【作者】 徐璐璐；

【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， 2009， 硕士

【摘要】 目的:观察切割大鼠右侧穹窿海马伞后,切割侧与正常侧海马内脑脂结合蛋白(brain lipid binding protein, BLBP)的表达变化。方法:(1)半定量RT-PCR:36只SD大鼠随机分成6组,每组6只。1组为正常对照组,其余5组分别为切割右侧穹窿海马伞后1、3、5、7和14d组。取各组大鼠的海马组织,提取总RNA,采用半定量RT-PCR法分析右侧穹窿海马伞切割后海马内BLBP mRNA表达水平的变化。以各泳道中BLBP与GAPDH条带的光密度比值表示BLBP mRNA的相对表达量,使用Stata8.0统计学软件进行单因素方差分析和两两比较。(2)Western Blot: 36只SD大鼠随机分成6组,每组6只。1组为正常对照组,其余5组分别为切割右侧穹窿海马伞后1、3、5、7和14d组。取各组大鼠的海马组织,制备成提取液,进行SDS-PAGE、转膜,用BLBP多抗进行免疫印迹检测。对BLBP阳性条带和内参β-actin条带分别进行积分光密度分析,二者的积分光密度比值作为BLBP蛋白不同时相点的相对表达量,用Stata8.0统计软件进行单因素方差分析和组间比较。(3)免疫组织化学方法:36只SD大鼠随机分成6组,每组6只。1组为正常对照组,其余5组分别为切割右侧穹窿海马伞后1、3、5、7和14d组。各组大鼠灌注固定,取脑、冰冻切片,行BLBP免疫组织化学染色。每只动物海马的前、中、后各取1张切片共3张切片,每张切片对海马齿状回门区和颗粒下层0.075㎜ 2区域中的BLBP阳性细胞数和灰度值进行检测分析,应用Stata8.0统计软件进行配对t检验。结果:1.RT-PCR:正常组右侧海马内BLBP mRNA的相对表达量为0.057±0.019。切割后1d时表达量为0.065±0.013,3d(0.441±0.107)开始升高,5d(0.803±0.128)达到最高水平,7d(0.459±0.099)时开始下降,14d(0.082±0.016)时恢复至接近正常水平。统计学分析表明:切割后3d组、5d组、7d组与正常对照组相比较差异均有显著性意义(P<0.01),并且切割后5d组与3d组、7d组、14d组相比较差异有显著性意义(P <0.01)。2.Western Blot:BLBP相对表达量在切割后1d略升高(0.602±0.051),但与正常对照组(0.5714±0.085)相比无显著性意义(P>0.05),切割后3 d(1.151±0.078)继续升高,至切割后5 d(1.458±0.132)达到最高水平,切割后7 d(1.043±0.058)开始下降,切割后14 d(0.869±0.088)继续下降至接近正常水平。统计学分析表明:除切割后1d组外,其余切割后各d组与正常对照组相比较差异均有显著性意义(P均<0.01)。切割后各d组之间除3d组与7d组,7d组与14d组之间差异无显著性意义(P>0.05),其余各组间的差异均有显著性意义(P <0.01或=0.01)。3.免疫组织化学染色:正常大鼠双侧海马各区和齿状回中细胞仅有BLBP微量表达,切割穹窿海马伞后1 d两侧差异不明显;3 d时切割侧颗粒下层阳性细胞及染色深度较正常侧增多加深;5 d时切割侧颗粒下层阳性细胞数量明显增多,染色较深,胞体变大,突起增长,并达到最高水平;7 d后BLBP阳性细胞数量和染色深度开始降低,14d时接近正常侧水平。而切割后3 d、5 d时两侧门区BLBP阳性细胞的数量差异不大,但切割侧染色较深,7 d后也逐渐降低,14 d时降至正常侧水平。结论:切割穹窿海马伞后的海马组织中BLBP mRNA和蛋白的表达明显上调,其表达量呈现由低到高再逐渐恢复正常、切割后5d时表达量最高的时相性特点; BLBP阳性细胞主要位于切割侧和正常侧海马锥体细胞层和齿状回颗粒下层及门区中,切割侧表达强度明显高于正常侧;切割穹窿海马伞侧海马齿状回门区中的BLBP阳性放射状胶质细胞与正常侧相比未见明显增多,而颗粒下层中BLBP阳性的放射状胶质细胞明显增多,且胞体增大,突起变粗增长,呈现“激活”现象,提示其构成的支架不仅有助于神经干细胞的迁移,可能还与神经干细胞的分化有关。更多还原

【Abstract】 Objective: To observe the change of the brain lipid binding protein (BLBP) expression in dentate gyrus(DG)region of transected and normal hippocampi after the right side of rats′fimbria-fornix transection. Methods: (1)RT-PCR: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group. One group served as normal control and the others served as fimbria-fornix transected 1st, 3rd, 5th, 7th and 14th day group, respectively. Then hippocampi were isolated and total RNA was extracted. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used in detection of the expression of BLBP mRNA in hippocampus after the right side of rats′fimbria-fornix transection. The relative expression level of BLBP mRNA was indicated by the ratio of the optical density value of BLBP to that of GAPDH. The Stata8.0 software was used in one-factor analysis of variance and comparison between every two groups. (2)Western Blot: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group. One group served as normal control and the others served as fimbria-fornix transected 1st, 3rd, 5th, 7th and 14th day group, respectively. Then hippocampi were isolated and the extracts were gained from the fimbria-fornix transected and the normal hippocampi. The expression product of BLBP was detected by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. The relative expression level of BLBP protein was indicated by the ratio of the integral optical density value of BLBP to that ofβ-actin. The Stata8.0 software was used for one-factor analysis of variance and comparison between every two groups. (3)Immunohistochemistry: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group. One group served as normal control and the others served as fimbria-fornix transected 1st, 3rd, 5th, 7th and 14th day group, respectively. The rats were perfused and fixed, then cryostat sections of hippocampus were prepared for BLBP immunohistochemistry. Three sections were taken from each rat in first, middle and back hippocampus.The number and gray scale volume of BLBP positive cells in 0.075㎜ 2 aera of hilus and subgranular layer of dentate gyrus were measured, respectively. Paired t-test was used for the number and the gray scale volume of BLBP positive cells with Stata8.0 software. Results: (1)RT-PCR: In normal group, The relative expression level of BLBP mRNA was 0.057±0.019. On the 1st day, the expression level of BLBP mRNA was 0.065±0.013. It started to increase on the3rd day (0.256±0.54) after transection, and the peak appeared on the 5th day (0.719±0.124). Then it started to decrease on the 7th day(0.459±0.099)and closed to the normal level on the 14th day(0.082±0.016). Statistical analysis showed that there were not only statistically significant differences between the normal control group and the 3rd, 5th and 7th day group after transection (P<0.01), but also between the 5th day group and the 3rd, 7th and 14th day group after transection (P<0.01). (2)Western Blot: On the 1st day, the expression level of BLBP protein was 0.602±0.051, which appeared no statistically significant differences by comparing with the normal control group (P>0.05). It started to increase on the 3rd day (1.151±0.078) after transection, and the peak appeared on the 5th day (1.458±0.132). Then it started to decrease on the 7th day(1.043±0.058)and closed to the normal level on the 14th day(0.869±0.088). Statistical analysis showed that except the 1st day, there were statistically significant differences between all the other groups and the control group group (P<0.01); there were also statistically significant differences between each two groups (P<0.01 or =0.01), except between the 3rd day group and the 7th day group , between the 7th day and the 14th day (P>0.05). (3) Immunohistochemistry: The present results showed that few BLBP positive cells were expressed in both sides of each region and subgranular layer of hippocampus in control group. After the fimbria-fornix transection, no significant difference was observed in both sides on the 1st day. Compared with the normal side, more BLBP positive cells with deeper staining were found in the subgranular layer of the transected side on the 3rd day. The number and the color of the larger-body positive cells with growing neurite in subgranular layer of transected side were detected much more than those in normal side and appeared to the peak on the 5th day. Then it started to decrease 7 days later and closed to the normal level on the 14th day. But, the number of the BLBP positive cells showed little difference in both sides of hilus on the 3rd and 5th day after transection, while the color was more deeper in the fimbria-fornix transected side. Seven days later, it also decreased slowly and closed to pre-transection leve1 on the 14th day. Conclusion: The expression level of BLBP mRNA and protein increase after fimbria-fornix transection. The expression of BLBP mRNA and protein are changed with time, that is, those increase at first and then decrease to normal level, and reach peak on the 5th day. BLBP positive cells are expressed in pyramidal layer of hippocampus , subgranular layer and hilus of dentate gyrus in both sides, which are significantly higher in transected side than those in normal side; there are few increased BLBP-positive radial glial cells in hilus of fimbria-fornix transected side by compareing with the normal side, while BLBP-positive radial glial cells markedly increase in subgranular layer, the cell bodys become larger, and the processes grow longer, which show an "activation" phenomenon. The results suggest that the scaffolds of BLBP-positive radial glial cells might conduce to the neural stem cells migration and the differentiation to the neurons.更多还原