骨髓间质干细胞移植在脑梗死大鼠脑内分化及对神经功能恢复的影响

背景间质干细胞的多向潜能性研究目前多处于动物实验阶段,至不同定位组织后是否有向其组织分化并产生相应的功能?目的观察骨髓间质干细胞移植于脑皮质缺血性梗死灶的周围,观察其向神经分化及其对神经功能恢复的作用.设计随机对照实验.单位中山大学基础医学院解剖教研室,中山大学附属第一医院神经内科,暨南大学医学院病理教研室,广州医学院医学检验系.材料实验于2002-11/2003-11在中山大学医学院完成.选择清洁级雄性SD大鼠48只,随机分为造模的梗死组32只和不造模的对照组16只.梗死组又随机分成移植组16只和磷酸盐缓冲液组16只;以前囟为参考点,尾侧3mm旁开1.5 mm,深度2.0～3.0 mm,分别移植5 μL(5×104 L-1)间质干细胞或磷酸盐缓冲液.方法从非血液系统疾病的胸外科手术中摘取的肋骨骨髓中分离、纯化而获得的间质干细胞,经体外培养、扩增、鉴定.各组大鼠在移植后第2,6周末麻醉,对标本移植部位进行25μm连续冰冻切片.用免疫组织化学方法检测神经元特异性烯醇化酶、神经丝蛋白、胶质纤维酸性蛋白、巢蛋白的表达,评估间质干细胞向神经元、神经胶质细胞分化的能力.随机抽取移植组8只大鼠,磷酸盐缓冲液组8只大鼠,在移植前和移植后2,6周末用横木行走试验评分法观察大鼠全身状态和反应能力,对照组16只大鼠与移植组和磷酸盐缓冲液组同时测评.主要观察指标①大脑皮质神经元特异性烯醇化酶、神经丝蛋白、胶质纤维酸性蛋白、巢蛋白的表达.②横木行走试验评定大鼠运动功能.结果48只大鼠全部进入结果分析.①第2周末移植间质干细胞位置可见胶质纤维酸性蛋白、巢蛋白表达呈现阳性.第6周末移植间质干细胞位置神经元特异性烯醇化酶、神经丝蛋白表达也呈出阳性.而磷酸盐缓冲液组相应注射部位神经元特异性烯醇化酶、神经丝蛋白、胶质纤维酸性蛋白、巢蛋白表达均为阴性.②对照组无明显神经症状,评分均为9分.移植2周后,移植组运动功能评分高于磷酸盐缓冲液组[(6.7±0.9)分,(5.3±1.0)分,(P＜0.05)].移植6周后,移植组运动功能评分也高于磷酸盐缓冲液组[(8.9±1.1)分,(7.2±0.8)分,(P＜0.05)].结论骨髓间质干细胞移植后在中枢神经系统有趋向神经元和神经胶质细胞分化的能力,表达了某些神经元胶质细胞的特性.移植组大鼠横木行走试验运动功能评分高于磷酸盐缓冲液组,并显示出明显的神经功能修复作用.

Differentiation of bone marrow mesenchymal stem cells transplanted into the brain in rats with cerebral infarction and its effect on the recovery of nerve functions

BACKGROUND: The study about the multiple differentiation potentials of the mesenchymal stem cells is still on the stage of the animal experimentation. Can mesenchymal stem cells have the ability to differentiate into certain tissues and develop the corresponding functions after they are transplanted into certain tissues?OBJECTIVE: To observe the differentiation of the bone marrow mesenchymal stem cells into the nerve and its effect on the nerve functional recovery after they are transplanted into the peripheral zone of the ischemic infarction focus of the cerebral cortex.DESIGN: A randomized controlled study.SETTING: The Department of Anatomy of the School of Basic Medical College of Sun Yat-sen University; the Department of Neurology of the First Hospital of Sun Yat-sen University; Department of Pathology of the Medical College of Jinan University.MATERIALS: The experiment was conducted at the Medical College of Sun Yat-sen University from November 2002 to November 2003. Forty-eight male SD rats were chosen and randomly divided into two groups, with 32 rats in cerebral infarction model group and 16 in the non-model control group. In the cerebral infarction group, the rats were randomly divided again into two groups: 16 rats in the transplantation group and 16 in the phosphate buffered fluid group. The anterior fontanel taken as the reference point, 5 μL(5 × 104 L-1) of the bone marrow mesenchymal stem cells or the phosphate buffered fluid was respectively transplanted at the site 3 mm away on the caudal side and 1.5 mm aside at the depth of 2. 0 - 3. 0 mm.METHODS: The bone marrow mesenchymal stem cells were obtained through the separation and purification of the bone marrow of the ribs taken away in the thoracic surgery from the patients without the hematological diseases, and then the cells underwent in vitro culture, the amplification and the identification. At the 2nd and 6t1 weekend after the transplantation,the rats of every group were anesthetized, and the samples were taken from the transplantation site and made into the 25 μm of continuous frozen section. Then, the immunohistochemical method was used for the detection of the expressions of neuron specific enolase, neurofibril protein, glial fibrillary acidic protein, and nidogen to evaluate the ability of the bone marrow mesenchymal stem cells to differentiate into neurons and glial cells. Eight rats of the transplantation group and 8 rats of the phosphate buffered fluid group were randomly taken out, and 2 and 6 weeks before and after the transplantation the bar walking test evaluation method was used to identify the general status and reaction ability of the rats. Sixteen rats of the control group were assessed at the same time.enolase, neurofibril protein, glial fibrillary acidic protein and nidogen in the bar walking test.2nd weekend after the transplantation, there were positive expressions of glial fibrillary acidic protein and nidogen at the transplantation site of the bone marrow mesenchymal stem cells. At the 6th weekend there were positive expressions of neuron specific enolase and neurofibril protein at the transplantation site of the bone marrow mesenchymal stem cells. While in the phosphate buffered fluid group, there were negative expressions of neuron specific enolase, neurofibril protein, glial fibrillary acidic protein and nidosymptoms in the control group and the evaluation scores were all 9. 2 weeks after the transplantation, and the evaluation scores of the motor function in the transplantation group were higher than the ones in the phosphate buffered fluid group, [(6.7±0.9), (5.3-±1.0), (P ＜0.05)]. Six weeks after the transplantation, the evaluation scores of the motor function in the transplantation group were also higher than those in the phosphate buffered fluid group[(8.9±1. 1),(7.2±0.8),(P ＜0.05)].CONCLUSION: After their transplantation into the central nervous system,the bone marrow mesenchymal stem cells showed the ability to differentiate into neurons and glial cells, in which the characteristics of some neurons and glial cells were found. Bar walking test found that the evaluation scores of the motor function in the transplantation group were higher than those in the phosphate buffered fluid group, which suggests that the bone marrow mesenchymal stem cells have a significant effect on restoration of the functions of the nerves.