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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 explore the feasibility of long-term tracing of bone marrow mesenchymal stem cells by CM-DiI during osteogenesis of tissue engineered bone to provide a marker for the in vivo study of seed cells. Methods Beagle BMSCs were isolated and induced into osteoblasts for the second generation. The cells were labeled with fluorescent dye CM-DiI. Fluorescence inverted microscope was used to observe the morphology of the labeled cells. MTT assay was used to detect the proliferation of BMSCs before and after labeling. The expression of Col-Ⅰ, Bone morphogenetic protein (BMP-2), osteocalcin (BGLAP) And osteonectin (SPARC) expression. Finally, CM-DiI fluorescent-labeled BMSCs were co-cultured with β-TCP and then implanted subcutaneously in the back of beagle dogs. After 8 weeks, the BMSCs were observed under a fluorescence microscope and the ectopic osteogenesis was observed by HE staining. Results The CM-DiI-labeled cells showed red fluorescence in early stage, and the fluorescence increased after 48 hours, but the fluorescence intensity did not decrease in 72 hours. The morphology of BMSCs before and after CM-DiI labeling was similar. The proliferation rate of the two groups showed no significant difference. After labeling, the expression of Col-Ⅰ, BMP-2, BGLAP and SPARC was detected by RT-PCR. Osteogenic differentiation. After the labeled cells were detected by scanning electron microscopy, the cells proliferated well on β-TCP and the cell matrix secreted abundantly. Cell-scaffold complexes were implanted subcutaneously in beagle dogs. After 8 weeks, the labeled cells were still observed by fluorescence microscopy. Hematoxylin-eosin staining showed the deposition of bone-like matrix in the scaffold pores. Conclusion CM-DiI has no significant effect on the proliferation, osteogenic differentiation of BMSCs. The tracing time of BMSCs in tissue engineering bone can be as long as 8 weeks and can be used as a long-term tracer in vivo.