以CM-DiI作为骨髓干细胞移植示踪剂的可行性

背景：供体细胞的示踪始终是干细胞移植研究中面临的一个重要问题，以往用于干细胞移植的示踪技术主要有核素、5-溴脱氧尿嘧啶核苷、绿色荧光蛋白、Hoechst和荧光原位杂交等。目的：探讨CM-DiI作为骨髓干细胞移植示踪剂的可行性和示踪效果。设计、时间及地点：细胞学体内观察，于2004-10/2007-12在中山大学附属第一医院神经科实验室完成。材料：四五周龄雄性DL小鼠80只为供体，2月龄遗传性肝功能障碍模型TX小鼠40只为受体，均由澳大利亚Dean教授馈赠。CM-DiI为Molecular Probes公司产品。方法：全骨髓法体外分离培养供体鼠骨髓干细胞，加入CM-DiI行荧光标记，无菌条件下经尾静脉注入已放疗的受体鼠体内，注射悬液量为0.2 mL/只，细胞数1.2×107个/只，于移植后不同时间取肝脏标本制备切片。主要观察指标：荧光倒置显微镜下观察CM-DiI标记细胞情况，流式细胞仪测定荧光标记率。供体细胞移植后在受体鼠肝脏的示踪。PCR半定量分析供体细胞在受体鼠肝脏中的比例。结果：荧光显微镜下CM-DiI标记的骨髓干细胞呈红色荧光，CM-DiI标记率为99.9%。在受体鼠肝脏组织内，CM-DiI的荧光在移植后第1天即可看到，渐增多，第4周达高峰，后渐下降。移植后第7天可检测到Sry基因，随后其变化趋势同CM-DiI荧光。结论：CM-DiI是良好的骨髓干细胞移植示踪剂，第4周时示踪效果最为显著。

Feasibility of CM-DiI as a tracer in bone marrow stem cell transplantation

BACKGROUND: Tracing of donor cells is always a key problem of stem cell transplantation. The tracer technique of stem cell transplantation includes nuclide, 5-Bromo-21-deoxyuridine (BrdU), green fluorescent protein, Hoechst and fluorescence in situ hybridization. OBJECTIVE: To investigate the feasibility and tracing effect of CM-DiI as a tracing marker in bone marrow stem cell transplantation.DESIGN, TIME AND SETTING: The cytology in vitro study was performed at the Laboratory, Department of Neurology, First Affiliated Hospital, Sun Yat-sen University from October 2004 to December 2007. MATERIALS: A total of 80 male DL mice aged 4-5 weeks served as donors, and 40 TX mice of hereditary liver dysfunction aged 2 months served as recipients. All mice were presented by Professor Dean from Australia. CM-DiI (Molecular Probes) was used in this study. METHODS: Bone marrow stem cells (BMSCs) were harvested from donor mice by the whole bone marrow method in vitro, labeled by CM-DiI, and then sterilely injected into recipient mice via the caudal vein, 0.2 mL per mouse, 1.2×107 cells per mouse. Liver samples were selected to prepare into sections at various time points after transplantation. MAIN OUTCOME MEASUERS: CM-DiI-labeled cells were observed under the fluorescence inverted microscope. Fluorescence labeling rate was detected by flow cytometry. Tracing in the liver of recipient mice was measured after transplantation. Proportion of donor cells in the liver of recipient mice was quantified by semiquantitative analysis of PCR. RESULTS: The label rating of CM-DiI-labeled BMSCs (red fluorescence) was 99.9%. At day 1 after transplantation, the fluorescence of CM-DiI could be found in the recipient liver, and increased with time to the peak at the week 4, then decreased gradually. Sry gene could be detected at day 7 following transplantation, and the change tendency was identical to CM-DiI fluorescence. CONCLUSION: CM-DiI is an excellent tracer in BMSC transplantation. The tracing efficiency is the most significant at week 4.