羰花青荧光染料示踪在大鼠胚胎皮肤成纤维细胞移植中的应用

Application of carbocyanines fluorescent dye for tracing fetal fibroblast transplant： experiment with rats

目的探讨羰花青荧光染料CM-Dil示踪大鼠胚胎皮肤成纤维细胞的可能性。方法体外培养孕16d的Wistar大鼠胚胎皮肤成纤维细胞，用10μg/ml的CM-Dil标记，流式细胞仪观察CM-Dil对细胞生长的影响，计算细胞增殖指数（PI）。将标记细胞注射移植到同种异体大鼠背部深Ⅱ度烫伤创面，分别于注射后0、1、3、7、14、28d取创面全层皮肤标本，荧光显微镜观察CM-Dil示踪效果。结果标记后15min，细胞呈现圆形红色荧光，高倍镜下可见双层膜结构。标记与未标记大鼠皮肤胚胎成纤维细胞P1分别为76．9±2．8和75．8±2．0，差异无统计学意义（t=0．80，P〉0．05）。CM-Dil标记细胞异体移植后第1天取材的大鼠创面皮肤标本中荧光信号较少；第3天标本荧光信号明显增多，荧光信号区域呈“烧杯形”分布；第7天标本可见“烧杯形”区域底部变宽，显示细胞增殖；第14天标本仍能检测到中等强度的荧光信号；第28天标本能检测到微弱的荧光信号。结论适宜浓度的CM-Dil对胚胎皮肤成纤维细胞没有毒性。CM-Dil在皮肤组织内淬灭较慢，4周内保持良好的荧光示踪稳定性，是一种比较理想的胚胎皮肤成纤维细胞荧光标记物。

Objective To investigate the feasibility of applying carbocyanines fluorescent dye chloromethyl-benzamidodialkyl carbocyanine （CM-Dil） in tracing fetal fibroblast transplant. Methods Sixteen-day-gestation fetal rat skin tissue samples were harvested and digested with trypsin to isolate fibroblasts. The fibroblasts proliferated in logarithmic phase and were incubated with CM-Dil for 15 minutes. Flow cytometry was used to calculate the proliferate index （PI） so as to determine the effect of CM-Dil on cell proliferation. Labeled fibroblasts were transplanted to the deep partial thickness burn wounds in the back of allogenic rats. Samples were harvested on 0,1,3,7,14, and 28 days post burns （DPB） and observed under fluorescence microscope in 6 μm section. Results The cultured fibroblasts were labeled with CM-Dil within 15 minutes, appearing as red fluorescent discs with bilayer membrane structure. The PI of the labeled fibroblasts was （76.9 ± 2.8 ）, not significant different from that of the cells not labeled （75.8±2.0, t=0.80,P〉0.05）. The area with fluorescence markers was small on 1 DPB, began to expand and be distributed in a beaker shape on 3 DPB, and then the bottom of the ＂beaker＂ broadened indicating cell proliferation significantly on 7 DPB. Fluorescence signals determined were moderate on 14 DPB, and can be still determined feebly on 28 DPB. Conclusion Proper concentration of CM-Dil shows no cytotoxicity to fetal fibroblasts. CM-Dil is an ideal fluorescent label to fetal fibroblasts for its fairly stable fluorescent tracing and slow quenching in skin tissue within 4 week＇s duration.