【作者】 施金洪；

【导师】 金国华；

【作者基本信息】 南通大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【摘要】 目的:研究Brn-4在海马神经干细胞向神经元分化中的作用,探讨穹窿海马伞切割后海马神经再生过程中NSCs向神经元分化的生物学机制。方法:实验一免疫荧光双标:分别制备切割穹窿海马伞侧和正常侧海马提取液,将体外培养的鼠胚海马神经干细胞球分成切割组、正常组和对照组,其中前两组中分别加入切割穹窿海马伞侧海马提取液和正常侧海马提取液。各组培养细胞分别于第3、7、14和21d行Brn-4/MAP-2免疫荧光双标检测。用图像处理系统分别计数每视野下Brn-4阳性细胞和Brn-4/MAP-2双标神经元数,并测量双标神经元的胞体面积、细胞周长及Brn-4的免疫荧光强度。用Stata8.0软件对数据进行方差分析和组间比较。实验二RNA干扰:将加入切割穹窿海马伞海马提取液、体外培养的鼠胚海马神经干细胞球分别接种入6孔培养板和24孔培养板中。筛选有效序列、优化转染条件后将接种于6孔培养板和24孔培养板中的细胞均随机分成沉默组和未沉默组,沉默组中加入针对Brn-4的有效siRNA序列。转染后第3、7、14和21d时分别提取6孔培养板中的细胞总RNA,行半定量RT-PCR,以各泳道中Brn-4与GAPDH条带的光密度比值表示Brn-4 mRNA的相对表达量;24孔培养板中的细胞于第3、7、14和21d时分别行时分别行Brn-4/MAP-2免疫荧光双标检测,用图像处理系统计数Brn-4阳性细胞和Brn-4/MAP-2双标神经元数,测量双标神经元的胞体面积、细胞周长以及Brn-4的免疫荧光强度。用Stata8.0软件对数据进行方差分析和组间比较。结果:实验一各组中Brn-4/MAP-2双标神经元数、胞体面积和细胞周长随时间推移均呈现由低到高再逐渐降低、Brn-4的免疫荧光强度也呈现由低到高再逐渐降低的趋势,且两者均于培养后第14d达到高峰的时相性特点;同一时间点的三组中,切割组双标神经元最多、胞体最大、突起最丰富,且Brn-4的免疫荧光也最强,正常组次之,而对照组最差。统计分析表明,除3d时各组Brn-4免疫荧光强度相比差异无统计学意义(P>0.05)外,其余数据组间比较均有统计学意义(P<0.01)。实验二本实验优化的转染条件合适,转染试剂无明显毒副作用,转染效率为95%。6孔培养板中第3、7、14d时同一时间点的沉默组和未沉默组相比,沉默组中Brn-4 mRNA的相对表达量明显低于未沉默组,组间比较P均<0.01,第21d时组间差异缩小,P<0.05。24孔培养板中同一时间点的两组比较,沉默组Brn-4/MAP-2双标细胞数少,胞体小,突起不丰富,且Brn-4的免疫荧光弱,与未沉默组比较,P均<0.01。结论:在切割穹窿海马伞海马提取液促进体外培养的海马神经干细胞向神经元分化过程中,Brn-4的表达明显增强;加入针对Brn-4的siRNA后Brn-4 mRNA和蛋白表达量明显降低,且神经干细胞向神经元的分化明显受到影响。提示Brn-4在大鼠海马神经干细胞向神经元分化过程中可能起着重要作用。更多还原

【Abstract】 Objective: To study the role of Brn-4 on the neural stem cells from hippocampus differentiating into neurons, and to investigate the biology mechanism of neural regeneration and reparation after fimbria/fornix transection. Methods: Immunofluorescence double-labeling in experiment one: NSCs isolated and expanded from the hippocampus of embryonic rat were divided into 3 groups: the transection group, the normal group and the control group. Then the extracts from the fimbria/fornix transected and intact hippocampi of adult SD rats previously obtained were respectively added into the transection and the normal group. The neurons differentiated from NSCs were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after culturing. The number of Brn-4 positive cells, the intensity of immuno- fluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double- labeling positive neurons were analyzed by image processing software respectively. Stata8.0 statistical software was adopted to analyze the results. RNA interference in experiment two: NSCs isolated and expanded from the hippocampus of embryonic rat and cultured in vitro were planted into 6-well and 24-well cell culture plate respectively with the extract from the fimbria/fornix transected hippocampi. Then the cells were divided into the silenced group and the unsilenced group from two plates respectively after siRNA sequence filtered and transfection condition optimized, and the effective fragment was added into the silenced group. The total RNA was extracted from cells of 6-well cell culture plate on the 3rd, 7th, 14th and 21st day after transfection. The relative level of Brn-4 mRNA expression was indicated by the ratio of the optical density value of Brn-4 to that of GAPDH by RT-PCR. The cells of 24-well cell culture plate were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after transfection. The number of Brn-4 positive cells, the intensity of immunofluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double-labeling positive neurons were measured respectively by image processing system. The analysis of variance and group comparison were applied with Stata8.0 statistical software. Results: In experiment one, the number of Brn-4/MAP-2 double-labeling positive neurons and its body area, perimeter increased gradually after transfection. And the peak appeared on the 14th day, then the growing status degraded later. The change of the intensity of immunofluorescence of Brn-4 showed the same tendency. Among three groups, the number, body area and perimeter of double-labeling cells were the best in the transection group. Compared with the control group, the growing status was better in the normal group. The differences were significant between any two groups except the intensity of immunofluorescence of Brn-4 on the 3rd day among three groups (P<0.01). The results in the experiment two showed that the optimized transfection condition was suitable and toxico side effect of transfection reagent was indistinct. It also suggested that the efficiency of transfection was 95 percent. The relative level of Brn-4 mRNA expression of the silenced group was obviously lower than the unsilenced group on the 3rd, 7th, 14th day respectively(P<0.01), and the difference decreased on the 21st day between the two groups(P<0.05). The Brn-4/MAP-2 double-labeling positive neurons were fewer, the bodies were smaller, and the intensity of immunofluorescence of Brn-4 was thinner in the silenced group compared with the unsilenced group significantly(P<0.01). Conclusion: The expression level of Brn-4 was enhanced obviously when the neural stem cells differentiated into neurons facilitated by the extracts from the fimbria/fornix transected hippocampi. The expression of Brn-4 mRNA and protein were degraded after the siRNA aimed directly at the Brn-4 gene, and the differentiation of the neural stem cells into neurons was influenced obviously. The results suggested that Brn-4 may play an important role in the process of neural stem cells differentiating into neurons.更多还原