[兔脂肪来源间充质干细胞的体外溴脱氧尿苷标记]
[In vitro bromodeoxyuridine labelling of rabbit adipose-derived stromal stem cells].
作者信息
Li Hongmian, Gao Jianhua, Lu Feng, Li Hua
机构信息
Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong, 510515, P.R. China.
出版信息
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2008 Jan;22(1):97-101.
OBJECTIVE
To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibility for stem cells labelling and tracer means.
METHODS
Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro. The 3rd passage of ADSCs was incubated withBrdU at 5, 10, 15 and 20 microg/mL (groups A, B, C and D) for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labelling. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labelling index (positive rate) and the cells' activity for different time after BrdU labelling. The ADSCs without BrdU labelling were used as control (Group E).
RESULTS
The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblasts and adipocytes under corresponding inductive medium. The ADSCs' nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labelling ratio increased in groups A, B, C and D after incubating 12 hours, the mean labelling ratio were 30.6% +/- 2.3%, 32.4% +/- 1.9%, 45.8% +/- 1.8%, 50.8% +/- 3.1%, respectively, and the labelling ratio of Group E was 0. There were significant differences between groups C, D and Group A (P < 0.01). The labelling ratio of groups A, B, C and D were 45.9% +/- 2.0%, 87.9% +/- 3.3%, 90.6% +/- 2.9%, 91.7%+/- 3.2%, respectively after 24 hours and the labelling ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P < 0.01). The results of all groups after incubating 48 hours and 72 hours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences (P > 0.05).
CONCLUSION
The most appropriate time for BrdU labelling ADSCs is 48 hours, the most appropriate concentration is 10 microg/mL. The labelling rate is high and cytotoxicity is little.
目的
探讨5-溴脱氧尿嘧啶核苷（BrdU）对兔脂肪来源基质干细胞（ADSCs）体外标记的最佳剂量、时间及细胞毒性，以确定其用于干细胞标记及示踪方法的可行性。
方法
本实验选用6只8-12周龄、体重1.5-2.0 kg、雌雄不限的家兔。取1-2 mL脂肪，采用贴壁法体外分离培养ADSCs。将第3代ADSCs分别用5、10、15和20 μg/mL的BrdU孵育12、24、48和72小时（A、B、C和D组），以确定细胞标记的最佳BrdU浓度和孵育时间。分别进行免疫组织化学和台盼蓝染色，计算BrdU标记后不同时间的标记指数（阳性率）和细胞活性。未用BrdU标记的ADSCs作为对照（E组）。
结果
原代ADSCs主要呈短梭形，第3代ADSCs呈长梭形。第3代ADSCs在相应诱导培养基下可分化为成骨细胞和脂肪细胞。BrdU标记后，荧光显微镜下ADSCs细胞核呈绿色荧光。孵育12小时后，A、B、C和D组的标记率升高，平均标记率分别为30.6%±2.3%、32.4%±1.9%、45.8%±1.8%、50.8%±3.1%，E组标记率为0。C、D组与A组之间差异有统计学意义（P<0.01）。孵育24小时后，A、B、C和D组的标记率分别为45.9%±2.0%、87.9%±3.3%、90.6%±2.9%、91.7%±3.2%，E组标记率为0。B、C、D组与A组之间差异有统计学意义（P<0.01）。孵育48小时和72小时后所有组的结果与孵育24小时后相似。A、B、C和D组的细胞计数优于E组，但差异无统计学意义（P>0.05）。
结论
BrdU标记ADSCs的最合适时间为48小时，最合适浓度为10 μg/mL。标记率高且细胞毒性小。
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