体内构建组织工程骨的示踪观察:CM-DiI长效示踪骨髓间充质干细胞的可行性研究

The Study on Biological Change of BMSCs During the Osteogenesis of Tissue-Engineered Bone in Vivo: the Feasibility of CM-DiI as a Long-Term Tracer of BMSCs

目的探索组织工程骨体内成骨时,CM-DiI长效示踪骨髓间充质干细胞的可行性,为种子细胞的体内转归研究提供标记方法。方法分离比格犬BMSCs,成骨诱导至第2代,用荧光染料CM-DiI进行细胞标记。荧光倒置显微镜观察标记后的细胞形态,MTT比色法测定标记前后BMSCs的增殖状况,检测标记前后Ⅰ型胶原(Col-Ⅰ)、骨形态发生蛋白(BMP-2)、骨钙素(BGLAP)和骨黏连蛋白(SPARC)的表达。最后,CM-DiI荧光标记后的BMSCs与β-TCP复合共培养后植入比格犬背部皮下,8周后取材,荧光显微镜下观察BMSCs体内转归,HE染色观察异位成骨情况。结果CM-DiI标记后早期细胞呈现红色荧光,48 h后荧光增强,72 h内荧光强度无明显减弱。BMSCs在CM-DiI标记前后细胞形态基本一致,两组间细胞的增殖率无明显差别;标记后RT-PCR检测Col-Ⅰ、BMP-2、BGLAP、SPARC表达,显示标记后的BMSCs亦可向成骨方向分化。扫描电镜检测到标记后细胞在β-TCP上增殖良好,细胞基质分泌丰富。细胞-支架复合物植入比格犬皮下,8周后荧光显微镜观察到标记细胞仍存在,且HE染色可见支架孔隙中有类骨基质沉积。结论CM-DiI对BMSCs的生长增殖、成骨分化无明显影响,对体内组织工程骨中BMSCs的示踪时间可长达8周,可用作体内细胞的长效示踪剂。

Objective To investigate the feasibility of CM-DiI on long-term tracing the biological change of bone mesenchymal stem cells （BMSCs） during osteogenesis of tissue engineered bone （TEB） in vivo, and to offer an alternative labeling method for tracing seed cells in vivo in future. Methods After BMSCs were separated from bone marrow in Beagle dogs and osteo-induced to the second generation （P2）, the cells were labeled with the fluorescent dye CM-DiI. The morphologic change of BMSCs after labeling was observed by fluorescence convert microscope. The proliferative ability was tested by MTr colorimetric between the labeling and un-labeling groups. The expression of Collagen I, BMP-2, BGLAP, and SPARC were detected through RT-PCR between the two groups. Lastly, CM-DiI-labeling BMSCs and beta-TCP were cocultured into TEB which was implanted subcutaneously into Beagle dogs. Eight weeks later, the implant was harvested and the biological change of BMSCs was observed by fluorescence convert microscope. At the same time, the ectopic osteogenesis of the TEB was tested by HE staining, Results Shortly after being labeled with CM-DiI, ringlike red fluorescence was present in the cytoplasm of BMSCs. 48 hours later, the fluorescence was enhanced and was not weaken during 72 hours. The morphology of labeled BMSCs was consistent with unlabeled ones. There was no significant difference in the proliferative ability between the labeled and unlabeled groups. The expression of Col- I , BMP-2, BGLAP, SPARC revealed that the CM- DiI labeled BMSCs were induced into osteoblasts successfully either. After seeding into β-TCP, the proliferation of labeledBMSCs was normal and abundant extracellular matrix （ECM） was observed in the pores of the ceramic by scanning electron microscope （SEM）. After harvesting the composite of labeled cells and ceramic 8 weeks later, the cells with red fluorescence were observed by fluorescent microscope. HE staining revealed deposited osteoid in the pores of ceramic. Conclusion CMDiI has no obvious impact on the growth, proliferation and osteogenic differentiation of BMSCs. The BMSCs in the composite of TEB could be traced as long as 8 weeks. It suggests a feasible method in monitoring of cells in vivo.