RT-PCR／Southern杂交法检测穹窿海马伞切割大鼠海马Brn-4 mRNA.docVIP

RT-PCR／Southern杂交法检测穹窿海马伞切割大鼠海马Brn-4 mRNA 　 作者：董传明 秦建兵，金国华，王 磊，朱蕙霞，田美玲 【摘要】 目的:探讨切割穹窿海马伞大鼠海马与正常海马内Brn-4 mRNA表达的差异。方法：42只SD大鼠随机分成正常对照组和切割穹窿海马伞后1、3、7、14、21及28 d 组。取各组大鼠的海马组织，提取总RNA，采用RT-PCR/Southern 杂交方法分析穹窿海马伞切割后海马内Brn-4 mRNA的表达变化。结果：海马内Brn-4 mRNA的表达量在切割后第3 d开始升高，14 d达到最高水平，随后下降，28 d左右恢复至正常水平。结论：切割穹窿海马伞后海马内Brn-4 mRNA表达明显上调，可能与促进海马内移植的或自体的神经干细胞向神经元分化有关。 【关键词】 穹窿海马伞切割；海马；Brn-4；逆转录聚合酶链反应；Southern 杂交；大鼠 [Abstract] Objective: To investigate the difference of Brn-4 mRNA expression between the fimbria fornix transected rats and normal ones． Methods: 42 SD rats were randomly divided into 7 groups，6 rats in each group．One group served as normal control and the others served as the 1st，3rd，7th ，14th ，21st and 28th day group after fimbria fornix transection, respectively. Then hippocampi were isolated and total RNA was extracted. RT-PCR/Southern blot was used to detect the change of Brn-4 mRNA expression in hippocampus after fimbria fornix transection. Results: The expression level of Brn-4 mRNA started to increase on the 3rd day after fimbria fornix transection．The peak appeared on the 14th day，and then the expression decreased slowly to pre-transection level on the 28th day. Conclusion: The process that the expression of Brn-4 mRNA increases significantly after fimbria fornix transection, perhaps，might be related to the transplanted or endogenous neural stem cells differentiating into neurons in hippocampus． [Key words] Fimbria fornix transection；Hippocampus；Brn-4；Reverse transcription-PCR；Southern blot; Rat 我们在近年的研究发现，将神经干细胞分别移植到切割穹窿海马伞侧海马和正常侧海马中，神经干细胞在切割侧海马中更易于存活、迁移和向神经元分化[1，2]。体外诱导分化发现，切割穹窿海马伞侧的海马提取液促进神经干细胞向神经元分化的作用也明显强于正常侧海马提取液[3]。提示切割穹窿海马伞后，海马中某些诱导神经干细胞迁移和向神经元分化的物质表达增强。 新近我们还证实切割穹窿海马伞后也能促进切割侧海马中的自体神经干细胞向神经元分化。POU同源盒基因在中枢神经系统的发育过程中起重要作用，已证实其家族成员Brn-4作为重要的转录因子在胚胎纹状体神经干细胞向神经元分化过程中起调控作用[4]。本文使用RT-PCR/Southern 杂交方法观察了切割穹窿海马伞后不同时间点大鼠海马Brn-4 mRNA表达量的变化，以期阐明Brn-4的表达变化与海马中植入的或自体的神经干细胞向神经元分化的相关性。 1 材料和方法 1.1 实验动物及分组 SD大鼠42只(南通大学实验动物中心提供)，雌雄不拘，体重220～250 g，随机分成7组，其中1组为正常对照组，其