5-BrdU标记的骨髓间充质干细胞对大鼠创面愈合的作用研究

Experimental Study on the Effect of Rat Wound Healing with 5-BrdU Labled BMSCs

目的探讨以5-溴脱氧尿嘧啶(5-BrdU)标记的骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)在大鼠创面愈合中的作用。方法分离培养大鼠BMSCs并用流式细胞仪技术鉴定。在大鼠背部脊柱一侧制作全层皮肤缺损模型,实验组经尾静脉注入经5-BrdU标记的BMSCs,移植细胞数为2×107。对照组为5-BrdU标记的BMSC移植的正常大鼠。术后3d、7d、14d、21d、28d分批处死动物,观察移植细胞参与创面愈合的情况。结果流式细胞仪鉴定细胞表达CD29、CD90;CD45表达呈阴性。实验组:经5-BrdU体外标记的BMSCs进行细胞移植14d后,大鼠创面局部及边缘可见5-BrdU标记的阳性细胞聚集,免疫组化染色可见基底层、真皮层、新生血管周围有散在红棕色标记细胞;对照组中未发现明显的聚集现象;抗BrdU/FⅧ、BrdU/波形蛋白免疫双标技术检测结果显示:在创伤部位有部分BMSCs分化为血管内皮细胞和成纤维细胞参与创面愈合。大鼠正常皮肤中未见明显的BMSCs聚集和增殖分化情况。结论经尾静脉注射移植的BMSCs参与皮肤创面愈合。

Objective To explore the contribution of rat bone marrow - derived cells to skin wound repair, and provide a new thought for mechanism study of wound healing and clinical treatment in the future. Methods Bone marrow-derived BMSCs of SD rats were isolated and cultured in vitro routinely and the fifth passage as taken for identification of specific surface antigens by flowcytometry, then were labled with 5-BrdU in vitro. Full thickness dermal wounds were established on the back of rats＇ skin. 5-BrdU labled BMSCs were implanted through tall vein to model rats. At 3, 7, 14, 21 and 28 days after BMSCs transplantation, immunohistochemical examinations were used to investigate BMSCs aggregate around the wound site and identify the contribution of bone marrow - derived cells to skin wound repair in different time during wound healing. Results Flow cytometry showed that BMSCs expressed CD29, and CD90, but no CD45. At 1, 2, 3, 4 weeks after the cells transplantation, the implanted cells were detected in the wound site in the model rats, which is not founded in normal skin of the rats＇. Part of the implanted cells were detected to differentiate into vascular endothelia cells and fibroblast, while there were not found in the normal skin through BrdU/FⅧ,BrdU/Vimentin double-labelling technique of immunofluoreseenee. Conclusion Wound is one mechanism by which these bone marrow derived cells are recruited to contribution of wound healing.