5-溴脱氧尿嘧啶核苷体外标记肌源性干细胞:最佳浓度及时间

Bromodeoxyuridine labeling of muscle-derived stem cells in vitro:Optimal concentration and time

背景:肌源性干细胞移植入动物体内可以修复骨、肌肉、软骨或神经系统的损伤,但要了解其在体内的迁移、增殖及分化的情况,还需要一种有效、安全的标记方法来追踪其在体内的变化。目的:观察5-溴脱氧尿嘧啶核苷标记肌源性干细胞的效果,探讨其最佳标记浓度和时间。方法:采用差速贴壁法体外分离SD大鼠肌源性干细胞,免疫细胞化学染色法鉴定第2代细胞并进行细胞计数。以终浓度5,10,15,20μmol/L的5-溴脱氧尿嘧啶核苷标记肌源性干细胞,检测其最佳标记浓度,免疫细胞化学染色法分析其标记率;通过MTT法、锥虫蓝排斥实验观察5-溴脱氧尿嘧啶核苷对细胞增殖的影响。结果与结论:免疫细胞化学染色显示肌源性干细胞呈Desmin阳性,阳性率为(85.7±6.0)%;经5-溴脱氧尿嘧啶核苷标记的肌源性干细胞与未进行标记细胞在活力和增殖上相比未见明显差异(P>0.05),标记后的肌源性干细胞活细胞率可达(92.3±2.1)%。其中浓度为10,15,20μmol/L标记组标记效果均优于5μmol/L标记组,10μmol/L为最佳工作浓度,最佳标记时间为3d,标记率为(70.9±4.5)%,随时间延长无明显变化。证实应用5-溴脱氧尿嘧啶核苷标记肌源性干细胞后,对细胞的活力及增殖情况无影响,标记率也能达到细胞移植对其示踪的要求。

BACKGROUND：The muscle-derived stem cells transplanted into the body of the animals can repair the defects of bone,muscle,cartilage and neural system.An efficient and safe labeling method is needed to understand the migration,proliferation and differentiation of the cells.OBJECTIVE：To observe the effect of 5-bromodeoxyuridine（Brdu） labeling of muscle-derived stem cells in vitro,to study the optimal dosage and timing.METHODS：Muscle-derived stem cells from Sprague Dawley rats were isolated in vitro using differential adhesion method.The second passage of cells was counted and identified by immunocytochemical staining.The cells were labeled by BrdU at 5,10,15,20 μmol/L to identify the optimal BrdU concentration.Immunocytochemistry was performed to calculate the labeling index.By MTT testing and trypan blue testing,we surveyed the influence of BrdU on the growth of muscle-derived stem cells.RESULTS AND CONCLUSION：Immunocytochemical staining results showed positive expression of Desmin in MDSCs with the percentage of（85.7±6.0）%.The viability and proliferation between the group labeled by BrdU and the control group were similar（P 0.05）.The rate of the living cells labeled by BrdU was（92.3±2.1）%.The labeling rate was better in the 10,15,20 μmol/L groups compared with 5 μmol/L group.The optimal dosage was 10 μmol/L,and the optimal time was 3 days,and the labeling index was（70.9±4.5）% with no significant change over time.It was proved that after labeled by BrdU,no difference in the viability and proliferation was found in muscle-derived stem cells,and the labeling index can come up to the required standards of the transplantation.