PEBP在切割穹窿海马伞大鼠海马中的表达变化
发布时间：2018-08-03 13:03
【摘要】：目的：探讨切割穹窿海马伞大鼠海马与正常海马内磷脂酰乙醇胺结合蛋白(Phosphatidylethanolamine-binding protein, PEBP)的表达差异。方法：(1) Real-time 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28 d组。取各组大鼠的海马组织,提取总RNA并合成第一链cDNA,应用Real-time PCR的方法分析切割穹窿海马伞后大鼠海马内PEBP mRNA的表达变化。检测切割穹窿海马伞后各组及正常对照组PEBP和GAPDH基因表达的CT值,采用2-ΔΔCT法进行分析,所得的结果作为PEBP基因在不同时间点的相对表达量；(2)原位杂交：取6只SD大鼠,于切割右侧穹窿海马伞后第7d,灌注固定,制备海马部位冰冻切片,采用地高辛标记的PEBP RNA探针,在海马的前、中、后各取1张切片,进行原位杂交,观察两侧海马组织内PEBP mRNA的表达变化。分别对每张切片切割侧和正常侧锥体细胞层、齿状回颗粒层及门区、颗粒下层0.03mm2区域内的PEBP mRNA阳性细胞进行计数和平均灰度值检测；(3) Western Blot: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组分为切割双侧穹窿海马伞后1、3、7、14、21和28 d组。取各组大鼠的海马组织,提取总蛋白并测定浓度,随后行SDS电泳并转膜,用兔抗PEBP抗体进行免疫印迹检测。对各时间点PEBP和β-actin阳性条带分别进行平均灰度值检测,二者比值的倒数作为PEBP蛋白不同时间点的相对表达量；(4)免疫组化：36只SD大鼠随机分成6组,每组6只。分为切割右侧穹窿海马伞后1、3、7、14、21和28d组。各组大鼠经心灌注固定后,取脑、冰冻切片,在海马的前、中、后各取1张切片行PEBP免疫组化染色,分别计数切割侧和正常侧大鼠海马锥体细胞层、齿状回颗粒细胞层及门区、颗粒下层0.06mm2区域内的PEBP阳性细胞的数目,并检测其灰度值；(5)用SPSS11.5统计学软件对各时间点数据进行方差分析和组间比较,对原位杂交和免疫组化切片数据进行配对t检验。结果：(1) Real-time PCR:海马内PEBP mRNA的相对表达量在正常组为1.16±0.10,在切割后第3d(1.51±0.08)开始升高,7d(1.64±0.07)达到最高水平,随后下降,21d(1.14±0.13)左右恢复至正常水平。各组PEBP mRNA相对表达量方差分析和两两比较结果表明,3d、7d组与其余各切割组之间存在显著性差异(P0.05),3d、7d组之间及其余各组之间比较无显著性差异(P0.05)；(2)原位杂交：切割侧和正常侧海马CA1～CA3锥体细胞层和齿状回颗粒层细胞均有PEBP mRNA表达,PEBP mRNA阳性细胞数无显著差异,但切割侧PEBP mRNA阳性细胞的平均灰度值为78.47±4.45,正常侧为102.58±15.99,切割侧杂交信号明显强于正常侧(P0.01)。在切割侧齿状回门区和颗粒下层中观察到较多的PEBP mRNA阳性细胞(60.67±6.71),平均灰度值为67.16±9.28；而正常侧阳性细胞较少(50.33±8.52),平均灰度值为99.62±13.44,两侧阳性细胞数和平均灰度值比较有显著性差异(P0.05);(3) Western Blot: PEBP蛋白的相对表达量正常对照组为0.0551±0.0093,在切割后第3d(0.077±0.0083)开始升高,7d(0.0965±0.0089)达到最高水平,随后下降,28d(0.0505±±0.0066)左右恢复至正常水平。各组PEBP相对表达量方差分析和两两比较结果表明,各切割组与正常组比较,1d、28d组与正常组间无显著性差异(P0.05),其余各切割组与正常组间均存在显著性差异(P0.05);各切割组间比较,切割后7d组与其余各组均存在显著性差异(P0.05),3d、14d、21d与1d、28d间存在显著性差异(P0.05),而3d、14d、21d间无显著性差异(P0.05),1d、28d间也无显著性差异(P0.05)；(4)免疫组化：PEBP免疫组化结果显示,PEBP主要表达于海马CA1～CA3区锥体细胞层、齿状回颗粒层及门区、颗粒下层的细胞胞浆中,各时间点切割侧海马锥体细胞层和齿状回颗粒层的PEBP阳性细胞数与正常侧相比无显著性差异(P0.05),但切割侧PEBP阳性细胞染色加深,7d时最为明显,平均灰度值为68.41±10.53,而正常侧平均灰度值为91.52±7.77,两侧比较有显著性差异(P0.01)。切割侧海马齿状回门区和颗粒下层中可见较多染色加深的PEBP阳性细胞,7d时最为明显,阳性细胞数为53.33±4.27,平均灰度值为65.34±15.84,而正常侧阳性细胞数为37.50±4.04,平均灰度值为97.16±11.62,切割侧细胞数及平均灰度值与正常侧相比均有显著性差异(P0.05)。14d后锥体细胞层、齿状回颗粒层及门区、颗粒下层PEBP的表达开始下降,28d时接近正常侧。结论：(1)切割穹窿海马伞后海马组织中PEBP mRNA和蛋白的表达水平均明显上调,二者的表达水平均存在一个由低到高再到低的表达过程,且二者的升降趋势基本一致,切割后7d时达到最高峰；(2) PEBP mRNA和蛋白主要表达于海马CA1～CA3锥体细胞层、齿状回颗粒细胞层、门区及颗粒下层的细胞之中；(3)切割侧海马CA1～CA3锥体细胞层、齿状回颗粒细胞层与正常侧比较仅有染色加深；而齿状回门区及颗粒下层与正常侧相比,不仅染色加深,而且阳性细胞数增多。提示切割穹窿海马伞后海马中PEBP mRNA和蛋白的表达增高可能与海马中自体或植入的神经干细胞向神经元或胆碱能神经元的分化有关。
[Abstract]:Objective: To investigate the difference in expression of phosphatidylethanolamine binding protein (Phosphatidylethanolamine-binding protein, PEBP) in hippocampus and normal hippocampus of fornix fornix in rats. Methods: (1) Real-time PCR:42 SD rats were randomly divided into 7 groups, 6.1 groups in each group were normal control group, and the other 6 groups were divided into bilateral fornix fornix hippocampus parachute. After 1,3,7,14,21 and 28 d groups, the hippocampus of rats in each group was taken, the total RNA was extracted and the first chain cDNA was synthesized. The expression of PEBP mRNA in the hippocampus of the cut fornix fornix was analyzed by Real-time PCR. The CT value of the expression of PEBP and GAPDH genes in each group and the normal control group after the parachute of the fornix fornix was detected by the 2- delta delta method. Analysis, the results were used as the relative expression of PEBP gene at different time points; (2) in situ hybridization: 6 SD rats were taken after cutting the right fornix fornix parachute at 7d, the frozen section of the hippocampus was prepared, and the PEBP RNA probe labeled with digoxin was used for 1 slices in the front and after the sea horse, and in situ hybridization was carried out to observe in situ hybridization. The changes in the expression of PEBP mRNA in the bilateral hippocampal tissues, the count and the mean gray value of PEBP mRNA positive cells in the cutting side and the normal lateral pyramidal layer, the dentate gyrus granular layer and the portal area, the 0.03mm2 region of the subgranular layer; (3) the Western Blot:42 rats were randomly divided into 7 groups, 6.1 groups in each group were normal pairs. In the group, the other 6 groups were divided into group 1,3,7,14,21 and 28 d after cutting bilateral fornix fornix. The hippocampus tissue of each group was taken, the total protein was extracted and the concentration was measured. Then SDS electrophoresis and transmembrane were carried out. The Rabbit anti PEBP antibody was detected by immunoblotting. The average gray value of the PEBP and the positive bands of the beta -actin at each time point was detected, and the ratio of the two was respectively. The reciprocal expression was used as the relative expression of PEBP protein at different time points; (4) immunohistochemistry: 36 SD rats were randomly divided into 6 groups, each group was divided into group 1,3,7,14,21 and 28d after cutting the right fornix fornix. After the heart perfusion was fixed, the brain was taken, the frozen section was taken, and 1 slices were taken in the hippocampus before and after the PEBP immunohistochemical staining. Do not count the hippocampal pyramidal layer, the granular cell layer of the dentate gyrus and the number of PEBP positive cells in the 0.06mm2 region of the subgranular layer, and detect its gray value. (5) the data of each time point were analyzed and compared with the data of SPSS11.5 statistics, and the data of in situ hybridization and immunohistochemistry were used. Paired t test. Results: (1) the relative expression of PEBP mRNA in the hippocampus of Real-time PCR: was 1.16 + 0.10 in the normal group, and began to rise at 3D (1.51 + 0.08) after cutting, and 7d (1.64 + 0.07) reached the highest level, then decreased, and 21d (1.14 + 0.13) returned to the normal level. The relative expression of PEBP mRNA in each group was compared with 22 The results showed that there was significant difference between the 3D, 7d group and the other cutting groups (P0.05). There was no significant difference between the 3D, 7d group and the other groups (P0.05). (2) in situ hybridization: the CA1 to CA3 pyramidal and granular layer cells of the hippocampus and the dentate gyrus were expressed in PEBP mRNA, and there was no significant number of PEBP mRNA positive cells in the cutting side and the normal side of the hippocampus. The mean gray value of PEBP mRNA positive cells on the cutting side was 78.47 + 4.45, the normal side was 102.58 + 15.99, the cross signal of the cutting side was significantly stronger than the normal side (P0.01). More PEBP mRNA positive cells (60.67 + 6.71) were observed in the cutting side of the dentate return door and the subgranular layer, and the average gray value was 67.16 + 9.28, while the normal side was positive. The cells were less (50.33 + 8.52), the average gray value was 99.62 + 13.44, and there was a significant difference between the number of positive cells on both sides and the average gray value (P0.05). (3) the relative expression of Western Blot: PEBP protein was 0.0551 + 0.0093 in the normal control group, and began to rise at 3D (0.077 + 0.0083) after cutting, and 7d (0.0965 +) reached the highest level, followed by the following. The reduction of 28d (0.0505 + 0.0066) returned to the normal level. The analysis of the relative expression of PEBP and the results of 22 comparison showed that there was no significant difference between the cutting groups and the normal group (P0.05), and there were significant differences (P0.05) between the other cutting groups and the normal groups (P0.05), and the comparison between the cutting groups and the 7d after the cutting group was 7d. There was significant difference between the group and the other groups (P0.05), and there was a significant difference between 3D, 14d, 21d and 1D and 28d (P0.05), but there was no significant difference between 3D, 14d and 21d (P0.05), and there was no significant difference between 1D, and 21d. (4) immunohistochemistry: the immunohistochemical results showed that it was mainly expressed in the pyramidal cell layer of the hippocampus and the dentate gyrus There was no significant difference in the number of PEBP positive cells between the hippocampal pyramidal layer and the dentate gyrus in the subgranular layer and the subgranular layer. The number of PEBP positive cells in the hippocampal pyramidal layer and the dentate gyrus at each time point had no significant difference (P0.05), but the PEBP positive cells in the cutting side were dyed deeper and the 7d was the most obvious. The average gray value was 68.41 + 10.53, while the average gray value of the normal side was 91.52. There was a significant difference between the two sides (P0.01). The PEBP positive cells with more dyeing and deepening in the dentate and granular layer of the cutting side were seen, the most obvious in 7d, the number of positive cells was 53.33 + 4.27, the average gray value was 65.34 + 15.84, while the number of positive cells in the normal side was 37.50 + 4.04, the average gray value was 97.16 + 11.62, the cutting side was in the cutting side. The number of cells and the average gray value were significantly different from that of the normal side (P0.05) the pyramidal layer, the granular layer and the portal area of the dentate gyrus, the expression of PEBP in the subgranular layer began to decline, and the 28d was close to the normal side. Conclusion: (1) the expression level of PEBP mRNA and protein in the hippocampus of the fornix fornix hippocampus were obviously up-regulated, and the expression of the two were expressed. There is a process of expression from low to high to low, and the ascending and lowering trend of the two is basically the same, and the highest peak is reached after 7d. (2) PEBP mRNA and protein are mainly expressed in the hippocampal CA1 to CA3 pyramidal layer, the granular cell layer of the dentate gyrus, and the cells in the portal and subgranular layers; (3) the hippocampal CA1 to CA3 pyramidal cells in the cutting side of the hippocampus In the dentate gyrus, the granular cell layer was only stained with the normal side, while the dentate return door and the subgranular layer were not only dyed, but also the number of positive cells increased. It suggested that the increase of PEBP mRNA and protein in the hippocampus in the hippocampus of the fornix fornix may be associated with the autologous or implanted neural stem cells in the hippocampus to the nerve. Differentiation of meta or cholinergic neurons.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341
本文编号：2161828
[Abstract]:Objective: To investigate the difference in expression of phosphatidylethanolamine binding protein (Phosphatidylethanolamine-binding protein, PEBP) in hippocampus and normal hippocampus of fornix fornix in rats. Methods: (1) Real-time PCR:42 SD rats were randomly divided into 7 groups, 6.1 groups in each group were normal control group, and the other 6 groups were divided into bilateral fornix fornix hippocampus parachute. After 1,3,7,14,21 and 28 d groups, the hippocampus of rats in each group was taken, the total RNA was extracted and the first chain cDNA was synthesized. The expression of PEBP mRNA in the hippocampus of the cut fornix fornix was analyzed by Real-time PCR. The CT value of the expression of PEBP and GAPDH genes in each group and the normal control group after the parachute of the fornix fornix was detected by the 2- delta delta method. Analysis, the results were used as the relative expression of PEBP gene at different time points; (2) in situ hybridization: 6 SD rats were taken after cutting the right fornix fornix parachute at 7d, the frozen section of the hippocampus was prepared, and the PEBP RNA probe labeled with digoxin was used for 1 slices in the front and after the sea horse, and in situ hybridization was carried out to observe in situ hybridization. The changes in the expression of PEBP mRNA in the bilateral hippocampal tissues, the count and the mean gray value of PEBP mRNA positive cells in the cutting side and the normal lateral pyramidal layer, the dentate gyrus granular layer and the portal area, the 0.03mm2 region of the subgranular layer; (3) the Western Blot:42 rats were randomly divided into 7 groups, 6.1 groups in each group were normal pairs. In the group, the other 6 groups were divided into group 1,3,7,14,21 and 28 d after cutting bilateral fornix fornix. The hippocampus tissue of each group was taken, the total protein was extracted and the concentration was measured. Then SDS electrophoresis and transmembrane were carried out. The Rabbit anti PEBP antibody was detected by immunoblotting. The average gray value of the PEBP and the positive bands of the beta -actin at each time point was detected, and the ratio of the two was respectively. The reciprocal expression was used as the relative expression of PEBP protein at different time points; (4) immunohistochemistry: 36 SD rats were randomly divided into 6 groups, each group was divided into group 1,3,7,14,21 and 28d after cutting the right fornix fornix. After the heart perfusion was fixed, the brain was taken, the frozen section was taken, and 1 slices were taken in the hippocampus before and after the PEBP immunohistochemical staining. Do not count the hippocampal pyramidal layer, the granular cell layer of the dentate gyrus and the number of PEBP positive cells in the 0.06mm2 region of the subgranular layer, and detect its gray value. (5) the data of each time point were analyzed and compared with the data of SPSS11.5 statistics, and the data of in situ hybridization and immunohistochemistry were used. Paired t test. Results: (1) the relative expression of PEBP mRNA in the hippocampus of Real-time PCR: was 1.16 + 0.10 in the normal group, and began to rise at 3D (1.51 + 0.08) after cutting, and 7d (1.64 + 0.07) reached the highest level, then decreased, and 21d (1.14 + 0.13) returned to the normal level. The relative expression of PEBP mRNA in each group was compared with 22 The results showed that there was significant difference between the 3D, 7d group and the other cutting groups (P0.05). There was no significant difference between the 3D, 7d group and the other groups (P0.05). (2) in situ hybridization: the CA1 to CA3 pyramidal and granular layer cells of the hippocampus and the dentate gyrus were expressed in PEBP mRNA, and there was no significant number of PEBP mRNA positive cells in the cutting side and the normal side of the hippocampus. The mean gray value of PEBP mRNA positive cells on the cutting side was 78.47 + 4.45, the normal side was 102.58 + 15.99, the cross signal of the cutting side was significantly stronger than the normal side (P0.01). More PEBP mRNA positive cells (60.67 + 6.71) were observed in the cutting side of the dentate return door and the subgranular layer, and the average gray value was 67.16 + 9.28, while the normal side was positive. The cells were less (50.33 + 8.52), the average gray value was 99.62 + 13.44, and there was a significant difference between the number of positive cells on both sides and the average gray value (P0.05). (3) the relative expression of Western Blot: PEBP protein was 0.0551 + 0.0093 in the normal control group, and began to rise at 3D (0.077 + 0.0083) after cutting, and 7d (0.0965 +) reached the highest level, followed by the following. The reduction of 28d (0.0505 + 0.0066) returned to the normal level. The analysis of the relative expression of PEBP and the results of 22 comparison showed that there was no significant difference between the cutting groups and the normal group (P0.05), and there were significant differences (P0.05) between the other cutting groups and the normal groups (P0.05), and the comparison between the cutting groups and the 7d after the cutting group was 7d. There was significant difference between the group and the other groups (P0.05), and there was a significant difference between 3D, 14d, 21d and 1D and 28d (P0.05), but there was no significant difference between 3D, 14d and 21d (P0.05), and there was no significant difference between 1D, and 21d. (4) immunohistochemistry: the immunohistochemical results showed that it was mainly expressed in the pyramidal cell layer of the hippocampus and the dentate gyrus There was no significant difference in the number of PEBP positive cells between the hippocampal pyramidal layer and the dentate gyrus in the subgranular layer and the subgranular layer. The number of PEBP positive cells in the hippocampal pyramidal layer and the dentate gyrus at each time point had no significant difference (P0.05), but the PEBP positive cells in the cutting side were dyed deeper and the 7d was the most obvious. The average gray value was 68.41 + 10.53, while the average gray value of the normal side was 91.52. There was a significant difference between the two sides (P0.01). The PEBP positive cells with more dyeing and deepening in the dentate and granular layer of the cutting side were seen, the most obvious in 7d, the number of positive cells was 53.33 + 4.27, the average gray value was 65.34 + 15.84, while the number of positive cells in the normal side was 37.50 + 4.04, the average gray value was 97.16 + 11.62, the cutting side was in the cutting side. The number of cells and the average gray value were significantly different from that of the normal side (P0.05) the pyramidal layer, the granular layer and the portal area of the dentate gyrus, the expression of PEBP in the subgranular layer began to decline, and the 28d was close to the normal side. Conclusion: (1) the expression level of PEBP mRNA and protein in the hippocampus of the fornix fornix hippocampus were obviously up-regulated, and the expression of the two were expressed. There is a process of expression from low to high to low, and the ascending and lowering trend of the two is basically the same, and the highest peak is reached after 7d. (2) PEBP mRNA and protein are mainly expressed in the hippocampal CA1 to CA3 pyramidal layer, the granular cell layer of the dentate gyrus, and the cells in the portal and subgranular layers; (3) the hippocampal CA1 to CA3 pyramidal cells in the cutting side of the hippocampus In the dentate gyrus, the granular cell layer was only stained with the normal side, while the dentate return door and the subgranular layer were not only dyed, but also the number of positive cells increased. It suggested that the increase of PEBP mRNA and protein in the hippocampus in the hippocampus of the fornix fornix may be associated with the autologous or implanted neural stem cells in the hippocampus to the nerve. Differentiation of meta or cholinergic neurons.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341
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