放射性碘标记转铁蛋白体外示踪脊髓内移植的间充质干细胞

Transferrin receptor expression of human mesenchymal stem cells and in vitro tracking by autoradiography after transplantation in spinal cord

目的 :检测间充质干细胞转铁蛋白受体 (TfR)在体外培养及移植入脊髓后的表达 ,进行体外放射性核素示踪研究。方法 :从胎儿血分离培养人间充质干细胞 ,应用流式细胞分析、免疫荧光染色和受体放射性分析鉴定转铁蛋白受体的表达 ,用放射性碘 (12 5I)标记的铁饱和转铁蛋白 [12 5I Tf(Fe) 2 ]作为示踪剂 ,进行体外放射自显影 ,示踪移植于兔正常脊髓内的间充质干细胞 ,并对12 5I Tf(Fe) 2 在脊髓实质的弥散动力学进行研究。结果 :流式细胞分析、免疫荧光染色证实体外培养的人间充质干细胞表达转铁蛋白受体 ,受体放射性分析表明12 5I Tf(Fe) 2 与其受体结合的平衡解离常数 (KD)为 (0 .98± 0 .12 )nmol/L ,最大结合位点 (Bmax)为每个细胞 (10 770 2± 6 2 2 6 )个。免疫荧光染色结果表明间充质干细胞移植于脊髓 2d后转铁蛋白受体表达 ,10d后不表达 ,移植 2d后以12 5I Tf(Fe) 2 为示踪剂进行脊髓切片体外放射自显影 ,获得移植细胞阳性影像。离体脊髓在12 5I Tf(Fe) 2 溶液中体外孵育 ,16h后12 5I Tf(Fe) 2 弥散入全部脊髓实质。结论 :12 5I Tf(Fe) 2 作为示踪剂可以在体外示踪到脊髓内移植 2d后的间充质干细胞 ,TfR和12 5I Tf(Fe) 2 是应用核医学影像进行间充质干细胞移植初期活体示踪可能的生物学标志和示踪剂?

Objective: To determine transferrin receptor (TfR) expression of human menschymal stem cells (MSCs) in vitro and after transplantion in rabbit spinal cord,and to detect implanted MSCs by in vitro autoradiography. Methods: Human mesenchymal stem cells (hMSCs) were isolated from fetal blood. Flow cytometry assay, immuno-fluorescent staining and receptor binding assay were used to determine TfR expression of hMSCs. Radioiodinated transferrin saturated with iron [ 125 I-Tf(Fe) 2] was used as tracer. The hMSCs transplanted in rabbit spinal cord was tracked by in vitro autoradiography. Diffusion of 125 I-Tf(Fe) 2 in spinal cord was examined with autoradiography. Results: TfR expression of MSCs was demonstrated by flow cytometry assay, immuno-fluorescent staining and receptor binding assay in vitro. 125 I-Tf(Fe) 2 bound to hMSCs with a equilibrium dissociation constant (KD) of (0.98±0.12) nmol/L and a maximal density of binding sites (B max ) of (107 702±6 226) sites per cell. Immuno-fluorescent staining showed that TfRs were expressed on hMSCs on the 2nd day but not be expressed on the 10th day post transplantation. Autoradiography showed distinct accumulation of 125 I-Tf(Fe) 2 but not 125 I-HSA at hMSCs implantation sites of spinal cord sections on the 2nd day post transplantation. 125 I-Tf(Fe) 2 had diffused into spinal cord 16 hours after incubation. Conclusion: Implanted hMSCs could be detected by in vitro autoradiography with 125 I-Tf(Fe) 2 on the 2nd day after being transplanted in spinal cord. To track implanted hMSCs with radionuclide imaging techniques in vivo, TfR was a suitable target for imaging and radioiodinated Tf(Fe) 2 was a feasible tracer.