自体骨髓内皮祖细胞移植治疗动脉粥样硬化大鼠急性脑缺血的实验研究

目的 探讨自体骨髓内皮祖细胞移植治疗动脉粥样硬化大鼠急性局灶性脑缺血的有效性.方法 高脂膳食制备20只动脉粥样硬化大鼠模型,采集骨髓,分离血管内皮祖细胞(EPCs)并扩增培养,检测其表面标记物的表达;第7天采用线栓法制作急性局灶性脑缺血模型,造模后3 h进行移植,其中实验组经颈静脉自体移植BrdU标记的EPCs,对照组给予等量的PBS.急性脑缺血术后6 h和第1、3、7、10、14天通过神经功能缺损评分(mNSS)量表行行为学评价,免疫组化染色观察BrdU标记的EPCs在缺血脑组织的分布和血管密度.结果体外培养大鼠骨髓来源的EPCs,细胞数目可达到5×106;CD34免疫荧光和FLK-1免疫组化鉴定呈阳性,并能特异性吸附FITC-UEA和内吞DIL-Ac-LDL.第14天实验组mNSS得分为6.13±0.30.对照组为8.50±0.46,比较差异具有统计学意义(P<0.05),实验组神经功能的恢复优于对照组.第28天实验组脑缺血区和血管壁可见BrdU标记的EPCs,而对照组则为阴性.免疫组化染色显示实验组大鼠缺血脑组织血管数为16.87±5.52,对照组大鼠缺血脑组织血管数为12.76±4.94,比较差异具有统计学意义(P<0.05).结论 从活体大鼠骨髓中分离的EPCs通过自体移植后可以进入脑缺血区并长期存活,其能够促进神经功能恢复,这可能与血管再生有关.

Treatment of acute cerebral ischemia in atherosclerotic rats with autologous transplantation with bone marrow-derived endothelial progenitor cells

Objective To explore the effeteness of autologous transplantation of bone marrow-derived endothelial progenitor cells in promoting the neovascularization and improving the neurological functional recovery in atherosclerotic rats with acute cerebral infarction. Methods Male Sprague-Dawley rat models of atherosclerosis were established by fat-rich diet feeding. Endothelial progenitor cells (EPCs) were obtained from bone marrow of all rats; the cells were cultured in vitro in Ml99 with VEGF, bFGF and EGF in it Assays were used to detect the expression of FLK-1 and CD34. on the 7th d, middle cerebral artery occlusion (MCAO) rat models were established by the method of thread thrombus. Three h after MCAO, all of the animals were randomized into experimental group (the autologous endothelial progenitor cells labeled with BrdU were injected into the carotid vein) and control group (same volume of PBS were injected into the carotid vein). Behavioral tests (modified neurological severity scale, mNSS) were performed 6 h and 1, 3, 7, 10 and 14 d after MCAO. Besides, immunohistochemical examinations were employed to observe the distribution of EPCs (labeled by BrdU) in the brain tissue and to measure the microvessel density. Results EPCs from bone marrow were isolated, induced and cultured successfully in vitro, which positively stained for FLK-1 by immunocytochemistry and partly positively expressed CD34 by immunofluorescence. The cells of FITC labeled UEA adsorption and DiL-acLDL internalization were positive under fluorescence confocal microscopy. These cells possessed robust proliferative potential and their number reached 5×106. On the 14th d, the neurological function recovery in the experimental group (mNSS scores: 6.13±0.30) was significantly improved as compared with that in the control group (mNSS scores: 8.50±0.46, P<0.05). On the 28th, some positive EPCs stained by BrdU were found in the experimental group and the numbers of blood vessels in the experimental group (16.87±5.52) were obviously larger than those in the control group (12.76±4.94,P<0.05). Conclusion EPCs from bone marrow can be autologously transplanted into the brain tissue of cerebral ischemia and survive. Cerebral ischemia can improve neurological functional recovery after MCAO, which were probably by promoting the neovascularization.