骨髓间充质干细胞经 Survivin 修饰后移植对大鼠实验性脑卒中的影响。
Effects of transplantation with bone marrow-derived mesenchymal stem cells modified by Survivin on experimental stroke in rats.
机构信息
Department of Neurology, Union Hospital, Fujian Medical University, Fuzhou 350001, PR China.
出版信息
J Transl Med. 2011 Jul 6;9:105. doi: 10.1186/1479-5876-9-105.
BACKGROUND
This study was performed to determine whether injury induced by cerebral ischemia could be further improved by transplantation with bone marrow-derived mesenchymal stem cells (MSCs) modified by Survivin (SVV).
METHODS
MSCs derived from bone marrow of male Sprague-Dawley rats were infected by the self-inactive lentiviral vector GCFU carrying green fluorescent protein (GFP) gene and SVV recombinant vector (GCFU-SVV). In vitro, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were detected in infected MSCs supernatants under hypoxic conditions by ELSIA. In vivo, experiments consisted of three groups, one receiving intravenous injection of 500 μl of phosphate-buffered saline (PBS) without cells (control group) and two groups administered the same volume solution with either three million GFP-MSCs (group GFP) or SVV/GFP-MSCs (group SVV). All animals were submitted to 2-hour middle cerebral artery occlusion (MCAO) and then reperfusion. Differentiation and survival of the transplanted MSCs were determined by confocal microscope. Western blot was used to detect the expression of VEGF and bFGF in ischemic tissue. A 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to assess the infarct volume. Evaluation of neurological function was performed using a modified Neurological Severity Score (mNSS).
RESULTS
In vitro, modification with SVV further increased secretion of VEGF and bFGF under hypoxic condition. In vivo, only very few transplantated cells co-expressed GFP and NeuN. The survival transplanted cells in the group SVV was 1.3-fold at 4 days after transplantation and 3.4-fold higher at 14 days after transplantation, respectively, when compared with group GFP. Expression of VEGF and bFGF in the ischemic tissue were further up-regulated by modification with SVV. Moreover, modification with SVV further reduced the cerebral infarct volume by 5.2% at 4 days after stroke and improved post-stroke neurological function at 14 days after transplantation.
CONCLUSION
Modification with SVV could further enhance the therapeutic effects of MSCs possibly through improving the MSCs survival capacity and up-regulating the expression of protective cytokines in the ischemic tissue.
背景
本研究旨在探讨 Survivin（SVV）修饰的骨髓间充质干细胞（MSCs）移植是否能进一步改善脑缺血引起的损伤。
方法
从雄性 Sprague-Dawley 大鼠的骨髓中分离出 MSCs，并用携带绿色荧光蛋白（GFP）基因和 SVV 重组载体（GCFU-SVV）的自我失活慢病毒载体 GCFU 感染。在体外，通过 ELSIA 检测缺氧条件下感染 MSCs 上清液中血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）的含量。在体内，实验分为三组，一组静脉注射 500μl 无细胞磷酸盐缓冲液（PBS）（对照组），两组分别注射相同体积的 300 万个 GFP-MSCs（GFP 组）或 SVV/GFP-MSCs（SVV 组）。所有动物均接受 2 小时大脑中动脉闭塞（MCAO），然后再灌注。通过共聚焦显微镜观察移植 MSCs 的分化和存活情况。采用 Western blot 检测缺血组织中 VEGF 和 bFGF 的表达。采用 2,3,5-三苯基氯化四氮唑（TTC）染色评估梗死体积。采用改良神经功能缺损评分（mNSS）评估神经功能。
结果
体外实验表明，SVV 修饰进一步增加了缺氧条件下 VEGF 和 bFGF 的分泌。体内实验仅发现极少数移植细胞共表达 GFP 和 NeuN。与 GFP 组相比，SVV 组移植细胞在移植后 4 天的存活率分别增加了 1.3 倍，在移植后 14 天的存活率增加了 3.4 倍。SVV 修饰进一步上调了缺血组织中 VEGF 和 bFGF 的表达。此外，SVV 修饰进一步减少了脑梗死体积，在中风后 4 天减少了 5.2%，并改善了中风后 14 天的神经功能。
结论
SVV 修饰可通过提高 MSCs 的存活能力和上调缺血组织中保护性细胞因子的表达，进一步增强 MSCs 的治疗效果。
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