【作者】 邹琳清；

【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:观察切割穹窿海马伞侧和非切割侧大鼠海马自体NSCs的增殖、迁移和向神经元分化的情况,以及NSCs向神经元分化过程中与Brn-4表达的关系,探讨穹窿海马伞切割后海马内神经再生与修复的机制。方法:实验一:36只SD大鼠随机分成6组,每组6只,分别为切割右侧穹窿海马伞后3、5、7、14、21和28d组。各组大鼠于处死前1d腹腔注射BrdU,每隔8h一次,共注射3次。取脑冰冻切片,行BrdU免疫荧光检测,计数切割侧和非切割侧海马齿状回内BrdU阳性细胞,应用Stata 7.0统计软件进行配对t检验。实验二:36只SD大鼠随机分成6组,每组6只,分别为切割右侧穹窿海马伞后3、5、7、14、28和42d组。各组大鼠于切割术后2d开始,腹腔注射BrdU,每天1次,连续5 d。取脑冰冻切片,进行BrdU免疫荧光、BrdU/NF-200和BrdU/GFAP免疫荧光双标检测,用Leica Qwin图像处理软件进行处理,计数切割侧和非切割侧海马齿状回内BrdU阳性细胞、BrdU/NF-200双标细胞和BrdU/GFAP双标细胞,观察其迁移和分布。应用Stata7.0统计学软件对两侧BrdU/NF-200双标细胞和BrdU/GFAP双标细胞的数量进行配对t检验,不同时相点之间的数据进行方差分析和SNK检验。实验三:36只SD大鼠随机分成6组,每组6只,分别为切割右侧穹窿海马伞后3、7、14、21、28和35d组。各组大鼠于切割术后2d开始,腹腔注射BrdU,每天1次,连续5 d。取脑冰冻切片,进行BrdU/Brn-4/β-TubulinⅢ免疫荧光三标检测,用Leica Qwin图像处理软件进行处理,分别计数海马齿状回门区和颗粒下层中BrdU/Brn-4阳性双标细胞和检测Brn-4荧光强度、计数BrdU/Brn-4/β-TubulinⅢ阳性三标细胞。用Stata7.0统计软件,分别对切割侧和非切割侧齿状回门区和颗粒下层中BrdU/Brn-4阳性双标细胞数和Brn-4的荧光强度、以及BrdU/Brn-4/β-TubulinⅢ阳性三标细胞数进行配对t检验,对切割侧各时相点数据进行方差分析和SNK检验。结果:实验一:切割后3d,切割侧海马内BrdU阳性的自体NSCs较非切割侧开始增加,5d时增加明显,7d时达到高峰, 14d后增殖开始下降,但仍高于非切割侧,28d时两侧均接近于正常水平;实验二:切割后3d时BrdU阳性的自体NSCs主要位于门区,5d时开始沿颗粒下层迁移,7d时迁移增多,14d时部分阳性细胞进入颗粒层,28d后进入颗粒层的细胞增多;术后5d,切割侧海马内BrdU/GFAP阳性细胞开始出现,7d时BrdU/NF-200阳性细胞开始出现,14d后双标的阳性细胞数均逐渐增加,28d后达高峰,42d时双标阳性细胞不再增加, BrdU/NF-200阳性细胞仅约占BrdU阳性细胞的7%,而BrdU/GFAP阳性的细胞较多,约占BrdU阳性细胞的70%,非切割侧未见到BrdU/NF-200阳性细胞,仅见少量BrdU/GFAP阳性细胞;实验三:切割侧海马齿状回门区和颗粒下层中,3d时可见到BrdU/Brn-4双标细胞,Brn-4荧光较弱,7d时BrdU/Brn-4双标细胞明显增多,Brn-4荧光增强,14d时BrdU/Brn-4双标细胞进一步增多并达到高峰,Brn-4荧光最强;21d后Brn-4阳性细胞未见明显减少,但荧光强度逐渐减弱,并接近于非切割侧;14d始切割侧海马内明显见到BrdU/Brn-4/β-TubulinⅢ三标细胞,21d时三标细胞增多,28d达到高峰。结论:切割穹窿海马伞后海马内自体NSCs明显增殖,其增殖强度呈现由低到高再逐渐恢复正常、切割后7d时最强的时相性特点;切割穹窿海马伞后,增殖的自体NSCs逐渐沿颗粒下层迁移,呈条带状排列,部分进入颗粒层;迁移过程中的自体NSCs少部分可分化为神经元,其数量约占增殖神经干细胞数的7%,大部分分化为胶质细胞;切割穹窿海马伞后,海马齿状回中自体神经干细胞向神经元的分化可能与Brn-4表达的增强有关。更多还原

【Abstract】 Objective: To observe the proliferation, migration and neuronal differentiation of the endogenous neural stem cells(NSCs) in the bilateral hippocampus of adult rat with unilateral fimbria/fornix transection, and the relationship between the expression of Brn-4 and the neuronal differentiation, and to investigate the mechanism of neural regeneration and reparation in the hippocampus after fimbria/fornix transection.Methods: Section One: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, which served as right fimbria/fornix transected 3rd, 5th, 7th, 14th, 21st and 28th day group, respectively. Each rat was injected intraperitoneally with 5-bromodeoxyuridine (BrdU) at 8 hours intervals, three times together, in the day before killed. The cryostat sections of the brain were prepared. Then, BrdU immunofluorescence detection was employed, and the number of BrdU immunoreactive cells in the bilateral dentate gyrus was counted using a fluorescent microscope. Paired t test analysis was used in statistic analysis of the number of BrdU positive cells with Stata7.0 software. Section Two: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, which served as right fimbria/fornix transected 3rd, 5th, 7th, 14th, 28th and 42nd day group, respectively. On the 2nd day after transection, BrdU was injected intraperitoneally once a day for 5 days. Then the rats were perfused and fixed. The brains were taken, and the cryostat sections of hippocampus were prepared. The BrdU immunofluorescence test was used to investigate the migration and distribution pattern of newborn cells. To detect the number of BrdU-labeled cells differentiating into neurons or astrocytes, BrdU/NF-200 and BrdU/GFAP double-labeled immunofluorescence test were employed. The Leica Qwin software was used for the image processing. Paired t test analysis was used for statistic analysis of the number of BrdU/NF-200 double-labeled cells and BrdU/GFAP double-labeled cells in bilateral hippocampus. Data between every two time points were analyzed using the ANOVA and SNK test. Section Three: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, which served as right fimbria/fornix transected 3rd, 7th, 14th, 21st, 28th and 35th day group, respectively. On the 2nd day after operation, BrdU was injected intraperitoneally once daily for 5 consecutive days. Then the cryostat sections of the brain were prepared. BrdU/Brn-4/β-TubulinⅢimmunofluorescence triple-labeled method, and the Leica Qwin image processing software were used to observe the number of BrdU/Brn-4 double-labeled cells, the number of BrdU/Brn-4/β-TubulinⅢtrible-labeled cells, and the Brn-4 fluorescent intensity in hilus and subgranular zone(SGZ) of dentate gyrus. Paired t test analysis, and one-way analysis of variance and comparison were used for above data in bilateral hippocampus and different time points respectively with stata 7.0 software. Results: Section One: Comparing with non-transection side, BrdU positive cells increased slightly on day 3 after operation, thereafter, increased markedly on day 5 and maximally on day 7 after fimbria/fornix transection in transection side. The number of BrdU positive cells in transection side decreased on day 14 after operation, but was still greater than non-transection side. The number of BrdU positive cells appeared to return to control levels on day 28. Section Two: The location of BrdU-labeled cells in the dentate gyrus changed as the time went on. At 3rd day after fimbria/fornix transection, most labeled cells were located in the hilus of dentate gyrus. At 5th day some labeled cells migrated out into the SGZ. At 7th day the majority cells were located in the SGZ or the edge of the granule cell layer(GCL). At 14th day after fimbria/fornix transection, many of the cells were still in the SGZ, but some were found throughout the GCL. At 28th day more newborn cells were found throughout the GCL. At 5 days after fimbria/fornix transection, some labeled cells expressed astrocyte marker—GFAP, but not expressed neuronal marker—NF-200. However, BrdU/NF-200 double-labeled cells could be observed at 7 days, increased gradually at 14 days, reached the peak at 28 days, and did not increase significantly at 42 days. A similar increase was observed as the time went on in the number of BrdU/GFAP double-labeled cells. Among the BrdU-labeled population in the dentate gyrus, about 7% cells were BrdU/NF-200 double-labeled neurons, and about 70% cells were BrdU/GFAP double-labeled astrocytes. Several BrdU/GFAP double-labeled cells were observed only, but no BrdU/NF-200 double-labeled neurons at any time point in non-transection side’s dentate gyrus. Section Three: In hilus and SGZ of dentate gyrus in transection side, BrdU/Brn-4 double-labeled cells could be observed on day 3, in which the intensity of Brn-4 labeling was less. The number of double-labeled cells and the fluorescent intensity of Brn-4 increased significantly on day 7, and reached peak on day 14. Then the double-labeled cells decreased slowly and the fluorescent intensity of Brn-4 was lost to leve1 of non-transection side on day 21. BrdU/Brn-4/β-TubulinⅢtriple-labeled cells appeared on day 14 after transection, increased on day 21 and reached peak on day 28.Conclusion: The endogenous NSCs in hippocampus proliferated significantly after fimbria/fornix transection. The intensity of proliferation changed as the time went on, that is, it increased at first and then decreased to normal level, and reached peak on day 7. The proliferating endogenous NSCs migrated and lined as a strip along the SGZ, and some migrated into the GCL. Approximately 7% of the proliferating cells could differentiate into neurons during migration. However, most endogenous NSCs differentiated into astrocytes. The increasing expression of Brn-4 might be related to the endogenous NSCs in the dentate gyrus differentiating into neurons after fimbria/fornix transection.更多还原