摘要
目的探讨荧光染料PKH26在构建组织工程骨中作为细胞示踪剂的可行性。方法取1周龄新西兰大白兔骨髓分离培养BMSCs,以PKH26标记第3代BMSCs,荧光显微镜观察并采用流式细胞仪检测标记率;以成骨诱导培养基诱导标记后BMSCs向成骨细胞分化,倒置相差显微镜观察诱导后7、14d细胞形态变化,并于14d采用钙钴法检测ALP活性,茜素红矿化结节染色观察诱导结果。将PKH26标记的BMSCs接种于生物衍生骨材料,48h后荧光显微镜观察细胞与材料的复合情况;将细胞-材料复合物植入成年新西兰大白兔双后肢肌袋,14、28d后荧光显微镜观察标记细胞的转归。结果荧光显微镜观察PKH26标记的BMSCs呈球形,呈现均匀分布的红色荧光;培养24h后细胞贴壁伸展,轮廓清晰可见。流式细胞仪检测细胞染色阳性率达97.2%。经成骨诱导后,细胞由长梭形变为多边形或立方形,ECM分泌增多,ALP及茜素红染色均呈阳性反应。PKH26标记细胞与生物衍生骨材料复合培养48h后,荧光显微镜观察可见材料表面及内部发出红色荧光。标记细胞-支架材料复合物植入肌袋后14d,在植入区可见发出红色明亮荧光的标记细胞;术后28d,在植入区仍可见发出红色荧光的细胞,但数量较14d时少,荧光强度也较弱。结论PKH26标记BMSCs可作为一种体外构建组织工程骨及短期体内示踪的有效手段。
Objective To explore the feasibility of using PKH26 as a cell tracer to construct tissue engineered bone. Methods BMSCs isolated from the bone marrow of 1-week-old New Zealand white rabbit were cultured. The BMSCs at passage 3 were labeled with PKH26 and were observed under fluorescence microscope. The percentage of the labeled cells was detected by Flow cytometer. The labeled cells were induced to differentiate into osteoblasts in vitro and the morphology of the cells after induction was observed under inverted phase contrast microscope. The osteogenic induction was evaluated by ALP staining and Alizarin red staining. The cells labeled with PKH26 were seeded on the bio-derived bone to construct tissue engineered bone in vitro. Then the compound of cells and material were observed under fluorescence microscope. The compound of labeled cells and material were implanted into the rabbit thigh muscle,and the transformation of the labeled cells was observed by fluorescence microscope 14 and 28 days later. Results Fluorescence microscope observation：the BMSCs labeled by PKH26 were spherical and presented with red and uniform-distributed fluorescence,and the contour of the cells were clearly observed when they were adherent 24 hours after culture. Flow cytometric detection revealed that the percentage of labeled cells was 97.2%. After osteogenic induction,the morphology of the cells changed from long-fusiform to polygon-shape or cube-shape,more ECM was secreted,and the ALP and the Alizarin red staining were positive. At 48 hours after culturing the PKH26 labeled BMSCs with bio-derived bone,the fluorescence microscope observation showed that there was red fluorescence on the surface and inside of the material. At 14 days after implantation,the labeled cells with red and light fluorescence were evident in the implantation area; while at 28 days,the cells withred fluorescence were still evident but less in quantity and weaker in fluorescence strength. Conclusion PKH26 can be used as BMSCs label for the construction of tissue engineered bone in vitro and the short-term tracing in vivo.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2009年第10期1246-1249,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
山西省自然科学基金资助项目(2006011132)~~
