【作者】 张志坚；

【导师】 刘春风；

【作者基本信息】 苏州大学 ， 神经外科学， 2007， 博士

【摘要】 目的观察经静脉移植骨髓间质干细胞(MSCs)治疗脑梗塞时,神经功能缺损的恢复情况,以及MSCs在脑梗塞灶的分布、迁移与分化情况。观察基质细胞衍生因子-1　(SDF-1　)在体内外对大鼠骨髓间质干细胞(rMSCs)的趋化诱导作用,探讨SDF-1　对rMSCs迁移影响的可能机制,为实现临床上对移植的rMSCs进行调控提供理论与实验依据。方法建立线栓法大鼠大脑中动脉栓塞(MCAO)模型,大脑中动脉血流阻断2小时后再灌注,再灌注后24h经尾静脉将标记的大鼠骨髓间质干细胞(rMSCs)植入脑梗塞鼠体内,于移植后24h、1W、2W时采用mNSS评分法对动物神经功能状况进行评分。应用荧光激发及免疫组化方法于移植后1W、2W时检测移植细胞在脑内存活、迁移及分化情况。细胞化学染色观察rMSCs体外培养时的分化情况。应用体外细胞迁移实验及大鼠脑梗塞模型体内移植,观察在施用和不施用抗SDF-1抗体时,SDF-1　对rMSCs迁移的影响。流式细胞术与RT-PCR检测rMSCs的CXC趋化因子受体4 (CXC chemokine receptor 4, CXCR4)表达。结果1.与对照组相比,移植组大鼠在移植后观察的2周内神经功能改善明显(p<0.001)。2.可观察到移植细胞在脑梗塞区及其周边区聚集并存活,这种聚集于第2周更为明显。在脑梗塞区及其周边区有部分植入细胞分化表达神经细胞表面标志物,而在体外同时程培养的rMSCs未见分化表达类似表面标志物。3.体外实验示在SDF-1　存在时,rMSCs迁移活跃,应用抗体封闭CXCR4受体后,这种迁移现象显著减弱。4.体内移植的rMSCs在脑内主要聚集在脑梗死灶及其周边区,但在封闭CXCR4受体后,这种聚集现象大为减弱。5.流式细胞术示仅小部分rMSCs表面表达CXCR4,但经Triton X-100处理后,表达CXCR4的rMSCs增加。RT-PCR检测显示rMSCs的Cxcr4基因表达CXCR4 mRNA。结论1. rMSCs经静脉移植后的2周观察表明移植的rMSCs可向宿主脑内病灶迁移、存活、并分化表达神经细胞表型,有促进脑梗塞后神经功能恢复的作用。2.经静脉路移植rMSCs不仅可达到治疗目的,而且可以避免直接穿刺移植带来的脑组织损伤,植入的rMSCs能迁移并更好地分布在脑梗塞灶。3. SDF-1可通过CXCR4受体对rMSCs起趋化作用,植入的rMSCs能聚集分布在脑梗塞灶及其周边区与这种趋化作用有关。利用这种趋化作用可望调控干细胞向靶组织的趋化聚集量,达到治疗目的。更多还原

【Abstract】 Objective To observe the therapeutic benefit of intravenous administration of rat mesenchymal stem cells(rMSCs)after cerebral ischemia in adult rats, which include the improvement of neurological function, the migration of rMSCs to infarct focus, and the differentiation of rMSCs, the effect of Stromal cell-derived factor-1 (SDF-1) on the migration of Mesenchymal Stem Cells(MSCs), and to explore the possible mechanism of this effect under the condition of brain ischemic injury.Methods Adult male SD rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAO). rMSCs or PBS were infused into tail vein at 24 h after MCAO respectively. Functional outcome measurements using the modified Neurological Severity Scores (mNSS) were performed at 24 h post-MCAO, 1 week and 2 weeks post-transplantation respectively. Immunohistochemical staining and immunocyto- chemistry were used synchronously to identify rMSCs and their differentiation markers in the brain sections and the cultured rMSCs in vitro. Effects of SDF-1 on the migration of cultural rat MSCs (rMSCs) and N9 microglial cells were observed by counting migrated cells and by calculating the migration indexes to show the difference of rMSCs migration under the conditions with or without the existence of SDF-1. CTO-labeled rMSCs were injected into the tail vein of rat with middle cerebral artery occlusion (MCAO). The migration and congregation of labeled cells in the injured region of brain sections were observed under fluorescence microscope. Anti- CXC chemokine receptor 4 (Anti-CXCR4) antibodies were used for blocking studies, and the expressions of CXC chemokine receptor 4(CXCR4)in rMSCs were observed with RT-PCR and flow cytometry respectively.Results There was significant neurological function improvement in rats treated with rMSCs compared with that of control group, as evidenced by mNSS scores (p<0.001). After transplantation, rMSCs were survived and localized around the ischemic core and its boundary zone, and a few rMSCs expressed nestin, NF-200 and GFAP, but there were no such expression in rMSCs cultured in vitro. In vitro, the migration indexes of rMSCs and N9 microglial cells in the SDF-1 groups were significantly higher as compared with the medium controls(P<0.01). The CXCR4 antibody blocking significantly impaired the SDF-1 chemotaxis. In vivo, labeled rMSCs mainly migrated and congregated around the injured region in the rMSCs transplantation groups. The CXCR4 antibody also significantly impaired both the migration and congregation of rMSCs.Conclusions Intravenous administration of rMSCs can promote the neurological functional improvement in rat stroke model. rMSCs can survive and localize to the ischemic area of the brain, and a few cells express the phenotypic protein marker of neural cells. The benefit of intravenous administration of rMSCs are no brain injury and better distribution of grafted cells.The results demonstrate that the chemotactic effect of SDF-1 on the MSCs is introduced by CXCR4. Although only small proportion of rMSCs expresses active extracellular CXCR4 receptor,there are higher level intracellular expression of CXCR4. SDF-1 secreted from injured brain tissue may be responsible for the chemotaxis and congregation of MSCs . This phenomenon will be significant in the therapeutical research of acute injury .更多还原