骨髓间质干细胞移植减轻犬急性脑梗死模型的缺血性脑损伤研究

Bone marrow mesenchymal stem cell transplantation reduces ischemic brain injury in a dog model of acute cerebral infarction

摘要:

目的 观察骨髓间质干细胞（BMSCs）对犬急性缺血脑组织的保护作用，并探讨其可能的机制。方法 将24只成年杂交犬随机分为治疗组及对照组，DSA引导下行自体血栓栓塞大脑中动脉闭塞缺血模型制作，并抽取骨髓提取BMSCs，传代并给予4'，6-二脒基-2-苯基吲哚（DAPI）标记；1周后开颅行多点脑内注射BMSCs移植；移植后1周行脑DWI序列扫描，计算梗死灶体积；4周后将犬处死，每组随机选择6只动物取脑标本行TTC染色测定梗死灶体积；另外6只动物进行HE染色、VG染色、TUNEL染色评价脑损伤情况；免疫荧光染色了解脑源性神经营养因子（BDNF）、碱性成纤维生长因子（bFGF）、胰岛素样生长因子1（IGF-1）和血管内皮生长因子（VEGF）的表达情况。结果 治疗组梗死灶内广泛存在DAPI阳性细胞。治疗组的梗死灶体积明显小于对照组，P<0.01。治疗组梗死灶范围、梗死灶内细胞坏死、胶质增生和胶质瘢痕均较对照组减轻。治疗组凋亡细胞明显少于对照组，P<0.05。治疗组细胞因子BDNF、BFGF、IGF-1和VEGF表达阳性的细胞均显著多于对照组。结论 脑梗死后给予BMSCs移植，BMSCs能存活并自行向梗死灶迁移，并发挥脑保护作用，其机制可能和分泌多种神经营养因子有关。

Abstract:

Objective To investigate the protective effect of bone marrow mesenchymal stem cells (BMSCs) on acute ischemic brain tissue and possible mechanisms.Methods A total of 24 hybridized adult dogs were randomized into treatment group and control group. A model of ischemia was established by digital subtraction angiography-guided middle cerebral artery occlusion using autologous blood clots. Bone marrow aspiration was performed to collect BMSCs, and then BMSCs were subcultured and labeled by 4',6-diamidino-2-phenylindole (DAPI). Craniotomy was performed at 1 week after modeling and BMSCs were transplanted via intracerebral injection at multiple sites. At 1 week after transplantation, brain diffusion-weighted imaging was performed to calculate infarct volume. The dogs were sacrificed at 4 weeks after transplantation, 6 dogs were randomly selected from each group to collect brain tissue, and TTC staining was performed to measure infarct volume; HE staining, Van Gieson staining, and TUNEL staining were performed for the other 6 dogs in each group to evaluate brain injury. Immunofluorescent staining was performed to measure the expression of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF).Results The treatment group had many DAPI-positive cells widely dispersed in infarct. The treatment group had a significantly smaller infarct volume than the control group (P<0.01). Compared with the control group, the treatment group had significant alleviation in infarct extent, cell necrosis in infarct, gliosis, and glial scar. The treatment group had a significantly lower number of apoptotic cells than the control group (P<0.05). Compared with the control group, the treatment group had significantly higher numbers of cells with positive expression of BDNF, bFGF, IGF-1, and VEGF.Conclusions When transplantation of BMSCs is performed after cerebral infarction, BMSCs can survive, migrate to infarct, and then protect the brain, possibly by secreting various neurotrophic factors.