绿色荧光蛋白标记骨髓基质干细胞在恒河猴体内构建组织工程骨的示踪作用

Green fluorescent protein as atracer of bone marrow stromal cells in bone tissue engineering in rhesus

目的用绿色荧光蛋白(GFP)标记恒河猴骨髓基质干细胞(rBMSC),以示踪其在体内参与组织工程骨形成的情况。方法用QBI-293A细胞对腺病毒Ad5.CMV-GFP进行扩增,用Ad5.CMV-GFP转染rBMSc,将转染成功的第三代BMSc在转染后48h消化制成细胞悬液,接种至块状可吸收HA上,体外培养3d后,将其植入恒河猴(同体移植)背阔肌的肌袋内,以未转染GFP的BMSC用同样的方法接种块状可吸收HA上,作为对照,术后6周取材,4%的中性多聚甲醛固定,塑料包埋,制作骨磨片PI染色在激光共聚焦显微镜下进行观测。结果转染GFP后,rBMSc仍贴壁生长,呈梭形或多角形,仍分裂增殖,但增殖速度有所降低。48h可见细胞发出强烈的荧光,呈全细胞分布,计数转染率达80%。在激光共聚焦显微镜下观察骨磨片,可见材料内有发出较强荧光的细胞结构,能同时被PI染液着色。结论绿色荧光蛋白能有效示踪组织工程骨种子细胞,种入体内的BMSC是组织工程骨骨组织形成的主要细胞来源。

Objective To observe the role of green fluorescent protein （GFP） in tracing rhesus bone marrow stromal cells （rBMSCs） during tissue-engineered bone formation in vivo. Methods AdS.CMV-GFP was amplified by infecting QBI-293A cells, and the bone marrow was harvested from the ilium of adult male rhesus to obtain rBMSCs, which were cultured and passaged in vitro. GFP was transfected into the third-passage rBMSCs via adenovirus vector and the labeled cells were inoculated into absorbable HA scaffold and cultured for 3 days, with untransfected rBMSCs as control, before the cell-matrix compounds were implanted into the latissimus dorsi muscles of rhesus. Samples were harvested at 6 week and embedded in paraform, and ground sections of the bone tissue were prepared to observe green fluorescence under laser scanning confocal microscope. Propidium iodide staining of the sections was also performed for observation. Results The rBMSCs grew well after GFP transfection, and green fluorescence could be seen 24 h after the transfection and became stronger till 48 h, with a positive transfection rate beyond 80%. Six weeks after cell implantation, the rBMSCs labeled by GFP-emitted green fluorescence were detected in the bone tissue under laser scanning confocal microscope. Conclusion GFP can effectively trace BMSCs during bone tissue engineering, and the transplanted BMSCs constitute the main source of bone-forming cells in bone tissue engineering.