CBP介导的组蛋白乙酰化修饰在VAD致学习记忆功能障碍中的作用及机制研究

发布时间：2018-06-29 12:03

本文选题：维生素A + 视黄酸核受体α　；参考：《重庆医科大学》2015年博士论文

【摘要】：第一部分孕期开始的VAD对成年仔鼠学习记忆功能及组蛋白乙酰化修饰的影响目的：探讨从孕期开始的VAD对仔鼠成年期学习记忆功能及组蛋白乙酰化修饰的影响,以及RA信号通路与组蛋白乙酰化修饰二者的联系。方法：建立从孕期开始VAD的大鼠模型,实时监测VAN与VAD组仔鼠血清中VA浓度的变化；采用水迷宫行为学检测VAN和VAD组实验大鼠在8W龄时的学习记忆功能；用real-time PCR 和 western blot技术分别检测大鼠海马区RARa的基因和蛋白表达,同时用HAT活性检测法检测大鼠海马区的HAT活性,western blot技术检测乙酰化组蛋白Ac-H3、Ac-H4的蛋白表达,real-time PCR检测学习记忆基因Zif268、 cFOS、FosB的基因表达,CHIP-qPCR对Ac-H3、Ac-H4在两组大鼠海马区学习记忆基因启动子区域的富集情况进行检测；进而分别用real-time PCR 和 western blot技术检测组蛋白乙酰转移酶CBP、p300、 PCAF的基因和蛋白表达情况；最后用Co-IP和激光共聚焦技术对两组大鼠海马区RARa和CBP蛋白之间的相互作用情况进行检测。结果：(1)实时监测VAN与VAD组仔鼠血清中视黄醇浓度的变化：可见VAD组血清VA浓度在生后1D、3D、7D、2W、4W、8W均明显低于VAN对照组(p0.001),并在8W龄进行水迷宫行为学检测：VAN组和VAD组大鼠均有学习记忆能力(p0.001),但从孕期开始的VAD大鼠在8W龄时学习能力明显差于VAN组(p0.05)；(2)大鼠海马区RARα的基因和蛋白表达：VAD组RARα表达较VAN组显著降低(p0.01/0.001)； (3)大鼠海马区HAT活性、乙酰化组蛋白表达：VAD组大鼠海马区的HAT活性、乙酰化组蛋白表达显著降低(p0.05/0.001)；大鼠海马区学习记忆基因表达：VAD组大鼠海马学习记忆基因(Zif268、cFOS、FosB)表达显著降低(p0.01/0.05)；并且富集在记忆相关基因启动子区域的乙酰化组蛋白Ac-H3、Ac-H4蛋白水平亦显著降低(p0.01/0.05)； (4)组蛋白乙酰转移酶(CBP、p300、PCAF)的基因和蛋白表达：VAD组CBP、p300的基因水平显著低于VAN组(p0.01/0.05), PCAF在两组间无显著差异(p=ns)；CBP蛋白水平在VAD组表达显著低于VAN组(p0.001),而p300、PCAF蛋白表达水平在两组间没有显著差异(p=ns)； (5)海马组织Co-IP和激光共聚焦检测RARα与CBP蛋白两者之间的相互作用：Co-IP实验发现RARα与CBP蛋白之间存在蛋白-蛋白相互作用,并且这种相互作用在VAD组要显著弱于VAN组(p0.001)；激光共聚焦实验证实VAD组的RARα与CBP蛋白在海马三个分区(CA1、CA3、DG)的表达均要明显弱于VAN组,两者之间存在共表达,并且这种共定位在VAD组也要显著弱于VAN组(p0.001)。结论： (1)成功建立了从孕期开始的VAD大鼠模型,并确证了孕期开始的VAD会引起成年期大鼠学习记忆功能的障碍； (2)VAD时大鼠海马区RARα的基因和蛋白表达显著降低； (3)VAD严重影响了大鼠海马HAT活性、乙酰化组蛋白Ac-H3、Ac-H4以及学习记忆相关基因的表达; (4)VAD时大鼠海马区组蛋白乙酰转移酶CBP、p300、PCAF表达不同程度下降,其中CBP表达严重受损,VAD时HAT活性、Ac-H3、Ac-H4表达以及学习记忆相关基因表达的下降可能与CBP表达的下降密切相关； (5)VAD引起RA信号通路和组蛋白乙酰化修饰的显著改变,大鼠海马区RARα和CBP表达均显著降低,二者之间存在相互作用,并且这种相互作用在VAD组也显著低于VAN组。LARα口CBP相互作用异常可能在VAD所致学习记忆功能障碍中起重要作用。第二部分 RARα介导的RA信号通路对学习记忆相关的CBP依赖性组蛋白乙酰化修饰作用的调控机制第一章 RARα对学习记忆相关的组蛋白乙酰化修饰作用的调控机制目的：研究激活或抑制RARα介导的RA信号通路对CBP、HAT活性、乙酰化组蛋白以及学习记忆相关基因表达的影响。方法：培养胎鼠原代海马神经元,于培养的第六天用NSE做标记对神经元纯度进行鉴定。设定①RFP、②RFP+RA、③RA+siRARα、④ RA+Ad-RARa四个组,于培养的第六天,分别加入相应的RA或RFP、siRARα、Ad-RARα的重组腺病毒进行干预,于培养的第七天采用Real-time PCR分别检测RARα、CBP、记忆相关基因的RNA水平；CHIP-qPCR检测四个组神经元中RARα在CBP启动子上的富集情况；于培养的第九天检测各组的HAT活性,CBP、Ac-H3、Ac-H4的蛋白表达。结果：(1)胎鼠原代海马神经元生长情况良好、胞体饱满,突触逐渐增长、形成相互连接,经NSE免疫荧光鉴定,NSE阳性率90%,纯度满足实验要求；(2)经RA处理后,RARa的基因和蛋白水平显著增高,RA+siRARα腺病毒作用后的神经元中RARα基因和蛋白表达水平显著低于RFP+RA组,经RA+Ad-RARα腺病毒处理后,RARα的mRNA和蛋白表达均达到最高,显著高于RFP+RA组(p0.001/0.01);(3)CBP基因(p0.001/0.01/0.05)和蛋白表达、HAT活性(p0.001/0.01/0.05)、乙酰化组蛋白Ac-H4表达、记忆相关基因表达在四个组的表达趋势均同RARα的变化趋势基本一致,即RFP+RA组的表达水平显著高于RFP组与RA+siRARα组,而显著低于RA+Ad-RARa腺病毒处理组； (4)RARα富集在CBP启动子区域,并且富集的水平与RARα的表达趋势基本一致(p0.001/0.01/0.05)。结论： (1)成功培养并鉴定了符合实验要求的胎鼠原代海马神经元 (2)获得了满足实验需求的相应的上调或下调的RARα信号；(3)RARα能够调控HAT活性、乙酰化组蛋白Ac-H3、Ac-H4和下游学习记忆相关基因的表达； (4)RARα不仅能够招募CBP成为辅激活子,还能直接调控CBP的表达水平,参与RA信号通路调控学习记忆基因的表达。第二章RARa介导的RA信号通路调控学习记忆相关信号中的CBP依赖性组蛋白乙酰化修饰机制目的：研究CBP信号对组蛋白乙酰化调控的特异性,以及其在RARa介导的RA信号通路调控学习记忆相关信号中的作用机制。方法：培养胎鼠原代海马神经元,于培养的第六天,设定(1)①NC、②siCBP两组； (2)③Ad-RARa+NC、④Ad-RARa+siCBP/C646两组； (3) ⑤RFP+NC、⑥RFP+RA+NC、⑦RFP+NC+siCBP三组。首先利用 (1) 中两个组别对四种 siCBP慢病毒(GR805/GR806/GR807/GR808)进行筛选和鉴定,选出具有最佳抑制效率和功能的siCBP慢病毒,然后在(2)、 (3)分组细胞中分别加入相应的RA、腺病毒、慢病毒或抑制剂进行干预,于培养的第七天采用Real-time PCR分别检测RARα、CBP、记忆相关基因的mRNA水平；于培养的第九天检测各组的HAT活性,CBP、Ac-H3、Ac-H4的蛋白表达。结果： (1)四种siCBP慢病毒感染原代海马神经元后CBP mRNA和蛋白表达有不同程度降低,抑制效果最佳的GR806对神经元的HAT活性及Ac-H3、Ac-H4表达均有显著抑制功能(p0.001/0.01/0.05)；(2)在用Ad-RARa将RA信号通路激活的基础上,siCBP能够特异性地将激活的CBP基因(p0.01)与蛋白表达显著抑制下来,并且神经元中HAT活性(p0.05)、乙酰化组蛋白Ac-H3、Ac-H4、记忆相关基因(p0.001/0.05)的表达亦均被显著下调； (3)siCBP慢病毒作用于神经元后,CBP的基因表达显著降低(p0.05),而RARα的基因表达未发生显著改变(p=ns)；CBP的抑制剂C646并没有使RARI-的蛋白产生显著下调； (4)用RA直接将RA信号通路激活后,CBP基因表达能被显著上调(p0.05),在RA的基础上siCBP作用后,上调的CBP表达又被显著下调(p0.05),相应地,下游乙酰化组蛋白Ac-H3、Ac-H4表达、记忆相关基因表达(p0.01/0.05)在三个组的表达趋势均同CBP的变化趋势基本一致。结论：(1)筛选并验证获得了最佳抑制效率的siCBP慢病毒；(2)获得了满足实验需求的相应变化的RARα、CBP信号 (3)CBP能够特异性地调节神经元中HAT活性、乙酰化组蛋白Ac-H3、Ac-H4和下游学习记忆相关基因的表达； (4)RARα在CBP的上游,RA信号通路对学习记忆相关信号的调节是通过RARα介导的CBP依赖性组蛋白乙酰化修饰的调控实现的。
[Abstract]:The effect of VAD on learning and memory function and histone acetylation in adult rats was investigated in the first part of pregnancy . The effects of VAD on adult learning and memory function and histone acetylation modification were discussed in the first part of pregnancy , and the relationship between RA signal pathway and histone acetylation modification was discussed .
The learning and memory function of the experimental rats at the age of 8 W was measured by the water maze behavioral study .
The gene and protein expression of RARa in rat hippocampus were detected by real - time PCR and western blot . The expression of Ac - H3 , Ac - H4 was detected by Western blot . The expression of Ac - H3 and Ac - H4 was detected by western blot .
Furthermore , real - time PCR and western blot were used to detect the gene and protein expression of histone acetyl transferase CBP , p300 and PCAF , respectively .
Results : ( 1 ) The changes of retinol concentration in serum of both groups of rat hippocampus were detected by co - IP and laser cofocus technique . Results : ( 1 ) The serum VA concentration in VAD group was significantly lower than that in control group ( p < 0.001 ) .
( 2 ) The gene and protein expression of RAR伪in hippocampus were significantly lower in VAD group than that in van group ( p0.01 / 0.001 ) .
( 3 ) In the hippocampus of rats , the expression of acetylated histone was significantly lower than that of VAD group ( p < 0.05 / 0.001 ) .
The expression of learning and memory genes in hippocampus was significantly lower in VAD group than in VAD group ( P 0.01 / 0.05 ) .
and the level of Ac - H3 and Ac - H4 protein enriched in the promoter region of the memory - related gene was also significantly reduced ( p0.01 / 0.05 ) ;
( 4 ) The gene and protein expression of histone acetyl transferase ( CBP , p300 , PCAF ) : The gene level of CBP and p300 in VAD group was significantly lower than that in the control group ( p0.01 / 0.05 ) , and there was no significant difference between the two groups ( p = ns ) .
The expression level of CBP protein was significantly lower in VAD group than that in control group ( p < 0.001 ) , while the expression level of p300 and PCAF was not significantly different between the two groups ( p = ns ) .
( 5 ) Co - IP and laser confocal microscopy showed that there was a protein - protein interaction between RAR伪and CBP protein .
Conclusion : ( 1 ) The VAD rat model started from pregnancy was successfully established , and it was confirmed that VAD induced by pregnancy could lead to the impairment of learning and memory function in adult rats .
( 2 ) The gene and protein expression of RAR伪in hippocampus were significantly decreased in VAD .
( 3 ) VAD significantly affects the expression of the activity , acetylated histones Ac - H3 , Ac - H4 and learning and memory - related genes in hippocampus of rats .
( 5 ) The effect of VAD on the expression of RAR伪and CBP in the hippocampus of rats was significantly decreased , and the expression of RAR伪and CBP was significantly lower in the hippocampus of rats .
The enrichment of RAR伪on CBP promoter was detected by CHIP - qPCR .
Results : ( 1 ) The growth of neurons in the primary hippocampal neurons of the fetal mice was good , the cells were full , the synaptic gradually increased , the formation of interconnection was formed , the positive rate of NSE was 90 % and the purity met the experimental requirements .
( 2 ) After RA treatment , RARa mRNA and protein level were significantly higher than that in RFP + RA group , and the expression of RAR伪mRNA and protein was highest after treatment with RA + Ad - RAR伪adenovirus , and the expression of RARa mRNA and protein was significantly higher than that in RFP + RA group ( p0.001 / 0.01 / 0.05 ) .
( 4 ) RAR伪was enriched in the promoter region of CBP , and the concentration of RAR伪was substantially identical to that of RAR伪( p0.001 / 0.01 / 0.05 ) . Conclusion : ( 1 ) Successful cultivation and identification of the primary hippocampal neurons ( 2 ) which meet the experimental requirements have obtained the corresponding up - regulated or down - regulated RAR伪signal that meets the experimental requirements ;
( 3 ) RAR伪was able to regulate the expression of the genes involved in the learning and memory related to the activity , histone Ac - H3 , Ac - H4 and downstream learning .
( 4 ) RARa can not only recruit CBP as a secondary activator , but also directly regulate the expression level of CBP and participate in the expression of learning and memory genes in the RA signal pathway .
( 2 ) ( 3 ) Ad - RARa + NC , ( 4 ) Ad - RARa + siCBP / C646 group ;
( 3 ) RFP + NC , ( 6 ) RFP + RA + NC , ( 7 ) RFP + NC + siCBP group .
Results : ( 1 ) The mRNA and protein expression of CBP , Ac - H3 , Ac - H4 decreased in different degrees after four siCBP slow virus infection in primary hippocampal neurons , and the best inhibitory effect of GR806 on the activity of the cells and the expression of Ac - H3 and Ac - H4 were significantly inhibited ( p0.001 / 0.01 / 0.05 ) .
( 2 ) On the basis of activation of RA signal pathway with Ad - RARa , siCBP could significantly inhibit the expression of active CBP gene ( p0.01 ) and protein expression , and the expression of CAP activity ( p0.01 ) , histone Ac - H3 , Ac - H4 and memory - related gene ( p0.001 / 0.05 ) in neurons were also significantly downregulated .
( 3 ) After the action of siCBP lentivirus on neurons , the gene expression of CBP decreased significantly ( p < 0.05 ) , while the gene expression of RAR伪did not change significantly ( p = ns ) ;
The inhibitor C646 of CBP did not significantly downregulate RARI - protein ;
( 4 ) After the RA signal pathway was directly activated by RA , the expression of CBP gene was significantly up - regulated ( p . 05 ) . After the action of siCBP on the basis of RA , the expression of the downstream acetylated histone Ac - H3 , Ac - H4 , and memory - related gene expression ( p0.01 / 0.05 ) were similar to that of CBP . Conclusion : ( 1 ) The siCBP lentivirus with the best inhibition efficiency was obtained and verified .
( 2 ) cbp signals ( 3 ) cbp which satisfy the corresponding changes of the experimental requirements can be obtained , and the cbp signals ( 3 ) cbp can specifically regulate the expression of hat activity , acetylated histones ac - H3 , Ac - H4 and downstream learning and memory related genes in the neurons ;
( 4 ) The regulation of RAR伪upstream of CBP and RA signal pathway to learning and memory related signals is realized by the regulation and regulation of CBP - dependent histone acetylation mediated by RAR伪.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R741

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