BrdU体外标记大鼠骨髓间充质干细胞的研究

In vitro bromodeoxyuridine labeling of rat bone marrow-derived mesenchymal stem cells

目的探讨连续培养的骨髓间充质干细胞(MSCs)的5-溴脱氧尿嘧啶核苷(BrdU)最佳标记时间、剂量。方法差速贴壁法对SD鼠MSCs进行体外传代培养;第6代细胞行流式细胞仪鉴定细胞的表面抗原;以终浓度分别为5、10、15μmol/L的BrdU检测最佳标记剂量;在细胞生长融合前72、48、36、24、12、3h加入BrdU,使终浓度为10μmol/L,并分别孵育至细胞融合,测定BrdU的标记率,找出最佳标记时间;免疫组化分析BrdU标记率。结果流式细胞仪检测所标记的细胞表达CD29和CD44,不表达CD11b和CD45;终浓度为10μmol/L和孵育48hBrdU标记率均>98%,并且连续传5代均可检测到。结论传代后贴壁生长的梭形细胞为MSCs,终浓度为10μmol/L和孵育48hBrdU标记率均>98%,且可以连续检测到,提示BrdU标记可用于MSCs移植入体内后存活、生长和分化的动态观察。

Objective To study the optimal dosage and timing for bromodeoxyuridine (BrdU) labeling of rat bone marrow-derived mesenchymal stem cells (MSCs) in vitro. Methods Bone marrow-derived MSCs of SD rats were cultured in vitro routinely and the sixth passage was taken for identification of specific surface antigens by flow cytometry. Before reaching cell confluence, the purified MSCs were incubated with BrdU at different concentrations (5, 10, and 15 μmol/L) for different incubating time (3, 12, 24, 36, 48, and 72 h) with 10 μmol/L BrdU, to identify the optimal BrdU concentration and incubating time for cell labeling. Immunohistochemistry was performed to calculate the labeling index (LI). Result Flow cytometry showed that MSCs expressed CD29 and CD44 but not CD11b or CD45. Incubation of the MSCs with BrdU at 10 μmol/L and for an optimal length of 48 h appeared to achieve the highest LI, both of which exceeded 98%, with the labeling identifiable in five consecutive passages. Conclusions The continually passaged cells are MSCs, the incubation of which with 10 μmol/L BrdU for 48 h may achieve a LI over 98% without producing obvious cell damages. The results suggest that BrdU labeling provides a feasible means for a dynamic in vivo observation of the survival, growth and differentiation of the implanted MSCs.