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背景:骨髓间充质干细胞移植技术对脑损伤修复具有重要的意义,但其作用途径尚需讨论。目的:观察脑缺血模型大鼠移植骨髓间充质干细胞后,皮质及海马区Bcl-2和Bax的表达变化,分析骨髓间充质干细胞颅内的移植抑制神经元凋亡的作用机制。设计:随机对照实验。单位:青岛大学医学院附属医院脑血管病研究所。材料:选用雄性成年Wistar大鼠24只,体质量180～240g,由山东大学实验动物中心提供实验大鼠随机分为对照组、损伤组、损伤移植组,每组8只。溴脱氧核苷尿嘧啶(BrdU,Sigma公司);TUNEL试剂盒,Bcl-2,Bax抗体试剂盒购自北京中山生物工程公司。方法:实验于2005-04/2006-09在青岛大学医学院附属医院脑血管病研究所完成。损伤组和移植组大鼠采用颈外动脉线栓法建立脑缺血模型,移植组大鼠造模成功7d后在脑皮质和纹状体区注射浓度为2×10~(12)L~(-1)的原代体外培养的骨髓间充质干细胞悬液。实验过程中对动物的处置符合动物伦理学要求。主要观察指标:分别于造摸成功后7和14d进行TUNEL染色及免疫组织化学检测大脑皮质、海马区神经元凋亡和Bcl-2,Bax表达的变化。结果:①神经元凋亡情况:造模成功后7d,对照组来发现凋亡细胞,移植组凋亡细胞的数量少于损伤组,差异有非常显著性意义(P<0.01)。②Bcl-2和Bax表达:造模成功后14d,损伤组大鼠皮质和海马区Bcl-2阳性神经元的表达低于对照组和移植组,差异有非常显著性意义(P<0.01)。损伤组大鼠皮质和海马区Bax阳性神经元的表达高于对照组和移植组,差异有非常显著性意义(P<0 01)。结论:大鼠脑缺血后,骨髓间充质干细胞能够促进Bcl-2表达并抑制Bax表达,从而减少神经元凋亡。
BACKGROUND: Bone marrow-derived mesenchymal stem cell transplantation is of great importance for the repair of brain injury. However, its mechanism of action needs to be discussed. OBJECTIVE: To observe the changes of Bcl-2 and Bax expression in cortex and hippocampus after transplantation of bone marrow mesenchymal stem cells in rats with cerebral ischemia, and to analyze the mechanism of intracerebral transplantation of bone marrow mesenchymal stem cells to inhibit neuronal apoptosis. Design: Randomized controlled experiment. Unit: Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College. MATERIALS: Twenty-four male adult Wistar rats weighing 180-240g were used. Experimental rats were randomly divided into control group, injury group and injury group. There were 8 rats in each group. BrdU (Sigma); TUNEL kit, Bcl-2, Bax antibody kit was purchased from Beijing Zhongshan Bioengineering Company. METHODS: The experiment was performed at the Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College from April 2005 to September 2006. The rats in the injury group and the transplantation group were subjected to the external carotid artery occlusion. The rats in the transplantation group were injected intracerebral cortex and striatum 7 days after the successful modeling, and the injection concentration was 2 × 10 ~ (12) L ~ (-1) -1) primary cultured in vitro bone marrow mesenchymal stem cell suspension. The handling of animals during the experiment conformed to the ethical requirements of animals. MAIN OUTCOME MEASURES: Neuronal apoptosis and changes of Bcl-2 and Bax expression in the cerebral cortex and hippocampus were detected by TUNEL staining and immunohistochemistry at 7 and 14 days after the successful establishment of the model. Results: ①Apoptosis of neurons: Apoptotic cells were detected in the control group 7 days after the model was established, the number of apoptotic cells in the transplantation group was less than that in the injury group (P <0.01). ②The expression of Bcl-2 and Bax in the injured cortex and hippocampus was lower than that in the control group and the transplantation group on the 14th day after model establishment, the difference was significant (P <0.01). The expression of Bax positive neurons in the cortex and hippocampus in the injury group was significantly higher than that in the control group and the transplantation group (P <0.01). CONCLUSION: Bone marrow mesenchymal stem cells can promote the expression of Bcl-2 and inhibit the expression of Bax after cerebral ischemia in rats, thereby reducing neuronal apoptosis.