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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。
Objective To investigate the in vitro labeling and in vivo tracing techniques of bone marrow mesenchymal stem cells (BMSCs). Methods Rat BMSCs were isolated and identified. BrdU labeling was used to culture labeled BMSCs and the BrdU labeling time and labeling efficiency were observed. The gastric mucosa Injury model, the purified BrdU labeled BMSCs were transplanted into the damaged gastric mucosa, and the tracing effect of BrdU on BMSCs transplanted in vivo was observed in vivo. Results Immunohistochemistry showed that the expression of CD44 and CD90 in rat BMSCs was negative, while the expression of CD14 and CD45 was negative. The microstructure showed the characteristics of stem cells. After BrdU labeling, the positive rate of the marker increased with the prolongation of labeling time, reached the peak at 48h, and maintained the high positive rate at 72h. BrdU incubated at a final concentration of 10μmol / L for 48h showed a significantly higher positive rate at 72h than that at 5μmol / L BrdU (P <0.05), but no significant difference was observed between BrdU and BrdU at 15μmol / L (P> 0.05) Subsequent subcultures can also be detected after 21 days. BrdU can be traced to BMSCs locally localized under the damaged gastric mucosa. Conclusion The BrdU labeling efficiency is high at a concentration of 10μmol / L for 48h to 72h. The BrdU labeling method can be used for dynamic tracing of BMSCs after implantation into the body for a certain period of time.