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

摘要/Abstract

摘要：

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 artery occlusion/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。

文章导读： 缺氧预处理条件下对脑缺血再灌注大鼠进行骨髓源性神经干细胞联合脑源性神经生长因子尾状核定向移植可以改善大鼠的神经功能缺损，但缺氧预处理未显示出促进骨髓源性神经干细胞移植后分化的效果。

关键词: 缺氧预处理; 骨髓源神经干细胞; 脑源性神经生长因子; 立体定向移植

Abstract:

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 ofmiddle 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 sacrifice 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 immunofluorescence 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 significantly 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 significantly 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 significantly 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 significantly improve the neurological function of rats. Hypoxic preconditioning treatment may promote the proliferation and differentiation of endogenous MSCs.

Key words: Hypoxia precondition; Source neural stem cells of bone marrow; BDNF; Stereotactic transplantation

雷延成，吴世政，张淑坤，侯倩，才鼎，肖宗宇，陈晓娟. 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[J]. 中国卒中杂志, 2016, 11(05): 360-367.

参考文献

1 He J，Gu D，Wu X，et a1. Major causes of death

among men and women in China[J]. N Engl J Med，

2005，353：1124-1134.

2 Lee J，Elkahloun AG，Messina SA，et al. Cellular and

genetic characterization of human adult bone marrowderived

neural stem-like cells：a potential antiglioma

cellular vector[J]. Cancer Res，2003，63：8877-8889.

3 钟小明，余鸿. 骨髓基质细胞及其上清液对缺氧缺血

脑损伤大鼠海马齿状回突触素表达的影响及机制[J].

实用医学杂志，2016，3：359-360.

4 Sobeih MM，Corfas G. Extracellular factors that

regulate neuronal migration in the central nervous

system[J]. Int J Dev Neurosci，2002，20：349-357.

5 Ahmed S，Reynolds BA，Weiss S. BDNF enhances the

differentiation but not survival of CNS stem cell derived

neuronal precursors[J]. J Neurosci，1995，15：5765-5778.

6 Shet ty AK，Turner DA. In vit ro survival and

differentiation of neurons derived from epidermal

growth factor responsive postnatal hippocampus stem

cells：Inducing effects of brain-derived neurotrophic

factor[J]. J Neurobiol，1998，35：395-425.

7 Kurozumi K，Nakamura K，Tamiya T，et al. BDNF

gene-modified mesenchymal stem cells promote

functional recovery and reduce infarct size in the rat

middle cerebral artery occlusion model[J]. Mol Ther，

2004，9：189-197.

8 Sa s a k i M，Radtke C，Tan AM，et a l. BDNFhypersecreting

human mesenchymal stem cells

promote functional recovery，axonal sprouting，and

protection of corticospinal neurons after spinal cord

injury[J]. J Neurosci，2009，29：14932-14941.

9 Rakofsky JJ，Dunlop BW. BDNF function as apotential mediator of bipolar disorder and postt

raumatic st ress disorder comorbidity[J]. Mol

Psychiatry，2012，17：22-35.

10 Hong CJ，Liou YJ，Tsai SJ. Ef fect s of BDNF

polymorphisms on brain function and behavior in health

and disease[J]. Brain Res Bull，2011，86：287-297.

11 周跃辉，袁海平. 低氧对大鼠大脑皮层细胞表达脑源

性神经生长因子的影响[J]. 神经解剖学杂志，2013，29：

35-39.

12 孟红旗，全亚萍. 神经干细胞在神经系统疾病中的应用

[J]. 中国组织工程研究与临床康复，2010，14：175-178.

13 Tian Y，Liu Y，Chen X，et al. Tetramethylpyrazine

promotes proliferation and differentiation of neural

stem cells from rat brain in hypoxic condition via

mitogenactivated protein kinases pathway in vitro[J].

Neurosci Lett，2010，474：26-31.

14 雷延成，吴世政. 改良法大鼠大脑中动脉阻塞再灌注

模型的制备与效果评价[J]. 青海医学院学报，2014，4：

276-279.

15 Longa EZ，Weinstein PR，Carlson S，et al. Reversible

middle cerebral artery occlusion without craniectomy

in rats[J]. Stroke，1989，20：84.

16 张颜波，杨明峰. 低氧预适应刺激脑海马区内源性神

经干细胞的增殖[J]. 中国组织工程研究与临床康复，

2010，14：6699-6702.

17 黄斐，马广文. 脑源性神经营养因子缓释胶原凝胶支

架对神经干细胞生长和分化的影响[J]. 安徽医科大学

学报，2014，49：586-589.

18 Shao G，Gong KR，Li J，et al. Antihypoxic effects

of neuroglobin in hypoxiaitioned mice and SH-SY5Y

cells[J]. Neurosignals，2009，17：196-202.

19 疏龙飞，刘家传. 缺氧预处理对颅脑损伤大鼠抗氧化

应激和神经功能的影响[J]. 中华神经医学杂志，2014，

13：576-580.

20 周跃辉. 袁海平. 低氧对大鼠大脑皮层细胞表达脑源

性神经生长因子的影响[J]. 神经解剖学杂志，2013，29：

35-39.

21 季丽莉，佟雷. 脑源性神经生长因子促进成年大鼠脑

海马神经干细胞定向分化的浓度选择[J]. 中国组织工

程研究与临床康复，2007，11：9255-9258.

22 刘光杰. 刘家传. 缺氧预处理对颅脑损伤大鼠皮质内

质网应激蛋白P-eIF2α表达的影响[J]. 中国微侵袭神

经外科杂志，2016，2：83-86.