自体骨髓来源内皮祖细胞移植改善脑缺血再灌注大鼠神经功能转归

Autologous bone marrow-derived endothelial progenitor cell transplantation improves neurological outcomes after cerebral ischemia-reperfusion in rats

目的 探讨目体骨髓米源内皮祖细肥（endothehalprogenitor cell,EPC）移植对脑缺血大鼠神经功能转归的影响及其可能机制.方法 体外分离培养自体骨髓来源EPC并用5-溴脱氧尿嘧啶核苷（5 -bromodeoxyuridine,BrdU）标记.线栓法制作大鼠大脑中动脉闭塞（middle cerebral arteryocclusion,MCAO）模型.EPC组大鼠经颈外静脉移植自体EPC[ 106/ml·kg）],对照组注射磷酸盐缓冲液（1 ml/kg）,假手术组不进行任何处理（n=15）.改良神经功能缺损严重程度评分（modifiedneurological severity score,mNSS）观察大鼠神经功能变化情况.BrdU免疫组化染色评价EPC增殖和分化.三维共聚焦图像分析检测脑缺血区血管结构和密度.TUNEL染色检测缺血脑组织凋亡细胞.酶联免疫吸附法检测血浆血管内皮生长因子（vascular endothelial growth factor,VEGF）浓度.结果 EPC组mNSS评分显著低于对照组[第8天时；（6.43±0.69）分对（8.86±0.95）分；q=2.673,P=0.035；第14天时：（4.55±0.89）分对（6.73±1.06）分；q=5.360,P=0.035].EPC组BrdU阳性细胞数量显著多于对照组[（42.2±5.76）对（25.67±5.49）；q=4.020,P=0.030].EPC组毛细血管直径显著小于对照组[（4.51±0.21）μm对（6.34±0.24） μm;q=3.980,P =0.003]；血管密度[（212.64±8.02）/0.002 mm^3对（153.60±7.21 ）/0.002 mn3；q =9.670,P=0.001]和微血管总表面积[（92 013±5 132）μm^3/0.002 mm^3对（71 366±4 538） μm2/0.002 mm^3；q=4.180,P=0.014]显著高于和大于对照组；EPC组凋亡细胞数量显著少于对照组[（36.26±6.91）对（78.34±7.21）；t=-4.834,P=0.003]；EPC组血浆VEGF浓度显著高于对照组[（54.91±5.71）pg/ml对（13.81±4.25）pg/ml;q=12.300,P=0.002].结论 自体EPC移植对大鼠缺血脑组织具有保护作用,可能与VEGF相关联的血管再生和神经保护有关,其在治疗缺血性脑血管病中具有重要的应用前景.

Objective To investigate the effect of autologous bone marrow-derived endothelial progenitor cell （EPC） transplantation on neurological outcomes in cerebral ischernia in rats and its poss le mechanisms.Methods Autologous bone marrow-derived EPC was cultured in vitro and it was labeled with 5-bromodeoxyuridine （BrdU）.A middle cerebral artery occlusion （MCAO） model was induced by the intraluminal suture method.The rats in a EPC group transplanted autologous EPC （106/ml/kg） via external jugular veins,those in a control group were injected with phosphate buffered saline （1 ml/kg）,and those in a sham operation group （n =15）were not treated.The modified neurological severity score （mNSS） was used to observe the neurological changes of the rats.BrdU immunohistochemical staining was used to evaluate EPC proliferation and differentiation.Three-dimensional confocal image analysis was used to detect the vascular structure and density in cerebral ischemic areas.TUNEL staining was used to detect the apoptotio cells in ischernic brain tissue.Enzyme-linked immunosorbent assay was used to detect the concentration of plasma vascular endothelial growth factor VEGF）.Results The mNSS in the EPC group was siginficantly lower than that in the control group （at day 8：6.43 ±0.69 vs.8.86 ±0.95,q =2.673,P=0.035; at day 14：4.55 ±0.89 vs.6.73 ± 1.06,q =5.360,P =0.035）.The number of BrdU positive cells in the EPC group was significantly higher than that in the control group （42.2±5.76 vs.25.67±5.49,q=4.020,P=0.030）.The capiilary diameter in the EPC group was significantly smaller than that in the control group （4.51 ± 0.21 μm vs.6.34 ± 0.24 μm,q =3.980,P =0.003）; the density of blood vessels （212.64 ± 8.02/0.002 mm3 vs.153.60 ± 7.21/0.002 mm3; q =9.670,P =0.001 ） and the total surface area of microvessel （92 013 ± 5 132 μm2/0.002 mm3 vs. 71 366 ±4 538 μm2/0.002 mm3; q=4.180,P=0.014） were significantly higher or more than those in the control group.The number of apoptotic cells in the EPC group was significantly less than that in the control group （36.26 ± 6.91 vs.78.34 ± 7.21; t =-4.834,P =0.003）.The plasma VEGF concentration in the EPC group was significantly higher than that in the control group （54.91 ± 5.71 pg/ml vs.13.81 ± 4.25 pg/ml,q =12.300,P=0.002）.Conclusions Autologous EPC transplantation has a protective effect on ischemic brain tissue in rats.It may be associated with VEGF related angiogenesis and neuroprotection.It has an important application prospect in the treatment of ischemic cerebrovascular disease.