骨髓间充质干细胞定向移植局灶性脑缺血大鼠的行为学变化

背景:骨髓间充质干细胞移植已经应用到神经损伤修复领域,脑内立体定位移植和经血管移植均为其移植途径.目的:经立体定位将骨髓间充质干细胞注入大鼠脑梗死灶周边区,通过前肢不对称应用试验及姿势反射试验观察大鼠神经功能障碍的改善情况. 设计:随机对照动物实验.单位:吉林大学第一医院神经内科.材料:实验于2006-10/2007-04在吉林大学人兽共患病研究所,人兽共患病教育部重点实验室进行.健康雄性Wistar大鼠由吉林大学实验动物中心提供,体质量250～280 g,清洁级,实验过程中对动物处置符合动物伦理学标准.方法:采用密度梯度分离法、贴壁筛选法体外培养扩增健康成人志愿者骨髓间充质干细胞,采用流式细胞仪鉴定其免疫表型.将Wistar大鼠随机分为正常对照组、假手术组、模型组、模型 无血清DMEM组、模型 骨髓间充质干细胞组,每组10只.采用Longa等改良线栓法制备大鼠右侧大脑中动脉闭塞脑缺血再灌注模型,正常对照组无任何处理,假手术组手术操作至暴露颈内外动脉后即缝合,其余处理同模型组.骨髓间充质干细胞立体定向移植:缺血90 min再灌注后1 h,采用立体定位仪取大鼠右侧大脑缺血周边区为移植点:前囟旁开3 mm、尾侧1 mm、深4 mm.缓慢注入5 μL经BrdU标记的骨髓间充质干细胞(4×1011 L-1)无血清培养基悬液于模型 骨髓间充质干细胞组大鼠,模型 无血清DMEM组大鼠注射5 μL无血清培养基,注射后留针5 min,缓慢退针,防止液体随针道返流.采用免疫组化技术检测骨髓间充质干细胞在大鼠体内存活情况,并于移植后1,3,7,28 d采用前肢不对称应用试验及姿势反射试验观察大鼠行为学变化.主要观察指标:①以流式细胞仪鉴定骨髓间充质干细胞免疫表型.②移植大鼠脑内骨髓间充质干细胞的存活情况.③大鼠行为学变化.结果:50只大鼠全部进入结果分析.①实验获得了高纯度的骨髓间充质干细胞,流式细胞仪检测显示,CD44、CD29均呈阳性,CD34、CD45、CD31均呈阴性.②模型 骨髓间充质干细胞组移植的骨髓间充质干细胞在脑缺血周边区聚集并存活.③模型 骨髓间充质干细胞组大鼠行为学评分较其他各组降低明显 (P < 0.05),并随着细胞移植后时间延长逐渐降低,并且移植后7 d行为学评分低于同组内其他时间点(P < 0.01).结论:除正常对照组外,其他组大鼠神经功能均有所恢复,但各组恢复情况明显不同,脑梗死周边区域注射骨髓间充质干细胞组大鼠功能恢复明显好于各对照组.

Behavioral changes in rats with focal cerebral ischemia after directional transplantation of bone marrow mesenchymal stem cells

BACKGROUND: Transplantation of bone marrow mesenchymal stem cells (MSCs) has been used in the field of repair of nerve injury. Brain stereotactic transplantation and transvascular transplantation are two transplantation methods. OBJECTIVE: We infused MSCs into rat peripheral cerebral infarct focus, in order to investigate the improvement of rat neurological dysfunction by forelimb use asymmetry test and postural reflex test.DESIGN: A randomized controlled animal experiment. SETTING: Department of Neurology, First Hospital of Jilin University.MATERIALS: This study was performed at the Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University between October 2006 and April 2007. Healthy male Wistar rats of clean grade, weighing 250-280 g, were provided by the Laboratory Animal Center of Jilin University. The protocol was performed in accordance with ethical guidelines for the use and care of animals.METHODS: MSCs from healthy adult volunteers were in vitro cultured and proliferated by density gradient separation and adherence screening method. Their immunophenotypes were identified by a flow cytometer. The Wistar rats were randomized into 5 groups with 10 rats in each: normal control group, sham-operated group, model group, serum-free DMEM-treated group (DMEM group) and MSCs -treated group (MSCs group). Rat models of cerebral ischemia/reperfusion were developed by occluding rat right middle cerebral artery following suture occlusion method modified by Longa et al. Rats in the normal control group were untouched. In the sham-operated group, operation was not ended till cervical interior and exterior arteries were exposed and sutured, and the other disposals were the same as those in the model group. At ischemia 90 minutes reperfusion 1 hour, a stereotaxic apparatus was used to take rat right peripheral cerebral ischemic region as transplantation site: 3 mm lateral to, 1mm caudal to and 4 mm posterior to Bregma. Rats in the MSCs group were slowly injected 5 μL BrdU-labeled MSCs (4×1011 L-1) serum-free medium. Rats in the DMEM group were injected 5 μL serum-free medium. After perfusion, inserted needle was retained for 5 minutes and then slowly withdrawn in order to avoid the back flow of liquid from needle pole. The survival of MSCs in rats was detected by immunohistochemical technique, and rat behavioral changes of observed on days1, 3, 7 and 28 after transplantation by forelimb use asymmetry test and postural reflex test.MAIN OUTCOME MEASURES: ① The immunophenotype of MSCs were identified by a flow cytometer. ② The survival of transplanted MSCs in the rat brain. ③ Rat behavioral changes. RESULTS: All the 50 rats were included in the final analysis. ① High purity of MSCs were harvested in the experiment. Flow cytometer detection showed that both CD44 and CD29 were positive, while CD34, CD45 and CD31 were negative. ② MSCs transplanted into the brains of rats in the MSCs group gathered in the peripheral cerebral ischemic region and survived. ③ Behavioral scores of rats in the MSCs group were significantly lower than those in the other groups (P < 0.05). They were gradually decreased with time after transplantation, and reached the valley value on day 7 after transplantation (P < 0.01). CONCLUSION: Neurological function of rats recovers in all the groups except normal control group. But the recovery differs in different groups, and neurological function of rats in the MSCs group recovers better than that in other groups.