BrdU标记家兔成纤维细胞的体外及体内实验观察

In Vitro and In Vivo Tests on BrdU-Labeled Rabbit Fibroblasts

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 optimize the method for labeling of rabbit fibroblasts with BrdU and explore the feasibility of BrdU as a tracer of rabbit fibroblasts. Methods Fibroblasts were isolated from the skin on necks of healthy rabbits aged 12 ～ 16 weeks by digestion with collagenase and cultured in vitro. The fibroblasts of passage 3 were co-cultured with BrdU at final concentrations of 2.5,5,10 and 15 μg / ml respectively,and the growth of fibroblasts were determined by MTT method. The fibroblasts of passage 3 were labeled with 2.5,5,10 and 15 μg / ml respectively,and the labeled rate was determined by IFA 6,12,24 and 48 h later. The rabbit fibroblasts were labeled with BrdU at optimal concentration for optimal time,then mixed with chitosan-sodium glycerophosphate hydrogel,injected into the carotid artery of rabbits and observed by IFA 1 week later. Results The BrdU at various concentrations showed no significant effect on the growth of rabbit fibroblasts within 48 h after labeling. Though both BrdU concentration and time for labeling affected the labeling rate,the time for labeling showed more significant effect. The labeling rate of fibroblasts with 5 μg / ml BrdU for 24 h reached （94. 50 ± 2. 08）%,while showed no significant decrease with increasing BrdU concentration and time for la-beling. The labeled fibroblasts at a large quantity were observed in carotid artery of rabbits 1 week after injection. Conclusion The optimal BrdU concentration and time for labeling of rabbit fibroblasts were 5 μg / ml and 24 h respectively. The method showed a high labeling rate but little effect on fibroblasts and was suitable for in vivo tracing of fibroblasts.