双重荧光标记验证静脉移植碱性成纤维细胞生长因子基因修饰骨髓间充质干细胞在脑缺血模型大鼠脑内的存活及分化

背景：碱性成纤维细胞生长因子可以促进骨髓间充质干细胞的增殖和向神经细胞方向分化，并被认为是胶质细胞的分裂原。目的：以双重荧光标记验证静脉移植碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞在脑缺血模型大鼠脑内的存活及分化情况，及其向神经元样细胞和神经胶质细胞分化的趋势。设计、时间及地点：随机对照动物实验，于2005-07/2006-03在中南大学实验动物中心实验室完成。材料：选用50只SD大鼠，按随机数字表法分为4组：假手术组（n=10），脑缺血/再灌注损伤模型组（n=10），骨髓间充质干细胞治疗组（n=15），碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞治疗组（n=15）。方法：除假手术组外，其余3组制备局灶性脑缺血再灌注模型。分别将骨髓间充质干细胞或碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞通过静脉移植至实验性脑缺血大鼠体内，脑缺血再灌注损伤组大鼠注入相同体积的DMEM培养基。主要观察指标：应用5-溴-2-脱氧尿苷-神经元特异核蛋白及5-溴-2-脱氧尿苷-胶质纤维酸性蛋白双重荧光标记法观察移植细胞在脑内的存活和分化情况，比较各组大鼠脑缺血后的神经功能评分及脑梗死体积变化。结果：移植7 d后，碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞组大鼠脑内5-溴-2-脱氧尿苷阳性细胞数、5-溴-2-脱氧尿苷-神经元特异核蛋白双标阳性细胞数均高于骨髓间充质干细胞治疗组（P < 0.05），两组间5-溴-2-脱氧尿苷-胶质纤维酸性蛋白双标阳性细胞数差异无显著性意义（P > 0.05）。再灌注7 d后，静脉移植骨髓间充质干细胞和碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞均能改善脑缺血后大鼠的神经功能、减少脑梗死体积，碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞的作用明显优于骨髓间充质干细胞。结论：碱性成纤维细胞生长因子诱导的骨髓间充质干细胞静脉移植后可在脑内缺血区存活，并分化为比例更合适的神经元和神经胶质细胞，发挥神经修复作用。

Survival and differentiation of basic fibroblast growth factor gene modified bone marrow mesenchymal stem cells following vein transplantation in cerebral ischemia model rats validated by double immunofluorescence staining

BACKGROUND: Basic fibroblast growth factor (bFGF) can accelerate the bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation into nerve cells, which is considered as a mitogen of glial cells. OBJECTIVE: To investigate the survival and differentiation of bFGF gene modified BMSCs transplanted on rat models of cerebral ischemia by double immunofluorescence staining, and to study the differentiation trend of BMSCs into neuron-like cells and glial cells.DESIGN, TIME AND SETTING: The randomized control animal experiment was completed in the central laboratory of Experimental Animal Center of Central South University from July 2005 to March 2006.MATERIAL: Fifty Sprague-Dawley rats were divided into 4 groups at random: sham operation group (n=10), cerebral ischemia-reperfusion injury group (n=10), BMSCs group (n=15) and bFGF modified BMSCs group (n=15).METHODS: Except sham operation group, rats in the other three groups were prepared for local cerebral ischemia-reperfusion models. Then BMSCs or bFGF modified BMSCs were intravenously transplanted into cerebral ischemic rats, and the same volume of DMEM were injected in the cerebral ischemia-reperfusion injury group. MAIN OUTCOME MEASURES: Survival rate and differentiation of grafted cells were observed by 5-bromo-2-deoxyuridine (BrdU)-NeuN, and BrdU-glial fibrillary acidic protein (GFAP) double immunofluorescence staining; the neurological scores and infarction volumes in each group. RESULTS: At 7 days after implantation, the number of BrdU/NeuN-positive cells and BrdU-GFAP-positive cells in the bFGF modified BMSCs group was higher than those on the BMSCs group (P < 0.05), but there were no significant differences in the co-expression cells by double immunofluorescence staining between the two groups (P > 0.05). At 7 days following reperfusion, neural function of cerebral ischemia rats were improved and infarction volume was reduced in both BMSCs group and the bFGF modified BMSCs group, and bFGF modified BMSCs group was superior to BMSCs group.CONCLUSION: BMSCs modified by bFGF can survive in cerebral ischemic region and differentiate into neuron and glial cells, which are more proper for repairing nerves.