不同浓度5-溴脱氧尿嘧啶核苷标记山羊骨髓基质干细胞的体外研究

In vitro bromodeoxyuridine-labeled goat bone marrow mesenchymal stem cells

目的利用5-溴脱氧尿嘧啶核苷（BrdU）标记山羊骨髓基质干细胞（BMSCs），检测其最佳标记浓度、时间及细胞毒性，探讨其作为山羊BMSCs标记及示踪方法的可行性。方法抽取10个月龄健康中国青山羊骨髓，贴壁培养并鉴定。以浓度分别为5、10、15和20μmol／L的BrdU标记第4代细胞，分别记为A、B、C、D组；未用BrdU标记的细胞作为空白对照组（E组）。分别标记12、24、48和72h后，免疫荧光法检测各组标记阳性率，锥虫蓝拒染法检测标记后细胞存活率。结果原代及传代培养的山羊BMSCs形态主要为梭形，经诱导后能向成骨细胞和软骨细胞分化。标记后，荧光显微镜下胞核呈绿色荧光。随着标记时间和浓度的增加，各实验组标记阳性率逐渐增高，于15μmlol／L孵育48h后，其标记率可达到（93．32±3．25）％，与15μmol／L孵育72h和20μmol／L孵育48、72h差异无统计学意义（P〉0．05），与其他各组各时间点差异有统计学意义（P〈0．05）；各时间点空白对照组标记阳性率均为0。锥虫蓝拒染实验示各组细胞存活率均在90％以上，差异无统计学意义（P〉0．05）。结论BrdU浓度为15μmol／L，标记时间为48h时，对山羊BMSCs可得到最佳的标记效果，且安全性较高。

Objective To explore the feasibility of labeling and tracing in vitro goat bone marrow mesenchymal stein cells （BMSCs） by bromodeoxyuridine （BrdU） on the basis of investigation of its optimal concentration, incubating time and cytotoxieity. Methods A healthy goat, aged 10 months old, male, weighing 32 kg, was used in this study. Bone marrow was aspirated. BMSCs were isolated and cultured using the adherence method in vitro. The fourth passage of BMSCs （P4） were incubated with BrdU at 5, 10, 15, 20 μmol/L as 5, 10, 15, 20 μmol/L BMSC groups. Cells were not labeled by BrdU as negative control. The following parameters were measured： induction, differentiation and determination of goat BMSCs; the optimal mass concentration and incubation time of 5-BrdU labeling; cell positive rate at 12, 24, 48 and 72 hours in each group using immunofluorescence; the cell survival rate after various concentrations of BrdU labeling by trypan-blue exclusion. Results The morphology of the primary and passage goat BMSCs was fusiform in shape. Goat BMSCs could differentiate into osteoblasts and ehondrocytes following induction. BMSC nucleus showed green fluorescence under fluorescence microscope after being labeled by BrdU. The mean labeling rate increased with the increase in the concentration and incubation time of BrdU, and reached to （93.32± 3.25）% after incubation in 15 μmol/L BrdU for 48 hours. There were no significant differences between 15 μmol/L BrdU for 72 hours, 20 μmol/L BrdU for 48 hours and 72 hours （ P 〉 0.05）, or between the other groups or time points （ P 〈 0.05）. The labeling rate of the blank control group was 0. The cell survival rate was all above 90% （ P 〉 0.05）. Conclusions BrdU can be used as a labeling marker for goat BMSCs. When the concentration is 15 μmol/L and the incubation time is 48 hours, the optimal labeling effect can be achieved. Goat BMSCs labeled with BrdU is of high efficiency and safety.