PEBP在切割穹窿海马伞大鼠海马中的表达变化
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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(P<0.05),3d、7d组之间及其余各组之间比较无显著性差异(P>0.05)；(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(P<0.01)。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(P<0.05);(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(P>0.05),其余各切割组与正常组间均存在显著性差异(P<0.05);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(P<0.05),3d、14d、21d与1d、28d间存在显著性差异(P<0.05),而3d、14d、21d间无显著性差异(P>0.05),1d、28d间也无显著性差异(P>0.05)；(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(P>0.05),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(P<0.01)。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(P<0.05)。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。
Objective:To observe the difference of PEBP expression in hippocampus between the fimbria-fornix-transected rats and normal ones. Methods:(1) Real-time PCR:Forty-two SD rats were randomly divided into 7 groups,6 rats in each group. One group served as normal control group and the others served as fimbria-fornix transected 1st,3rd,7th,14th,21st and 28th day group respectively. Then hippocampi were isolated and total RNA was extracted and then the first strand cDNA was synthesized, the method of Real-time PCR was used to observe the changes of PEBP mRNA expression in hippocampus on different time points. CT value of PEBP and GAPDH in normal group and transected groups were detected by the method of 2-△△CT T respectively. The relative expression level of PEBP mRNA was indicated by the result of the method of 2-△△CT. (2) In Situ Hybridization:Six SD rats'right fimbria-fornix were transected. On the 7th day after transection, the rats were perfused and fixed, then cryostat sections of hippocampus were prepared. DIG-labeled PEBP RNA probe was prepared and used in hybridization in order to observe the expression change of PEBP mRNA in hippocampus of both sides. One section was taken from anterior, middle and posterior location respectively of each rat. The number and the mean gray value of PEBP mRNA positive cells were measured in transected and normal sides of pyramidal layer, granular layer of dentate gyrus and on 0.03mm2 area of hilus, subgranular of dentate gyrus respectively. (3) Western Blot:Forty-two SD rats were randomly divided into 7 groups,6 rats in each group. One group served as normal control, and fimbria-fornix of the other groups rats were transected on the 1st,3rd,7th,14th,21st and 28th day respectively. Then hippocampi were isolated and the total protein was acquired from the fimbria-fornix transected and the normal hippocampus, then detected the concentration of each group, the protein sample was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the membrane. The expression was detected by PEBP polyclonal antibody immunoblot. The mean gray value of PEBP and B-actin positive strips was then detected, the relative expression level of PEBP protein was indicated by the reciprocal of the mean gray value of PEBP to B-actin. (4) Immunohistochemistry:Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, and served as right fimbria-fornix transected 1st,3rd,7th,14th,21st and 28th day group respectively. The rats were perfused and fixed, then cryostat sections of hippocampus were prepared for PEBP immunohistochemistry. One section was taken from anterior, middle and posterior location respectively of each rat. The number and gray value of PEBP positive cells in pyramidal layer and granular layer of dentate gyrus, in hilus of dentate gyrus and subgranular layer of 0.06mm2 area on both transected and normal sides were measured respectively. (5) Analysis of variance and mutual comparision of each group was analyzed by SPSS 11.5 software, and the both sides'results of in situ hybridization and immuohistochemisty were conducted by the method of paired t test. Results:(1) Real-time PCR:In normal group, the relative expression level of PEBP mRNA was 1.16±0.10. It started to increase on 3rd day (1.51±0.08) after transection, and the peak appeared on 7th day(1.64±0.07), then decreased slowly to pre-transection level on 21st (1.14±0.13). One-factor analysis of variance and comparison between each two groups of the PEBP mRNA relative expression manifested that there were significant difference between the fimbria-fornix transected 3rd,7th day groups and other groups (P<0.05). The difference was not significant between 3rd and 7th day group, and not significance existed among other groups (P>0.05). (2) In Situ Hybridization:PEBP mRNA was expressed in CA1～CA3 pyramidal layer of hippocampus and granular layer of dentate gyrus on both sides. The number of PEBP mRNA positive cells manifested no discrepancy between two sides. But the mean gray value of PEBP mRNA positive cells in transected side (78.47±4.45) was lower than that in normal side (102.58±15.99), the signal was higer in transected side than normal side. PEBP mRNA positive cells was also seen in hilus and subgranular layer of dentate gyrus, in transected side, the number of PEBP mRNA positive cells was 60.67±6.71, the mean gray value was 67.16±9.28. In normal side, there was less PEBP mRNA positive cells (50.33±8.52), and the mean gray value was 99.62±13.44. The positive cell numbers and mean gray value of two sides existed significant difference (P<0.05). (3) Western Blot:Western Blot showed that the expression of PEBP protein was 0.0551±0.0093 in control group. It started to increase on 3rd day (0.077±0.0083) and the peak appeared on 7th day (0.0965±0.0089), then decreased slowly to pre-transection level on 28th day (0.0505±0.0066). When transected groups compared with the normal group, statistic results showed no significant difference between the 1st,28th day group and the normal group (P<0.05), while significant difference existed between the normal group and the other five transected groups (P<0.05). When transected groups compared mutually, the difference between the 7th day and other transected groups was significant (P<0.05), the significant difference also existed between the 3rd,14th,21st day group and 1st、28th day groups, while the difference among 3rd,14th,21st day groups were not significant (P>0.05), the difference between the 1st and 28th day was not significan either. (P>0.05). (4) Immunohistochemistry:The result of PEBP immunohistochemistry showed that PEBP was majorly expressed in the kytoplasm of cells which located in the pyramidal layer of CA1～CA3 areas and granular layer, hilus, subgranular layer of dentate gyrus. The number of PEBP protein positive cells has no discrepancy between two sides in the pyramidal layer of CA1～CA3 areas and (P>0.05). But the stain of PEBP positive cells in transected side was deeper than normal side, especially on 7th day, the mean gray value was 68.41±10.53, while the mean gray value was only 91.52±7.77 in the normal side, the significant discrepancy existed between the two sides (P<0.01). More deeper stained PEBP positive cells could be seen in the hilus and subgranular of dentate gyrus, especially on the 7th day after transection, the number of positive cells was 53.33±4.27, the mean gray value was 65.34±15.84, while the number and mean gray value of positive cells in the normal side was 37.5±4.04 and 97.16±11.62 respectively, the significant discrepancy existed between the two sides (P<0.05). The expression of PEBP in pyramidal layer, hilus and subgranular of dentate gyrus was decreased on the 14th day after transection and reached pre-transection level on the 28th day. Conclusion:(1) The expression of PEBP mRNA and protein obviously increased after fimbria-fornix transection, the expression changed with time, it increased at first and then decreased to normal level, the ascending and descending tendency of PEBP mRNA and protein was generally coincident, it reached peak on the 7th day after transection. (2) The PEBP mRNA and protein majorly expressed in the kytoplasm which located in the CA1～CA3 areas of pyramidal layer, granular layer, hilus and subgranular layer of dentate gyrus. (3) Cells in the CA1～CA3 areas of pyramidal layer, granular layer of dentate gyrus were stained deeper in the transection side than the normal side. However, compared with the normal side, cells in the hilus and subgranular layer of dentate gyrus were not only stained deeper but also possessed more numbers of positive cells. The results indicated that the higher expression of PEBP mRNA and protein in the fimbria-fornix transection side hippocampus may involve the hippocampal neural regeneration which induce the neural stem cells differentiating into neurons or cholinergic neurons.
Objective:To observe the difference of PEBP expression in hippocampus between the fimbria-fornix-transected rats and normal ones. Methods:(1) Real-time PCR:Forty-two SD rats were randomly divided into 7 groups,6 rats in each group. One group served as normal control group and the others served as fimbria-fornix transected 1st,3rd,7th,14th,21st and 28th day group respectively. Then hippocampi were isolated and total RNA was extracted and then the first strand cDNA was synthesized, the method of Real-time PCR was used to observe the changes of PEBP mRNA expression in hippocampus on different time points. CT value of PEBP and GAPDH in normal group and transected groups were detected by the method of 2-△△CT T respectively. The relative expression level of PEBP mRNA was indicated by the result of the method of 2-△△CT. (2) In Situ Hybridization:Six SD rats'right fimbria-fornix were transected. On the 7th day after transection, the rats were perfused and fixed, then cryostat sections of hippocampus were prepared. DIG-labeled PEBP RNA probe was prepared and used in hybridization in order to observe the expression change of PEBP mRNA in hippocampus of both sides. One section was taken from anterior, middle and posterior location respectively of each rat. The number and the mean gray value of PEBP mRNA positive cells were measured in transected and normal sides of pyramidal layer, granular layer of dentate gyrus and on 0.03mm2 area of hilus, subgranular of dentate gyrus respectively. (3) Western Blot:Forty-two SD rats were randomly divided into 7 groups,6 rats in each group. One group served as normal control, and fimbria-fornix of the other groups rats were transected on the 1st,3rd,7th,14th,21st and 28th day respectively. Then hippocampi were isolated and the total protein was acquired from the fimbria-fornix transected and the normal hippocampus, then detected the concentration of each group, the protein sample was isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the membrane. The expression was detected by PEBP polyclonal antibody immunoblot. The mean gray value of PEBP and B-actin positive strips was then detected, the relative expression level of PEBP protein was indicated by the reciprocal of the mean gray value of PEBP to B-actin. (4) Immunohistochemistry:Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, and served as right fimbria-fornix transected 1st,3rd,7th,14th,21st and 28th day group respectively. The rats were perfused and fixed, then cryostat sections of hippocampus were prepared for PEBP immunohistochemistry. One section was taken from anterior, middle and posterior location respectively of each rat. The number and gray value of PEBP positive cells in pyramidal layer and granular layer of dentate gyrus, in hilus of dentate gyrus and subgranular layer of 0.06mm2 area on both transected and normal sides were measured respectively. (5) Analysis of variance and mutual comparision of each group was analyzed by SPSS 11.5 software, and the both sides'results of in situ hybridization and immuohistochemisty were conducted by the method of paired t test. Results:(1) Real-time PCR:In normal group, the relative expression level of PEBP mRNA was 1.16±0.10. It started to increase on 3rd day (1.51±0.08) after transection, and the peak appeared on 7th day(1.64±0.07), then decreased slowly to pre-transection level on 21st (1.14±0.13). One-factor analysis of variance and comparison between each two groups of the PEBP mRNA relative expression manifested that there were significant difference between the fimbria-fornix transected 3rd,7th day groups and other groups (P<0.05). The difference was not significant between 3rd and 7th day group, and not significance existed among other groups (P>0.05). (2) In Situ Hybridization:PEBP mRNA was expressed in CA1～CA3 pyramidal layer of hippocampus and granular layer of dentate gyrus on both sides. The number of PEBP mRNA positive cells manifested no discrepancy between two sides. But the mean gray value of PEBP mRNA positive cells in transected side (78.47±4.45) was lower than that in normal side (102.58±15.99), the signal was higer in transected side than normal side. PEBP mRNA positive cells was also seen in hilus and subgranular layer of dentate gyrus, in transected side, the number of PEBP mRNA positive cells was 60.67±6.71, the mean gray value was 67.16±9.28. In normal side, there was less PEBP mRNA positive cells (50.33±8.52), and the mean gray value was 99.62±13.44. The positive cell numbers and mean gray value of two sides existed significant difference (P<0.05). (3) Western Blot:Western Blot showed that the expression of PEBP protein was 0.0551±0.0093 in control group. It started to increase on 3rd day (0.077±0.0083) and the peak appeared on 7th day (0.0965±0.0089), then decreased slowly to pre-transection level on 28th day (0.0505±0.0066). When transected groups compared with the normal group, statistic results showed no significant difference between the 1st,28th day group and the normal group (P<0.05), while significant difference existed between the normal group and the other five transected groups (P<0.05). When transected groups compared mutually, the difference between the 7th day and other transected groups was significant (P<0.05), the significant difference also existed between the 3rd,14th,21st day group and 1st、28th day groups, while the difference among 3rd,14th,21st day groups were not significant (P>0.05), the difference between the 1st and 28th day was not significan either. (P>0.05). (4) Immunohistochemistry:The result of PEBP immunohistochemistry showed that PEBP was majorly expressed in the kytoplasm of cells which located in the pyramidal layer of CA1～CA3 areas and granular layer, hilus, subgranular layer of dentate gyrus. The number of PEBP protein positive cells has no discrepancy between two sides in the pyramidal layer of CA1～CA3 areas and (P>0.05). But the stain of PEBP positive cells in transected side was deeper than normal side, especially on 7th day, the mean gray value was 68.41±10.53, while the mean gray value was only 91.52±7.77 in the normal side, the significant discrepancy existed between the two sides (P<0.01). More deeper stained PEBP positive cells could be seen in the hilus and subgranular of dentate gyrus, especially on the 7th day after transection, the number of positive cells was 53.33±4.27, the mean gray value was 65.34±15.84, while the number and mean gray value of positive cells in the normal side was 37.5±4.04 and 97.16±11.62 respectively, the significant discrepancy existed between the two sides (P<0.05). The expression of PEBP in pyramidal layer, hilus and subgranular of dentate gyrus was decreased on the 14th day after transection and reached pre-transection level on the 28th day. Conclusion:(1) The expression of PEBP mRNA and protein obviously increased after fimbria-fornix transection, the expression changed with time, it increased at first and then decreased to normal level, the ascending and descending tendency of PEBP mRNA and protein was generally coincident, it reached peak on the 7th day after transection. (2) The PEBP mRNA and protein majorly expressed in the kytoplasm which located in the CA1～CA3 areas of pyramidal layer, granular layer, hilus and subgranular layer of dentate gyrus. (3) Cells in the CA1～CA3 areas of pyramidal layer, granular layer of dentate gyrus were stained deeper in the transection side than the normal side. However, compared with the normal side, cells in the hilus and subgranular layer of dentate gyrus were not only stained deeper but also possessed more numbers of positive cells. The results indicated that the higher expression of PEBP mRNA and protein in the fimbria-fornix transection side hippocampus may involve the hippocampal neural regeneration which induce the neural stem cells differentiating into neurons or cholinergic neurons.
引文
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