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

Effects of bone marrow stromal stem cell transplantation on growth-associated protein-43 expression in cerebral infarction rats

背景:关于骨髓干细胞移植修复神经损伤的研究已有一些报道,但对细胞移植的时间、方式以及检测指标各有不同观点。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3组,模型对照组、假手术组、干细胞移植组各20只。方法:另取2月龄SD大鼠4只用于制备骨髓基质细胞,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总动脉;其余大鼠制备右侧大脑中动脉缺血模型,造模后24h向干细胞移植组大鼠左侧侧脑室推注20μL5×105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和28d应用免疫组化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7d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14d增多达高峰,移植后28d逐渐减少并消失。免疫组化图像分析结果显示干细胞移植组大鼠在移植后7,14d脑梗死灶周边区生长相关蛋白43免疫活性显著高于模型对照组(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的神经功能评分逐渐降低,从移植后14d开始,干细胞移植组的神经功能评分都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。

BACKGROUND： There are studies on bone marrow mesenchymal stem cells in the repair of nerve injury, and varied studies addressing cell transplantation time, manner and detection index. OBJECTIVE： To explore the expression of growth-associated protein-43 in rats with focal cerebral ischemic brain damage following bone mesenchymal stem cell transplantation. DEISNG, TIME AND SETTING： A randomized controlled animal study was performed at the Laboratory of Department of Neurology, Renmin Hospital of 60 Wuhan University and Doctoral Scientific Research Work Station of C-BONS PHARMA, Hubei, China from March 2006 to October 2007. MATERIALS： A total of clean adult Sprague-Dawley rats were equally and randomly assigned into model control group, sham operation group and stem cell transplantation group. METHODS： Another four 2-month Sprague Dawley rats were used to prepare bone marrow stromal cells. Bone marrow stromal stem cell suspension was labeled with 5-bromodeoxyuridine. Following anesthesia, rats in the sham operation group were utilized to deligate fight common carotid artery. The remaining rats were employed to establish fight middle cerebral artery models. At 24 hours following model induction, 20 u L 5× 10^5 bone marrow stromal stem cells were infused into the rat left lateral ventricle in the stem cell transplantation group. An equal volume of phosphate buffer saline was injected into the rats in the model control group. MAIN OUTCOME MEASURES： Expression of growth associated protein-43, cell survival and migration surrounding the cerebral infarcted region was observed using the immunohistochemistry before transplantation, at 7, 14, 21 and 28 days following transplantation in each group, and score of neurologic impairment was recorded. RESULTS： Cultured bone marrow stromal stem cells labeled with 5-bromodeoxyuridine were transplanted into the left lateral cerebral ventricle, and could migrate surrounding the infarcted region. At 7 days following transplantation, 5-bromodeoxyuridine-labeled cells could be detected surrounding the infarcted region. At 14 days following transplantation, the increased number of cells reached a peak, but gradually disappeared at 28 days following transplantation. Immunohistochemistry imaging analysis results demonstrated that immunological activity of growth associated protein-43 was significantly higher in the stem cell transplantation group than in the model control group at 7 and 14 days following transplantation （P 〈 0.05）. No nerve injury was tested in rats from the sham operation group, and the score of neural function was 0 point. With time prolonged, the score of neural function was gradually reduced in the model control and stem cell transplantation groups. The score was significantly lower in the stem cell transplantation group than in the model control group at 14 days following transplantation （P 〈 0.05）. CONCLUSION： Bone marrow stromal stem cell transplantation can up-regulate growth associated protein-43 expressions surrounding the infarcted region after focal cerebral ischemia, which is accorded with the recovery of neural function in rats.