骨髓内皮祖细胞输注后在肝纤维化大鼠肝脏内的示踪研究

Intrahepatic tracking of bone marrow-derived endothelial progenitor cells after transplanting into rats with liver fibrosis

目的建立PKH26荧光标记肝纤维化大鼠骨髓内皮祖细胞（EPC）的方法，观察PKH26标记的EPC输注后在肝纤维化大鼠肝内的迁移和分化情况。方法分离和培养肝纤维化大鼠骨髓源性EPC，并在体外进行PKH26荧光标记，通过荧光共聚焦显微镜观察和采用流式细胞仪检测PKH26的标记率，并观察荧光标记对细胞生长状态的影响；将PKH26荧光标记的EPC经尾静脉输注入肝纤维化大鼠体内，观察其在大鼠肝内的迁移示踪情况，并用免疫荧光法检测内皮细胞标志物CD31和血管性假血友病因子（vWF）的表达。结果PKH26标记的细胞呈红色荧光，标记率为96．65％；PKH26标记的EPC生长状态良好，与未标记的EPC相比，生长曲线基本一致；EPC迁移入肝后主要存在于沿纤维分布的血管内皮和肝小叶内的窦内皮，并表达内皮细胞特异性抗原CD31和vWF。结论PKH26可在体外成功标记肝纤维化大鼠骨髓源性EPC，该细胞输注入肝纤维化大鼠体内后，可迁移至肝内血管周围，并向成熟的内皮细胞分化。

Objective To establish a method of PKH26-1abeled bone marrow-derived endothelial progenitor cells （EPCs） into rats with liver fibrosis and observe cell immigration and differentiation in the liver after transplantation. Methods Bone marrow-derived EPCs were isolated and cultured from rats with liver fibrosis, and then labeled with PKH26 in vitro. Under the scanning confocal microscopy and flow cytometry, PKH26 fluorescent labeling rate and ceil survival rate were measured. EPCs of PKH26 fluorescent labeling were transplanted into rats with liver fibrosis via the tail vein, and the migration situation was observed in the liver. Endothelial cell markers CD3I and yon willebrand factor （vWF） were detected by using immunofluorescence. Results The PKH26-1abeled EPCs appeared red fluorescence and the labeling rate was 96. 65%. As compared with unlabeled cells, the labeled cells grew well, and had no significant changes in the growth curve. After transplantation into the liver of rats, the PKH26-1abeled ceils were mainly distributed in blood vessel endothelium along fibers and hepatic sinusoids in hepatic lobule. Endothelial cell-specific antigens such as CD31 and vWF could be detected along the vascular walls. Conclusion PKH26 could be used to label and track EPCs in the liver of rats with liver fibrosis in vitro. The PKH26-1abeled cells may migrate to the surrounding of the hepatic vessels and differentiate into mature endothelial cells.