骨髓基质干细胞移植对脑梗死大鼠生长相关蛋白43表达的影响

背景:目前生长相关蛋白43作为构建中枢神经系统可塑性的基本物质,是研究神经生长及损伤修复的首选分子标记物.目的:通过建立大鼠局灶性脑缺血模型,观察骨髓基质干细胞移植内源性轴突再生标志物生长相关蛋白43的表达变化.设计、时间及地点:随机对照动物实验,于2006-03/2007-10在武汉大学人民医院神经科实验室和丝宝药业有限公司博士后科研工作站完成.材料:选取清洁级成年SD大鼠60只,按随机数字表法分为模型对照组、假手术组和干细胞移植组,每组20只.方法:另取2月龄SD大鼠4只用于制备骨髓基质干细胞,骨髓基质干细胞悬液用5-溴脱氧尿嘧啶核苷法标记.假手术组大鼠麻醉后分离结扎右侧颈内总动脉;其余大鼠制备右侧大脑中动脉缺血模型,造模后24 h向干细胞移植组大鼠左侧侧脑室推注20 μL5×105的骨髓基质干细胞,模型对照组推注等量磷酸盐缓冲液.主要观察指标:每组大鼠于移植前、移植后7,14,21,28 d应用免疫组织化学法检测脑梗死灶周边区生长相关蛋白43的表达和移植细胞的存活及迁移情况,并记录神经功能缺损评分.结果:培养的骨髓基质干细胞经5-溴脱氧尿嘧啶核苷标记后移植到大鼠左侧侧脑室,可迁移到梗死灶周围,移植后7 d在梗死灶能检测到5溴脱氧尿嘧啶核苷标记的阳性细胞,移植后14 d增多达高峰,移植后28 d逐渐减少并消失.免疫组织化学图像分析结果显示干细胞移植组大鼠在移植后7,14 d脑梗死灶周边区生长相关蛋白43免疫活性高于模型对照组(P<0.05).假手术组大鼠无神经损伤症状,神经功能评分均为0分;随时间的推移,模型对照组和干细胞移植组动物的神经功能评分逐渐降低,从移植后14 d开始,干细胞移植组的神经功能评分均明显低于模型对照组(P<0.05).结论:骨髓基质干细胞移植能上调局灶性脑缺血大鼠脑梗死灶周边区生长相关蛋白43的表达,与大鼠神经功能的恢复趋势相符.

Influence of bone marrow mesenchymal stem cell transplantation on growth-associated protein-43 expression in rats with cerebral infarction

BACKGROUND:Growth-associated protein-43 is the primary substance for constructing plasticity of central nervous system, which is recognized as the first molecular marker for studying growth and repairing injury of nerves. OBJECTIVE: To explore the expression of growth-associated protein-43 in rats with focal cerebral ischemia following bone marrow mesenchymal stem cells (MSCs) transplantation.DEISNG, TIME AND SETTING: A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Renmin Hospital of Wuhan University and Doctoral Scientific Research Work Station of C-BONS Pharmacy, Hubei, China, from March 2006 to October 2007.MATERIALS: Sixty adult Sprague Dawley rats, with clean grade, irrespective of genders, weighing (180±20) g, were randomly divided into the model, sham operated, and MSCs transplantation groups, with 20 animals in each group. METHODS: Additional 4 Sprague Dawiey rats, aged 2 months, were selected to isolate and culture MSCs, which was labeled with 5-Bromo-2,-deoxyuridine (BrdU). In the sham operated group, the right internal carotids were deligated after anaesthesia. The remained rats were prepared for models of right middle cerebral artery ischemia. At 24 hours after model preparation, 20 μL culture solution containing 5×105/L MSCs was injected into the left lateral ventricle of rats in the MSCs group, the same volume of phosphate buffer saline was injected in the model group.MAIN OUTCOME MEASURES: The rats were sacrificed prior to and at days 7, 14, and 21 after transplantation. The expression of growth associated protein-43 at the infarcted areas, the survival and migration of transplanted cells were examined by immunohistochemistry, at the same time, neurological deficit scores were recorded.RESULTS: The transplanted MSCs migrated from the left lateral ventricle to the infracted areas. Seven days after transplantation, the expression of BrdU-positive cells was found, reached a peak at day 14, and gradually decreased, until disappeared at 28 days after transplantation. Results of immunohistochemistry image analysis showed that immunological activity of growth associated protein-43 around the infarcted area of the MSCs group was obviously greater than that of the model group at days 7 and 14 after transplantation (P<0.06). There was no neurological deficit in the sham operated group. Moreover, with time prolonged, the neurological deficit scores gradually decreased in the model and MSCs groups, which were significantly lower in the MSCs group compared to the model group at day 14 after transplantation (P<0.05).CONCLUSION: MSCs transplantation up-regulates the expression of growth associated protein-43 around the infarcted area, which is consistent with the recovery of neurological function.