小鼠异基因造血干细胞移植中清髓照射对供者来源内皮祖细胞归巢的影响

Irradiation-induced Homing of Donor Endothelial Progenitor Cells in Mice Allo-HSCT Model

目的研究异基因造血干细胞移植预处理因素——清髓照射对供者来源内皮祖细胞（EPCs）归巢的影响。方法密度梯度离心和差速贴壁法获取小鼠骨髓单个核细胞，EGM-2培养基培养，5～7d检测CD133^＋CD31^＋CD45^k/w表面标记及吸食Dil-Ac-LDL和结合FITC-UEA-1鉴定，并行羟基荧光素一醋酸盐琥珠酰亚胺酯（CFSE）标记。实验分组：正常组，正常＋EPCs移植组，照射组，照射＋EPCs移植组。流式细胞术（FCM）分析外周血、脾脏和骨髓中内皮细胞（ECs）、EPCs和CFSE阳性细胞比例；病理、电镜和免疫荧光分析各组织的损伤情况及CFSE标记EPCs的分布。结果流式细胞术鉴定内EPCs为CD45^k/wCD133^＋CD31^＋；Dil-ac-LDL^＋、FITC-UEA-1^＋。照射后短时间内循环中ECs即开始升高，第5d达高峰，持续到第7d，与正常组相比差异有统计学意义（P〈0．05）；循环中EPCs照射后短时间内也增加，于第4d达到峰值，随后下降至正常水平，其增加与正常组相比差异有统计学意义（P〈0．05）。照射后3d光镜下肝脏弥漫性的炎症浸润，小肠大量炎症细胞浸润；电镜下肝脏中血小板聚集到内皮下暴露的胶原上，小肠中ECs和基底膜间被损坏。照射＋EPCs移植组，荧光显微镜下观察，移植后3h见CFSE阳性细胞归巢至损伤的肝脏和小肠，细胞少且不连续；36h细胞较多，且呈连续性。结论移植预处理中的清髓照射可引起严重的内皮损伤，该损伤启动了EPCs的动员，并驱动外源性EPCs归巢至损伤比较重的组织中。

Objective To investigate the effect of aIto-HSCT irradiation preconditioning on the homing of donor endothelial progenitor cells （EPCs）. Methods The mononuclear cells were collected form mice bone morrows, then cultured in EGM-2 medium. The cells were identified after 5-7 days and were labeled with CFSE. The mice were divided randomly into four groups： control, EPCs transplantation, irradiation, and irradiation EPCs transplantation. The circulating ECs, circulating EPCs and CFSE positive cells in the peripheral blood, spleen and bone morrows of the mice were measured by FCS. The histological examination, electron microscope and immunofluorescence analyses of those tissues were also performed. The distribution of labeled EPCs was observed. Results The cells were identified as CD45^k/w, CD133^＋ CD31^＋, Dil-ac-LDL^＋. The number of those cells in the irradiation group increased gradually after preconditioning and reached peak at day 3 or 5 and sustained the peak level for 2 days, which was significantly different from the control （P〈0.05）. Under the light microscopy and TEM, damaged endothelium was detected. In the irradiation and EPCs transplantation groups, CFSE labeled cells increased and reached peak at 18 hours, and then decreased till 72 hours. Green fluorescent ceils were observed in different tissues at different times, and the strongest fluorescence intensity occurred at 36 hours. Conclusions The EPCs can be induced from mice bone morrow mononuclear cells, with expressed phenotypic characterization. Irradiation in transplantation preconditioning can cause severe endothelium injury, which can start the mobilization of EPCs. The endothelium injury can induce extrinsic EPCs homing to the tissues which are badly injured.