PKH26标记法在组织工程神经种子细胞体内示踪中的应用

目的 探讨PKH26标记骨髓间充质干细胞(BMSCs)的效果及其应用于组织工程神经种子细胞体内示踪实验的可行性.方法 取Wistar大鼠股骨和胫骨的骨髓,用全骨髓贴壁的方法分离、培养BMSCs.用PKH26荧光染料进行标记,荧光显微镜下观察标记效果,流式细胞仪检测荧光标记率,MTT法测定细胞的活性,体外成脂和成骨诱导鉴定细胞的分化能力,并与未标记细胞进行比较;使用显微注射的方法将BMSCs植入去细胞神经支架内制备成组织工程神经,于体外培养3 d、5 d、7 d、14 d,行组织切片,观察BMSCs在支架内存活、迁移的情况.另外,将体外构建组织工程神经桥接Wistar大鼠坐骨神经15 mm的缺损,于术后1周、4周、6周、8周取出移植物,行组织切片观察植入的BMSCs在支架内的存活情况.结果 PKH26能有效地标记BMSCs,标记率达95%以上,标记后细胞活性及诱导成骨成脂分化能力与未标记细胞无明显差异.在体外培养的环境下,BMSCs能在去细胞神经支架里粘附生长,并沿着支架迁移;在体内外周围神经再生的微环境下,BMSCs可存活8周以上.结论 PKH26荧光染料标记法是一种有效的标记BMSCs的方法,可用于组织工程神经种子细胞体内示踪的研究.

PKH26 dye for tracing the seed cells of tissue-engineered peripheral nerve in vivo

Objective To assess the validity of PKH26 dye for labeling the bone marrow mesenchymal stem cells (BMSCs) and the feasibility of this method for tracing the seed cells of tissue engineering nerve in vivo. Methods BMSCs isolated from Wistar rat's bone marrow were labeled with the PKH26 dye, the marked validity was observed by the fluorescence microscopy and flow cytometer, the cellular vitality was de-tected by MTT, the differentiated abilities were tested by in vitro osteablastic and lipoblastic differentiation. After that, the labelld cells were micro-injected into the acellular nerve grafts(ANG) to build compound grafts and cultured for 3,5,7,14 days in vitro, then frozen sections were made respectively to observe the survival and migration of the implanted cells in ANG. Simultaneously, these compound grafts were used to bridge the 1.5 cm sciatic nerve defects of the Wistar rats, 1,4,6,8 weeks postoperation, the grafts were taken out and frozen section were made in order to observe the survival of the implanted stem cells in vivo inside the ANG. Results PKH26 dye was good for labelling the BMSCs, marked validity was higher than 95%. The cellular vitality and differentiated abilities were not affected compared with the unlabelled cells. Further more, BMSCs could adhere to the ANG, and suvive,magrate in vitro. In vivo, BMSCs was able to survive at least 8 weeks in the nerve regenerative microenvironment. Conclusion PKH26 dye could label the BMSCs effectively, and it's suitable for in vivo seed cells tracing in tissue engineering nerve model.