells were homogeneously spindle-shaped after 3 generations and grew in whirlpool manner, also they had osteogenic and adipogenic differentiation potential. The reconstructed baculovirus was successfully constructed, after Sf9 amplification, the tilter was 109v.g/ml after extreme dilution method；Bv-GFP could effectively transfected mAFSs, with the transfecting rate at 52.85%, and remained osteogenic and differentiation potential after gene modification. Conclusions mAFSs are ideal stem cells, baculovirus is a new type of viral genetic vector, which can effectively transfect mAFSs without changing the osteogenic differentiation potential. MAFSs, after genetically modified by reconstructed baculovirus, provides a new treating mode for bon