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目的用5-溴脱氧尿嘧啶核苷(BrdU)标记家兔皮肤成纤维细胞,确定最佳标记方法,探讨其作为成纤维细胞示踪方法的可行性。方法取12～16周龄健康中国大耳白兔颈部皮肤,胶原酶消化,分离并培养成纤维细胞。取第3代细胞,加入终浓度分别为2.5、5、10和15μg/ml的BrdU共培养,MTT法检测细胞的生长情况。分别用2.5、5、10和15μg/ml的BrdU标记第3代细胞,标记6、12、24和48h后,采用免疫荧光法检测各组细胞的标记阳性率。以最佳剂量及时间标记家兔成纤维细胞,与壳聚糖-甘油磷酸钠水凝胶混合后,注入兔颈内动脉,1周后,免疫荧光法观察植入细胞。结果加入BrdU48h内,各浓度的BrdU对家兔成纤维细胞生长的影响均不明显。标记剂量及标记时间均可影响标记率,但标记时间的影响更大。5μg/ml剂量标记24h,标记率可达(94.50±2.08)%,再加大标记剂量或延长标记时间,标记率提高不明显。以该方法标记的家兔成纤维细胞注入家兔颈内动脉1周后,可在兔颈内动脉观察到大量标记细胞。结论BrdU对家兔成纤维细胞的最佳标记方法为5μg/ml标记24h,该方法对细胞影响小,标记率高,适用于成纤维细胞体内示踪。
Objective To label rabbit skin fibroblasts with BrdU and determine the best labeling method to explore its feasibility as a fibroblast tracer method. Methods The neck skin of healthy Chinese white rabbits 12 to 16 weeks old were digested with collagenase and fibroblasts were isolated and cultured. The third generation of cells were taken and cultured with BrdU at the final concentration of 2.5, 5, 10 and 15 μg / ml respectively. The growth of the cells was detected by MTT assay. The 3rd generation cells were labeled with BrdU at 2.5, 5, 10 and 15μg / ml, respectively. After labeled for 6, 12, 24 and 48h, the positive rate of each group was detected by immunofluorescence. Rabbit fibroblasts were labeled with the best dose and time, mixed with chitosan-glycerophosphate hydrogel, then injected into the rabbit’s internal carotid artery. One week later, the cells were observed by immunofluorescence. Results BrdU 48h, the concentration of BrdU on the growth of rabbit fibroblasts were not obvious. The labeling dose and labeling time can affect the labeling rate, but the labeling time has a greater impact. 5μg / ml dose labeling 24h, labeling rate up to (94.50 ± 2.08)%, then increase the labeled dose or extend the labeling time, labeling rate increased not obvious. After rabbit fibroblasts labeled with this method were injected into rabbit internal carotid artery for 1 week, a large number of labeled cells could be observed in rabbit internal carotid artery. Conclusion The optimal labeling method of BrdU for rabbit fibroblasts is 5μg / ml for 24h, which has little effect on the cells and high labeling rate. It is suitable for in vivo tracing of fibroblasts.