BrdU标记家兔脂肪组织来源干细胞的体外研究

IN VITRO BROMODEOXYURIDINE LABELLING OF RABBIT ADIPOSE-DERIVED STROMAL STEM CELLS

目的通过采用BrdU标记连续培养的家兔脂肪组织来源干细胞(adipose-derived stromal stem cells,ADSCs),检测其最佳标记时间、标记剂量及毒性作用,探讨其作为干细胞标记示踪方法的可行性。方法8～12周龄健康新西兰大白兔6只,雌雄不限,体重1.5～2.0kg。切取腹股沟皮下1～2mL脂肪组织,采用贴壁法体外分离培养ADSCs,并进行鉴定。取第3代细胞以终浓度分别为5、10、15和20μg/mL的BrdU进行标记,分别记为A、B、C及D组;另1孔不含BrdU,作为空白对照(E组)。标记12、24、48和72h后,采用免疫组织化学检测各组细胞阳性率,苔盼蓝排染法行细胞计数观察标记后细胞活性。结果原代培养的ADSCs形态主要为宽大、扁平、短梭形细胞;传代后细胞形态主要为长梭形;第3代ADSCs经诱导均能向成骨细胞和脂肪细胞分化。免疫组织化学观察:第3代ADSCs经BrdU标记后,在荧光显微镜下胞核呈绿色荧光。孵育12h,A、B、C、D组细胞标记阳性率逐渐增高,分别为30.6%±2.3%、32.4%±1.9%、45.8%±1.8%、50.8%±3.1%,C、D组与A组比较,差异有统计学意义(P<0.01);24h,A、B、C及D组标记率分别为45.9%±2.0%、87.9%±3.3%、90.6%±2.9%及91.7%±3.2%;48h和72h,A、B、C、D组标记情况与24h相似;E组各时间点细胞标记阳性率为0。孵育24、48及72h,B、C及D组细胞阳性率与A组比较,差异均有统计学意义(P<0.01)。孵育12、24、48及72h细胞计数,各组细胞活性均在90%以上,A、B、C、D组与E组比较,差异无统计学意义(P>0.05)。结论BrdU标记ADSCs的最佳时间为48h,最佳浓度为10μg/mL;BrdU对细胞标记率及安全性高。

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 μg/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 adi pocytes 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 h ours 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）. Condusion The most appropriate time for BrdU labelling ADSCs is 48 hours, the most appropriate concentration is 10 μg/mL. The labelling rate is high and cytotoxicity is little.