绿色荧光蛋白标记结合激光共聚焦显微镜三维重建技术观测组织工程骨的构建和体内移植

COMBINED APPLICATION OF GREEN FLUORESCENT PROTEIN LABELING AND CONFOCAL LASER SCANNING MICROSCOPE THREE-DIMENSIONAL RECONSTRUCTION TO MONITOR CONSTRUCTION AND IN VIVO TRANSPLANTATION OF TISSUE ENGINEERED BONE

目的结合荧光蛋白标记技术和激光共聚焦显微镜三维重建技术观察体外构建的组织工程骨及其体内移植情况,为筛选优良的支架材料和三维构建方法提供技术支持。方法取2岁龄青山羊(体重约25kg)髂骨骨髓,利用密度梯度离心法结合贴壁培养法分离培养BMSCs,并通过流式细胞技术检测表面分子CD29、CD60L、CD45和CD44表达情况。将质粒pLEGFP-N1扩增、提取后酶切鉴定。用脂质体转染的方法将质粒转染到PT67包装细胞,G418筛选后扩增,获取病毒液。根据测定的滴度转染BMSCs,获得绿色荧光蛋白(green fluorescent protein,GFP)表达的BMSCs,扩增后作为种子细胞,以脱钙骨基质作为支架材料构建组织工程骨,并利用激光共聚焦显微镜观察种子细胞。将构建的组织工程骨移植到山羊股骨缺损处,利用激光共聚焦显微镜观察其存活和分布情况。结果获得的细胞呈成纤维细胞状,CD29和CD44呈阳性表达,CD60L和CD45呈阴性。质粒pLEGFP-N1用HindⅢ、BamHⅠ、SalⅠ和BglⅡ内切酶酶切后,凝胶电泳可见其相对分子质量约6900bp。将pLEGFP-N1转染到PT67包装细胞扩增后获取病毒液。根据测定的滴度(1.3×106cfu/mL)转染BMSCs,获得GFP表达阳性的BMSCs克隆。激光共聚焦显微镜三维立体成像技术观察到BMSCs在组织工程骨支架上分布、增殖和迁移。将构建的组织工程骨移植到山羊股骨缺损处,28d后激光共聚焦显微镜观察到GFP阳性细胞在移植区仍存在。结论荧光蛋白标记技术结合激光共聚焦显微镜三维重建技术可很好地观察支架上和移植体内的细胞,为组织工程产物三维培养的立体观察提供了可行方法 。

Objective The combined application of green fluorescent protein（GFP） and confocal laser scanning microscope three-dimensional reconstruction（CLSM-3DR） were used to monitor the construction and in vivo transplantation of tissue engineered bone（TEB）,to provide for technology in selection of scaffolds and three-dimensional constructional methods.Methods After bone marrow mesenchymal stem cells（BMSCs） were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture,and the expressions of CD29,CD60L,CD45,and CD44 in BMSCs were detected by flow cytometry.Plasmid of pLEGFP-N1 was amplified,digested by enzymes（Hind Ⅲ,BamH Ⅰ,Sal Ⅰ,and Bgl Ⅱ）,and identified.Transfection of pLEGFP-N1 into PT67 cells was performed under the help of liposome.Positive PT67 cells were picked out with G418,and proliferated for harvesting virus.Based on the titre of virus,after BMSCs were infected by virus containing pLEGFP-N1,GFP positive BMSCs were collected and proliferated for seeding cells.TEB was fabricated by GFP positive BMSCs and decalcified bone matrix（DBM） and observed by CLSM-3DR for the evaluation of the distribution and proliferation of seeding cells.After TEB was transplanted in the defect of goat femur,CLSM was used for observing the survival and distribution of GFP positive cells in the grafts.Results The isolated cells were fibroblast-like morphous,with the positive expression of CD29 and CD44,and negative expression of CD60L and CD45.The digested production of pLEGFP-N1 was collected for ionophoresis,whose results showed the correct fragment length（6 900 bp）.The virus of pLEGFP-N1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs.The proliferated GFP positive BMSCs and DBM were used for fabrication of TEB.The distribution,proliferation,and migration of BMSCs in TEB were observed by CLSM-3DR.GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation.Conclusion The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR.It suggests a wide use potency in monitoring of three-dimensional cultured TEB.