大鼠骨髓间质干细胞的BrdU体外标记和体内示踪研究

In vitro Analysis and in vivo Tracing of BrdU-labeled Rat Bone Marrow Mesenchymal Stem Cells

目的探讨骨髓间质干细胞(BMSCs)的体外标记和体内示踪技术。方法分离培养并鉴定大鼠BMSCs,进行5-溴脱氧尿嘧啶核苷(BrdU)标记后,体外培养被标记的BMSCs,观察其连续传代后的BrdU可标记时间和标记效率;建立大鼠胃黏膜损伤模型,将分离纯化的BrdU标记BMSCs移植到损伤的胃黏膜下,体内观察BrdU对活体移植BMSCs的示踪作用。结果免疫组化显示体外培养大鼠BMSCs的CD44、CD90表达阳性,CD14、CD45表达阴性,显微结构表现出干细胞特征。进行BrdU标记后,标记阳性率随标记时间延长而增高,48h达高峰,72h仍维持高阳性率。终浓度为10μmol/L的BrdU孵育48h、72h阳性标记率高于5μmol/L BrdU组(P<0.05),但与15μmol/L BrdU组阳性标记率差异无统计学意义(P>0.05),并且连续传代培养21d后也可检测到。BrdU可示踪BMSCs在损伤的胃黏膜下局部定植。结论 BrdU在浓度为10μmol/L、标记时间48h至72h标记效率较高;BrdU标记可用于一定时间段内BMSCs移植入体内后定植和生长的动态示踪观察方法。

Objectives To investigate the optimal condition of bromodeoxyuridine（BrdU） labeling for bone marrow mesenchymal stem cells（BMSCs） in vitro and the feasibility of in vivo tracing of BrdU-labeling BMSCs.Methods BMSCs were isolated from Wistar rats and were in vitro routinely cultured.The third passage BMSCs was used for identification of special surface antigens by immunohistochemical methods.The purified BMSCs were incubated with BrdU at different concentrations for different incubating time to investigate optimal BrdU concentration and incubating time for cell labeling.The cell labeling index of BrdU was calculated with immunohistochemical analysis.BMSCs labeled BrdU were injected into damaged gastric mucosa of rats by micro injector.The colonization of BMSCs labeled BrdU in gastric mucosa was viewed.Results After purification and proliferation,the primary cultured BMSCs were uniformly long spindle-shapped form and formed cell colony,which showed the characteristics of stem cell.Immunocytochemistry showed BMSCs were positive for CD44 and CD90,while negative for CD14,CD45.The labeling rate of BrdU increased with the labeling time lasting and reached its height at 48 h.After incubating 48 and 72 hours,the labeling rate of BrdU with a concentration of 10 μmol/L was higher than that of BrdU with a concentration of 5 μmol/L（P0.05） and similar with that of BrdU with a concentration of 15 μmol/L （P0.05）.In addition,the BrdU labeling could be detected after five consecutive passages and the labeling time could keep 21 d.The pathological observation demonstrated that BrdU-labeled BMSCs could colonize the damaged gastric mucosa with normal morphologic characteristics during observation period.Conclusion BrdU labeling might be a feasible method for dynamic observation of the migration,growth and differentiation of migrating BMSCs in colonizing sites.