静脉注射同种异体骨髓间充质干细胞对血管性痴呆大鼠海马区脑组织形态及微管相关蛋白2表达的影响

Effect of bone marrow mesenchymal stem cells by intravenous injection on morphology and expression of microtubule-associated protein-2 in hippocampus of vascular dementia rats

目的:骨髓间充质干细胞移植进入脑组织后,能否影响脑组织的形态及微管相关蛋白2的表达从而改善痴呆状态下的认知功能?观察静脉注射同种异体骨髓间充质干细胞后,血管性痴呆模型大鼠脑海马CA1区脑组织形态及微管相关蛋白2的变化。方法:实验于2004-08/2005-05在解放军第二军医大学完成。①实验动物:健康雄性SD大鼠60只,随机数字表法分为细胞移植组、模型对照组、假手术组,20只/组。②实验方法:另取20只大鼠用于体外分离、培养扩增骨髓间充质干细胞,传至第2代时加入BrdU进行标记,制备单细胞悬液,调整细胞浓度为3×109L-1。细胞移植组、模型对照组采用双侧颈总动脉结扎法建立血管性痴呆动物模型,假手术组仅暴露双侧颈总动脉但不结扎。造模后4周,细胞移植组尾静脉注射骨髓间充质干细胞悬液1mL,模型对照组注射等量磷酸盐缓冲液,假手术组不进行尾静脉注射。③实验评估:细胞移植后4周,苏木精-伊红染色检测各组大鼠脑海马CA1区脑组织形态变化,免疫荧光染色观察经BrdU标记的骨髓间充质干细胞示踪情况,免疫组织化学染色检测脑海马CA1区微管相关蛋白2的表达。结果:细胞移植组5只大鼠死亡,模型对照组3只大鼠死亡,假手术组均进入结果分析。①脑海马CA1区锥体细胞形态变化:细胞移植组和模型对照组CA1区锥体细胞较假手术组排列稀疏、紊乱,细胞肿胀、脱失,核固缩,但细胞移植组细胞排列较模型对照组规则,且细胞肿胀、脱失及核固缩较模型对照组减轻。②脑海马内骨髓间充质干细胞的示踪:细胞移植后4周,大鼠脑海马内可见被绿色荧光标记的骨髓间充质干细胞。③脑海马CA1区微管相关蛋白2的表达:与假手术组比较,细胞移植组及模型对照组脑海马CA1区锥体细胞层微管相关蛋白2阳性产物的吸光度值均明显降低(P<0.05);细胞移植组明显高于模型对照组(P<0.05)。结论:静脉注射骨髓间充质干细胞能够使血管性痴呆大鼠脑海马CA1区神经元损伤及脱失减轻,并使微管相关蛋白2表达增加。

AIM： To explore the effects of intravenous injection of xeno-isotype bone marrow mesenchymal stem cells （MSCs） on the morphology and the expression of microtubule-associated protein-2 （MAP-2） in CA1 region in hippocampus of vascular dementia rats, and improve the cognitive function of those rats. METHODS： The experiment was finished at the Second Military Medical University of Chinese PLA from August 2004 to May 2005. ①Experiment artimals： Sixty adult male SD rats were randomly divided into MSCs transplantation group, control group and sham group. Each group had 20 rats. ②Experiment methods： MSCs were isolated and expanded in vitro from another 20 rats, and the second lineage MSCs were labeled with BrdU. Then calls were suspended at the density of 3x10^9 L^-1. MSCs transplantation group and control group were received permanent bilateral carotid arteries ligation to establish vascular dementia models. Bilateral carotid arteries in sham group were exposed but not ligated. At 4 weeks after operation, MSCs （1 mL） were intravenously injected into rat tails of MSCs transplantation group; Phosphate buffer solution （PBS） was intravenously injected at the same volume in control group; Sham group received no injection of MSCs or PBS, ③Experiment evaluations： Rats in all the groups were killed four weeks after injection. Hematoxylin-eosin staining was used to observe＇ the morphology in CA1 region. Immuno-histochemistry was used to identify the trace of BrdU-labeled MSCs and the expression of MAP-2 in CA1 region. RESULTS： Five rats were dead in MSCs transplantation group and 3 rats in control group. Rats in sham group all entered the analysis. ①Morphology in CA1 region： Compared with sham group, the arrangement of the pyramidal neurons in CA1 region was sparse and tangled, and there were many cellular swelling, loss and pyknosis in MSCs transplantation group and control group. The pathologic impairments above appeared in MSCs transplantation group were much better than that in control group. ②The trace of MSCs in hippocampus： BrdU-labeled MSCs were found in hippocampus four weeks after transplantation. ③MAP-2 expression in CA1 region： The expression of MAP-2 was significantly decreased in the CA1 pyramidal cells of rats in MSCs transplantation group and control group compared with sham group （P〈 0.05）, but the expression was significantly increased in MSCs transplantation group than in control group （P 〈 0,05）. CONCLUSION= Intravenous injection of MSCs can protect neurons and increase the expression of MAP-2 in hippocampus CA1 region of vascular dementia rats.