缺氧预处理后骨髓源神经干细胞联合脑源性神经生长因子移植治疗大鼠脑缺血再灌注损伤的研究

目的 研究缺氧预处理后骨髓源性神经干细胞（source n eural s tem c ells o f b one m arrow，BMSCs-NSCs）联合脑源性神经生长因子（brain-derived neurotrophic factor，BDNF）立体定向移植治疗大鼠脑缺血再灌注损伤的疗效，为高原地区脑梗死的细胞移植治疗提供动物实验基础。方法 72只SD大鼠随机分为缺氧预处理组和常氧组，每组各36只，缺氧预处理组造模前3 d进行低氧预处理（hypoxic preconditioning，HPC）。两组均制作大脑中动脉阻塞再灌注（middle cerebral arteryocclusion/reperfusion，MCAO/R）模型。每组分为3个亚组（BMSCs-NSCs+BDNF组、BMSCs-NSCs组和对照组，每组各12只），分别梗死灶同侧尾状核内立体定向移植BMSCs-NSCs+BDNF、BMSCs-NSCs和DMEM/F12培养基。移植后3 d、7 d、14 d、21 d、28 d、35 d进行神经功能缺损评分，每个时间点每组取2只大鼠，断头取脑后行免疫组织化学染色，观察5-溴脱氧尿嘧啶（5-Bromo-deoxyuridine，Brdu）阳性细胞的迁移路径，行CD133、Nestin、微管相关蛋白2（microtubule-associated protein 2，MAP-2）、兔抗微管蛋白（β-tubulin）、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）、半乳糖神经酰胺（Galactosylceramidase，Galc）免疫荧光染色，了解骨髓源性神经球分化情况。结果 常氧BMSCs-NSCs+BDNF组、常氧BMSCs-NSCs组、缺氧预处理BMSCs-NSCs+BDNF组、缺氧预处理BMSCs-NSCs组7 d、14 d、21 d、28 d和35 d的神经功能评分均显著低于同组3 d时的神经功能评分。移植3 d时缺氧预处理对照组神经功能学评分显著低于常氧对照组（P =0.040）；移植7 d时缺氧预处理BMSCs-NSCs+BDNF组神经功能评分显著低于常氧BMSCs-NSCs+BDNF组（P =0.031）。无论缺氧预处理组还是常氧组，BMSCs-NSCs+BDNF组CD133、Nestin、MAP-2、β-tubulli n、GFAP、Gal c免疫荧光染色光密度值（integral optical density，IOD）均显著高于BMSCs-NSCs组（均P＜0.001）；BMSCs-NSCs+BDNF组、BMSCs-NSCs移植组各检测指标IOD均高于对照组（均P＜0.001）。结论 大鼠缺氧预处理后BMSCs-NSCs联合BDNF立体定向移植可显著提高BMSCs-NSCs的效果。缺氧预处理并不能促进外源性BMSCs-NSCs分化，但却能明显改善大鼠神经功能。

Research of the Treatment on Neural Stem Cells of Bone Marrow by Hypoxia Preconditioning Joint with BDNF on Cerebral Infarction in Rats

Objective To investigate the curative effect of joint treatment on source neural stem cells of bone marrow (BMSCs-NSCs) by hypoxic preconditioned and brain derived neurotrophic factor (BDNF) stereotactic transplantation on cerebral infarction in rats, so as to provide animal experimental bases on the therapy of cell transplantation on brain infarction in plateau areas. Methods A total of 72 SD rats were randomly divided into hypoxia precondition group (n=36) and normal oxygen group (n=36). Acute hypoxic preconditioning (HPC) was performed for 3 days before molding in hypoxia precondition group. Two groups of rats were made the model of
middle cerebral artery occlusion reperfusion (MCAO/R). Each group was divided into 3 subgroups: BMSCs-NSCs+BDNF group (n=12), BMSCs-NSCs group (n=12) and control group (n=12), and each subgroup was respectively transplanted with stereotactic MSCs-NSCs combined with BDNF, MSCs-NSCs, and DMEM/F12 culture medium to the ipsilateral caudate nucleus of rats brain infarction. After transplantation, neurological function scoring was performed at 3 d, 7 d, 14 d, 21 d, 28 d and 35 d. Tworats were sacriifce from each group after been scored. The migration path of positive cells of 5-Bromo-deoxyUridine (Brdu) of the tissue of brain by immunohistochemistry staining. The expressions of CD133, Nestin, MAP-2, beta-tubullin, GFAP, GalC were detected by immunolfuorescence staining to understand the neural differentiation in bone marrow-derived, and to observe its curative effect. Results At 7 d, 14 d, 21 d, 28 d and 35 d, neurological function scoring were signiifcantly lower than 3 d in BMSCs-NSCs+BDNF and BMSCs-NSCs group of hypoxia precondition group and normal oxygen group. At 3 d after transplantation, in subgroup of control, neurological function scoring in hypoxia precondition group were signiifcantly lower than normal oxygen group (P=0.040). At 7 d after transplantation, in subgroup of BMSCs-NSCs+BDNF, neurological function scoring in hypoxia precondition group were signiifcantly lower than normal oxygen group (P=0.031). Whether in hypoxia precondition group or normal oxygen group, each index of integral optical density (IOD) in BMSCs-NSCs+BDNF group was higher than BMSCs-NSCs group (P<0.001), and each index of IOD in BMSCs-NSCs+BDNF group and BMSCs-NSCs group was higher than the control group (P<0.001). Conclusion The curative effect was obvious improvement by the joint treatment on BMSCs-NSCs by hypoxic preconditioned and BDNF stereotactic transplantation. Hypoxia preconditioning does not promote the differentiation of exogenous MSCs-NSCs, but can signiifcantly improve the neurological function of rats. Hypoxic preconditioning treatment may promote the proliferation and differentiation of endogenous MSCs.