【摘要】：目的：探討切割穹窿海馬傘大鼠海馬與正常海馬內(nèi)磷脂酰乙醇胺結(jié)合蛋白(Phosphatidylethanolamine-binding protein, PEBP)的表達差異。方法：(1) Real-time PCR:42只SD大鼠隨機分成7組,每組6只。1組為正常對照組,其余6組分別為切割雙側(cè)穹窿海馬傘后1、3、7、14、21和28 d組。取各組大鼠的海馬組織,提取總RNA并合成第一鏈cDNA,應用Real-time PCR的方法分析切割穹窿海馬傘后大鼠海馬內(nèi)PEBP mRNA的表達變化。檢測切割穹窿海馬傘后各組及正常對照組PEBP和GAPDH基因表達的CT值,采用2-ΔΔCT法進行分析,所得的結(jié)果作為PEBP基因在不同時間點的相對表達量；(2)原位雜交：取6只SD大鼠,于切割右側(cè)穹窿海馬傘后第7d,灌注固定,制備海馬部位冰凍切片,采用地高辛標記的PEBP RNA探針,在海馬的前、中、后各取1張切片,進行原位雜交,觀察兩側(cè)海馬組織內(nèi)PEBP mRNA的表達變化。分別對每張切片切割側(cè)和正常側(cè)錐體細胞層、齒狀回顆粒層及門區(qū)、顆粒下層0.03mm2區(qū)域內(nèi)的PEBP mRNA陽性細胞進行計數(shù)和平均灰度值檢測；(3) Western Blot:42只SD大鼠隨機分成7組,每組6只。1組為正常對照組,其余6組分為切割雙側(cè)穹窿海馬傘后1、3、7、14、21和28 d組。取各組大鼠的海馬組織,提取總蛋白并測定濃度,隨后行SDS電泳并轉(zhuǎn)膜,用兔抗PEBP抗體進行免疫印跡檢測。對各時間點PEBP和β-actin陽性條帶分別進行平均灰度值檢測,二者比值的倒數(shù)作為PEBP蛋白不同時間點的相對表達量；(4)免疫組化：36只SD大鼠隨機分成6組,每組6只。分為切割右側(cè)穹窿海馬傘后1、3、7、14、21和28d組。各組大鼠經(jīng)心灌注固定后,取腦、冰凍切片,在海馬的前、中、后各取1張切片行PEBP免疫組化染色,分別計數(shù)切割側(cè)和正常側(cè)大鼠海馬錐體細胞層、齒狀回顆粒細胞層及門區(qū)、顆粒下層0.06mm2區(qū)域內(nèi)的PEBP陽性細胞的數(shù)目,并檢測其灰度值；(5)用SPSS11.5統(tǒng)計學軟件對各時間點數(shù)據(jù)進行方差分析和組間比較,對原位雜交和免疫組化切片數(shù)據(jù)進行配對t檢驗。結(jié)果：(1) Real-time PCR:海馬內(nèi)PEBP mRNA的相對表達量在正常組為1.16±0.10,在切割后第3d(1.51±0.08)開始升高,7d(1.64±0.07)達到最高水平,隨后下降,21d(1.14±0.13)左右恢復至正常水平。各組PEBP mRNA相對表達量方差分析和兩兩比較結(jié)果表明,3d、7d組與其余各切割組之間存在顯著性差異(P0.05),3d、7d組之間及其余各組之間比較無顯著性差異(P0.05)；(2)原位雜交：切割側(cè)和正常側(cè)海馬CA1～CA3錐體細胞層和齒狀回顆粒層細胞均有PEBP mRNA表達,PEBP mRNA陽性細胞數(shù)無顯著差異,但切割側(cè)PEBP mRNA陽性細胞的平均灰度值為78.47±4.45,正常側(cè)為102.58±15.99,切割側(cè)雜交信號明顯強于正常側(cè)(P0.01)。在切割側(cè)齒狀回門區(qū)和顆粒下層中觀察到較多的PEBP mRNA陽性細胞(60.67±6.71),平均灰度值為67.16±9.28；而正常側(cè)陽性細胞較少(50.33±8.52),平均灰度值為99.62±13.44,兩側(cè)陽性細胞數(shù)和平均灰度值比較有顯著性差異(P0.05);(3) Western Blot: PEBP蛋白的相對表達量正常對照組為0.0551±0.0093,在切割后第3d(0.077±0.0083)開始升高,7d(0.0965±0.0089)達到最高水平,隨后下降,28d(0.0505±±0.0066)左右恢復至正常水平。各組PEBP相對表達量方差分析和兩兩比較結(jié)果表明,各切割組與正常組比較,1d、28d組與正常組間無顯著性差異(P0.05),其余各切割組與正常組間均存在顯著性差異(P0.05);各切割組間比較,切割后7d組與其余各組均存在顯著性差異(P0.05),3d、14d、21d與1d、28d間存在顯著性差異(P0.05),而3d、14d、21d間無顯著性差異(P0.05),1d、28d間也無顯著性差異(P0.05)；(4)免疫組化：PEBP免疫組化結(jié)果顯示,PEBP主要表達于海馬CA1～CA3區(qū)錐體細胞層、齒狀回顆粒層及門區(qū)、顆粒下層的細胞胞漿中,各時間點切割側(cè)海馬錐體細胞層和齒狀回顆粒層的PEBP陽性細胞數(shù)與正常側(cè)相比無顯著性差異(P0.05),但切割側(cè)PEBP陽性細胞染色加深,7d時最為明顯,平均灰度值為68.41±10.53,而正常側(cè)平均灰度值為91.52±7.77,兩側(cè)比較有顯著性差異(P0.01)。切割側(cè)海馬齒狀回門區(qū)和顆粒下層中可見較多染色加深的PEBP陽性細胞,7d時最為明顯,陽性細胞數(shù)為53.33±4.27,平均灰度值為65.34±15.84,而正常側(cè)陽性細胞數(shù)為37.50±4.04,平均灰度值為97.16±11.62,切割側(cè)細胞數(shù)及平均灰度值與正常側(cè)相比均有顯著性差異(P0.05)。14d后錐體細胞層、齒狀回顆粒層及門區(qū)、顆粒下層PEBP的表達開始下降,28d時接近正常側(cè)。結(jié)論：(1)切割穹窿海馬傘后海馬組織中PEBP mRNA和蛋白的表達水平均明顯上調(diào),二者的表達水平均存在一個由低到高再到低的表達過程,且二者的升降趨勢基本一致,切割后7d時達到最高峰；(2) PEBP mRNA和蛋白主要表達于海馬CA1～CA3錐體細胞層、齒狀回顆粒細胞層、門區(qū)及顆粒下層的細胞之中；(3)切割側(cè)海馬CA1～CA3錐體細胞層、齒狀回顆粒細胞層與正常側(cè)比較僅有染色加深；而齒狀回門區(qū)及顆粒下層與正常側(cè)相比,不僅染色加深,而且陽性細胞數(shù)增多。提示切割穹窿海馬傘后海馬中PEBP mRNA和蛋白的表達增高可能與海馬中自體或植入的神經(jīng)干細胞向神經(jīng)元或膽堿能神經(jīng)元的分化有關。
[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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R341

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