【作者】 王磊；

【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， 2006， 硕士

【摘要】 目的：观察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，探讨穹窿海马伞切割后海马内神经再生与修复的分子生物学机制。方法：(1)RT-PCR：42只SD大鼠随机分成7组，每组6只。1组为正常对照组，其余6组分别为切割双侧穹窿海马伞后1、3、7、14、21和28d组。取各组大鼠的海马组织，提取总RNA，采用半定量RT-PCR法分析穹窿海马伞切割后海马内Brn-4 mRNA表达水平的变化。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，使用Stata7.0统计学软件进行单因素方差分析和两两比较。(2)原位杂交：取6只SD大鼠，切割右侧穹窿海马伞。切割后14d，灌注固定，制备海马部位冰冻切片；采用体外转录法制备地高辛标记的Brn-4 RNA探针，进行原位杂交，观察两侧海马组织内Brn-4 mRNA的表达变化。每只动物取前、中、后3张切片，分别对每张切片切割侧和正常侧Brn-4 mRNA阳性细胞进行计数和光密度值检测，并应用Stata7.0统计学软件对两侧Brn-4 mRNA阳性细胞的数量和平均光密度值进行配对t检验分析。结果：(1)海马内Brn-4 mRNA的表达量在正常组为0.138±0.06，在切割后第3d(0.256±0.54)开始升高，14d(0.719±0.124)达到最高水平，随后下降，28d(0.189±0.059)左右恢复至正常水平。(2)切割侧和正常侧海马锥体细胞层和齿状回颗粒层细胞均有Brn-4 mRNA表达，Brn-4 mRNA阳性细胞数量无明显差异，但切割侧Brn-4 mRNA阳性细胞的平均光密度值(0.27±0.06)较正常侧(0.14±0.02)明显增加，t值为11.4709，P<0.01。在切割侧齿状回门区和颗粒下层中观察到Brn-4 mRNA阳性细胞(40.5±9.37)，平均光密度值为0.28±0.05，而正常侧仅有少量阳性细胞(6.5±2.07)，且平均光密度值仅为0.09±0.02，两侧阳性细胞数和平均光密度值相比较，P均<0.01。结论：切割穹窿海马伞后的海马组织内Brn-4 mRNA表达明显上调，并且呈现由低到高再逐渐恢复正常，切割后14d时最高的时相性特点；Brn-4 mRNA的表达见于切割侧和正常侧海马锥体细胞层和齿状回颗粒层细胞，但Brn-4 mRNA的表达量切割侧明显高于正常侧；Brn-4 mRNA的表达还见于切割侧和正常侧齿状回门区和颗粒下层的细胞中，其阳性细胞数和表达量切割侧远较正常侧明显升高。提示切割穹窿海马伞后海马中Brn-4 mRNA表达的增高可能与促进其中的神经干细胞向神经元分化有关。更多还原

【Abstract】 Objective: To observe the difference of Brn-4 mRNA expression inhippocampus between the fimbria/fornix-transected rats and normal ones,and to investigate the molecular biology mechanism of neural regenerationand reparation after fimbria/fornix-transection. Methods: (1) RT-PCR:Forty-two SD rats were randomly divided into 7 groups, 6 rats in each group.One group served as normal control and the others served as fimbria/fornixtransected 1st, 3rd, 7th, 14th, 21st and 28th day group, respectively. Thenhippocampi were isolated and total RNA was extracted. Semi-quantitativereverse transcriptase-polymerase chain reaction (RT-PCR) method was usedin detection of the expression of Brn-4 mRNA in hippocampus. The relativeexpression level of Brn-4 mRNA was indicated by the ratio of the opticaldensity value of Brn-4 to that of GAPDH. The Stata7．0 software was used inone-factor analysis of variance and comparison between every two groups.(2) In Situ Hybridization: Six SD rats’ right fimbrias were transected. Onthe 14th day after transection, the rats were perfused and fixed, then cryostatsections of hippocampus were prepared. Digoxigenin-labeled Brn-4 RNAprobe was prepared and used in hybridization to observe expression changeof Brn-4 mRNA in hippocampus after operation. Three sections were takenfrom each rat in anterior, middle and posterior location, respectively. Thenumber and the mean optical density value of Brn-4 mRNA positive cells intransected and normal sides was measured, respectively. Paired t test analysiswas used in statistic analysis of the number and the mean optical densityvalue of Brn-4 mRNA positive cells with Stata7．0 software. Results: (1) RT-PCR: In normal group, The relative expression level of Brn-4 mRNAwas 0．138±0．06．It started to increase on day3(0．256±0．54) aftertransection, and the peak appeared on day 14(0．719±0．124) , then decreasedslowly to pre-transection level on day28(0．189±0．059) . (2) In SituHybridization: Brn-4 mRNA was expressed in pyramidal layer ofhippocampus and granular layer of dentate gyrus in both sides. The numberof Brn-4 mRNA positive cells has no discrepancy between two sides. But themean optical density value of Brn-4 mRNA positive cells in transectedside(0．27±0．06) was higher than that in normal side(0．14±0．02) , t=11．4709,P<0．01．Brn-4 mRNA positive cells was also seen in hilus and subgranularlayer of dentate gyrus. In transected side, the number of Brn-4 mRNApositive cells was 40．5±9．37, the mean optical density value was 0．28±0．05．In normal side, there was only few Brn-4 mRNA positive cells (6．5±2．07) , and the mean optical density value was 0．09±0．02．For the twoobjects, P values were both less than 0．01．Conclusion: The expressionlevel of Brn-4 mRNA increases after fimbria/fornix transection. Theexpression of Brn-4 mRNA is changed with time, that is, it increases at firstand then decreases to normal level, and reaches peak on day 14; Brn-4mRNA is expressed in pyramidal layer of hippocampus and granular layer ofdentate gyrus in both sides. The expression level of Brn-4 mRNA issignificantly higher in transected side than that in normal side; Brn-4 mRNAis also expressed in hilus and subgranular layer of dentate gyrus in both sides.The number and the mean optical density value of Brn-4 mRNA positivecells were both significantly higher in transected side than these in normalside. The results suggest that the process in which the expression of Brn-4mRNA increases after fimbria/fornix transection mights be related to theneural stem cells differentiating into neurons in hippocampus.更多还原