后基因组学研究：吗啡依赖的蛋白质组学分析DNA甲基化谱分析技术的建立与评价
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摘要
随着人类基因组计划(HGP)的实施和完成,基因组学研究的重心开始从揭示生命的基本遗传信息转移到在分子整体水平对功能研究的后基因组学时代,即从基因组整体水平上对基因表达和调控的活动规律进行阐述。后基因组学研究包括基因组SNP分析、转录组学、蛋白质组学以及最近发展起来的新兴学科——表观基因组学等。
蛋白质组学是应用高通量的技术对组织和细胞整体蛋白的组成、表达和相互作用进行研究的学科,在神经科学领域,它越来越多地被应用于各种疾病如帕金森病、阿尔茨海默病、药物成瘾和精神分裂症的蛋白表达谱和分子间相互作用的研究。药物成瘾是精神活性物质反复作用于大脑引发的一种长期适应性改变,并被认为是一种慢性复发性的脑病。目前的研究认为,多巴胺能和谷氨酸能神经的突触可塑性在药物成瘾中起了重要的作用,精神活性物质的强烈作用,使两类受体发生相互调控,神经信号在受体经整合后触发了受体后细胞内的级联反应,从而调控了突触的基因表达和蛋白质合成,长期的基因、蛋白的表达以及分子组成的变化,引起了突触结构和形态发生改变,从而导致神经功能的不可逆变化,这一系列过程在药物依赖的神经适应性过程中起了关键的作用。前额叶皮质(prefrontal cortex,PFC)是奖赏环路的重要组成部分,它对行为的执行起着判定和控制的作用,因而是成瘾相关的重要脑区之一。有研究表明,慢性吗啡作用会引起腹侧背盖核(ventral tegmental area,VTA)多巴胺神经元的体积缩小以及伏隔核(nucleus accumbens,NAc)和PFC树突联系和中间神经原的棘突数量的减少。为了对成瘾相关的突触分子作用机制进行更深入的研究,我们使用双向电泳(two dimensional gel electrophoresis,2-DE)结合质谱的蛋白质组学技术分析了吗啡诱导的条件性位置偏爱(Conditioned Place Preference,CPP)三个阶段大鼠PFC脑区突触后致密区(post synaptic density,PSD)蛋白表达谱的动态变化。
本论文的第一部分描述了吗啡诱导的CPP三个阶段大鼠PFC脑区的PSD蛋白表达谱的分析结果。我们使用CPP模型来评价大鼠的吗啡依赖效应,CPP训练程序包括:自然偏爱测试,条件性偏爱的建立、消退和复燃。基于吗啡诱导的CPP模型,我们使用2-DE技术对CPP三个阶段(建立、消退和复燃)大鼠PFC脑区的PSD蛋白进行分析,筛选了差异表达的蛋白。进而我们使用MALDI-TOF-MS质谱技术成功鉴定了部分差异蛋白,并基于免疫沉淀技术对差异蛋白表达谱的分析结果进行了验证。
结果:
1.建立吗啡诱导的CPP模型,包括条件性获得,消退和复燃三个阶段。吗啡组大鼠在CPP获得阶段表现出对吗啡伴药盒的显著偏爱,在消退阶段对吗啡伴药盒的偏爱消失以及在复燃阶段由于小剂量吗啡的诱导又表现为对伴药盒的显著偏爱。
2.使用2-DE技术对吗啡诱导CPP三个阶段的大鼠PFC脑区的PSD蛋白进行分析,共筛选到80个差异蛋白,其中代表性差异表达的蛋白—CNPasel(cyclicnucleotide phosphodiesterase 1)在CPP三个阶段都发生了显著的变化,其余79个蛋白在CPP的一个或两个阶段有差异表达。
3.使用MALDI-TOF-MS质谱技术对差异表达蛋白进行鉴定,质谱成功鉴定了CPP获得阶段21个差异蛋白,消退阶段14个差异蛋白和复燃阶段23个差异蛋白。通过生物信息学的分析,这些蛋白参与了多种重要的生物学功能如能量代谢,信号传导,突触传递,细胞蛋白骨架,分子伴侣和突触蛋白合成器等。
4.为了评价蛋白质组学分析结果的准确性,我们使用western blot验证了代表性差异蛋白CNPase1。Western blot的结果显示,CNPase1在吗啡诱导CPP的获得阶段上调了2.1倍,消退阶段下调了1.6倍,然而在复燃阶段又上调了1.4倍,该结果与2-DE分析中的变化趋势是一致的。Western blot验证结果表明使用蛋白质组学对吗啡诱导的CPP三个阶段大鼠PFC脑区PSD蛋白表达谱的分析结果是可信的。
结论:
我们对吗啡诱导的CPP三个阶段大鼠PFC脑区PSD蛋白表达谱的分析获得了大量差异表达的蛋白,这些蛋白参与了多种重要的生物学功能如能量代谢,信号传导,突触传递,细胞蛋白骨架,分子伴侣和突触蛋白合成器等。本研究第一次对吗啡诱导的条件性依赖的不同阶段一获得、消退和复燃,基于动物模型分析了成瘾相关重要脑区PFC的PSD蛋白分子的动态变化,从而为吗啡成瘾相关的突触分子作用机制的研究提供了许多重要的靶点。
表观遗传学是指DNA序列没有发生改变,但通过DNA甲基化、组蛋白修饰以及染色质结构的重塑来调控基因表达和功能的可遗传的变化。DNA甲基化是表观遗传学中研究得最为广泛的一种修饰,它参与了哺乳动物的多种生命活动过程,包括基因的沉默、染色体结构的稳定、X染色体的失活、基因印记和细胞分化,因而DNA甲基化的异常改变在人类多种重大疾病如肿瘤、精神性和免疫性等疾病中起了非常重要的作用。目前大多数研究对DNA甲基化修饰的分析都是基于单个候选基因途径,主要采用的检测技术为亚硫酸氢钠转化DNA结合测序、甲基化特异性酶切、甲基化特异性PCR(Methylation Specific PCR,MSP)和变性高效液相色谱(Denaturing High Performance Liquid Chromatography,DHPLC)等。但对于重大、复杂疾病的研究,小规模单基因的研究模式显然不能完整地解释其中表观遗传的调控网络和作用机制,这促使了表观基因组学的发展。表观基因组学则是在全基因组水平上对表观遗传学修饰的研究,基因组水平DNA甲基化修饰谱的研究是表观基因组学的重要组成部分。因而在本研究中,我们开发了高通量的DNA甲基化谱分析芯片—人9K CGI芯片,该芯片可以应用于人类各种生命活动和重大疾病的DNA甲基化修饰谱的分析。虽然9K CGI芯片为基因组DNA甲基化谱分析提供了一个高通量的检测工具,但DNA甲基化谱研究的瓶颈是全基因组甲基化DNA的富集,最大限度地获得纯化的甲基化DNA是后续基于高通量技术进行DNA甲基化分析的关键。目前国际上广泛采用的方法有:(1)基于甲基化限制性酶切技术,代表性的有差异甲基化杂交(Differential Methylation Hybridization,DMH)和基于微阵列芯片的单个样品甲基化评价(Microarray Based Methylation Assessment of Single Sample,MMASS);(2)甲基化结合蛋白(Methylation Binding Protein,MBD Protein)纯化甲基化DNA;(3)甲基化DNA免疫沉淀(Methylated DNA Immunoprecipitation,MeDIP)。已有研究表明MeDIP的灵敏性高于MBD层析柱纯化的方法,但没有研究对基于甲基化限制性酶切和基于免疫沉淀的富集方法进行过系统的评价。因此,在本研究中,我们以胃上皮细胞系Ges-1和胃腺癌细胞系MGC-803为研究对象,基于9K CGI芯片优化了DMH、MMASS和MeDIP三种方法中的一些重要的实验参数,进而对各种优化后的方法进行了灵敏性和准确性的系统评价,从而为9KCGI芯片更好地应用于各种基因组DNA甲基化修饰的表观遗传学研究打下了坚实的基础。同时对MMASS和MeDIP方法分析两种细胞系甲基化谱获得的差异CGI探针相关的基因进行GO分类,结果显示大量的基因参与了多种重要的生物学过程,从而证实了9K CGI芯片对人类的表观遗传学研究具有较大的应用价值。
本论文的第二部分阐述了DNA甲基化谱分析芯片的研制,基于该芯片优化和评价了全基因组甲基化DNA的富集方法,并对甲基化差异的CGI探针相关的基因进行GO分类。首先,我们对CGI文库进行了大规模的克隆测序,合并Wellcome Trust Sanger Institute对同一文库测序获得的克隆,与预测的全基因组CGI比对来鉴定两部分测序获得的CGI克隆。同时我们预测了部分肿瘤相关基因和印记基因第一个外显子和转录起始位点上游10kb范围内的CGI。CGI克隆和目的基因相关CGI进行PCR扩增后的产物作为探针点制成CGI芯片。我们基于CGI芯片,通过对胃腺癌细胞系MGC-803的甲基化谱分析优化了DMH、MMASS和MeDIP三种方法中的一些重要的实验参数,包括PCR退火温度和免疫沉淀反应中的抗体孵育时间。进而对基于胃上皮细胞系Ges-1和胃腺癌细胞系MGC-803的甲基化谱分析比较,系统评价了三种方法的灵敏性和准确性。并对正常和癌症细胞系的甲基化谱分析中筛选到的甲基化差异的CGI探针相关基因进行GO分类。
结果:
1.为了开发高通量的基因组DNA甲基化谱分析芯片,我们对人源性CGI文库进行了18,816个克隆(18K)的测序,序列经拼接、比对及聚类分析后,共获得10,749个代表性克隆,冗余度为37.02%;应用Wellcome Trust Sanger Institute对同一CGI文库进行部分测序获得的12,192个单克隆(12K),同样对12K数据的拼接、比对及聚类分析后,共获得6,445个代表性克隆,冗余度为37.84%。
2.使用CpGi130软件预测到人类全基因组共33,116个CGI。将18K克隆测序获得的10,749个代表性克隆和来自12K克隆的6,445个代表性克隆与全基因组预测得到的CGI进行比对,共鉴定了8,967个特异性CGI克隆。同时从已知的450个肿瘤相关基因和90个印记基因的第一个外显子和转录起始位点上游10K的区域预测了830个特异性CGI。对所有特异性CGI克隆和候选基因的CGI进行了注释,包括在基因组上的位置,邻近的基因,与邻近基因的位置关系以及对基因的描述。
3.为了制备CGI芯片,对8,967个特异性CGI克隆和830个候选基因的CGI进行PCR扩增,成功扩增的9,223个CGI探针用于9K CGI芯片的制备。对9K CGI芯片的质量评价表明芯片杂交的信号强度和重复性都非常高。
4.基于9K CGI芯片,优化了DMH、MMASS和MeDIP三种全基因组甲基化DNA富集方法中的一些重要的参数。对DMH和MMASS方法中不同PCR退火温度的比较表明,使用较高的退火温度(72℃)扩增甲基化和非甲基化酶切产物,大大提高了芯片检测CGI甲基化差异的灵敏性;对MeDIP方法中不同抗体孵育时间的比较表明,较长的一抗孵育时间(12小时)和合适的二抗孵育时间(3小时左右)既提高了甲基化DNA的结合效率,又降低了非甲基化DNA的非特异性结合。
5.基于9K CGI芯片,对优化后的DMH、MMASS和MeDIP三种方法的系统评价结果表明:1)三种方法能检测到共同的甲基化差异的CGI探针,但每种方法也能筛选到特异性的差异CGI探针;2)三种方法中MMASS的敏感性最高,MMASS和MeDIP的准确性类似,但DMH的敏感性和准确性都较低。
6.合并MMASS和MeDIP对Ges-1和MGC-803细胞系甲基化谱分析获得的差异CGI探针,基于探针的基因注释,对361个差异CGI探针相关的基因进行GO分类,包括细胞组分(Cellular Component)、分子功能(Molecular Function)和生物学过程(Biological Process),结果显示大量参与多种重要生物学功能的基因在癌症细胞系中可能发生了甲基化的调控。
结论:
基于对CGI文库的测序及部分目的基因CGI的预测,我们鉴定并扩增了9223个CGI探针,从而开发了9K CGI芯片,该芯片为基因组水平DNA甲基化修饰的研究提供了一个高通量的平台。基于该芯片我们对甲基化DNA的富集方法—DMH、MMASS和MeDIP进行了优化,从而提高了各种方法对基因组CGI甲基化差异评价的灵敏性和特异性。进而对优化后的三种方法的系统评价表明MMASS方法的敏感性最高,但MMASS和MeDIP两种方法的准确性类似,因而基于不同原理的两种方法MMASS和MeDIP对全基因组CGI的甲基化差异的评价是互为补充的。因此在实际的应用中,只有联合不同原理的两种或两种以上的方法,才能对全基因组CGI的甲基化水平进行更全面的分析。同时对差异CGI探针相关基因的GO分类显示,癌症细胞中潜在受甲基化调控的基因参与了多种重要的生物学功能,因而基于9K CGI芯片的甲基化谱分析为癌症的甲基化修饰机制的研究提供了许多重要的靶点,从而也表明9K CGI芯片将在人类生理和疾病的表观遗传学研究中具有广阔的应用前景。
With the completion of Human Genome Project (HGP) the genomic study has moved from genomic sequencing to functional study on genome-scale, emphasizing exploration of the expression and regulatory network of genes on genome-wide. The diversified post-genomic field includes, but not limits, genomic SNP analysis, transcriptomics, proteomics, and newly developed epigenomics.
Proteomics is to study protein expression and interaction of entire proteins in specific tissues and cells, and recently has been intensively used in human neurological diseases such as Parkinson's disease, Alzheimer disease, drug addiction and schizophrenia. Drug abuse is recognized as long-term adaptation in brain which is resulted from repeatedly usage of psychoactive substances. Therefore, drug addiction is also presumed as chronic and relapsing encephalopathy. The numerous lines of evidence have suggested that dopaminergic and glutamatergic neuronal synaptic plasticity play an important role in drug addition. In general, psychoactive substances strongly affect on the neuronal systems through dopamine D-1 and glutamate N-methyl-D-aspartate (NMDA) andα-amino-3-hydroxy-5-methylisoxazole -4-propionic acid (AMPA) receptors, which induce intracellular transcriptional and translational cascades, leading to adaptive changes in gene expression, protein expression and molecular constitute. The long-term adaptation of synaptic plasticity induced from the above molecular alteration, causes the alteration of synaptic morphology and structure which further result in irreversible neural dysfunction.
Prefrontal cortex (PFC) is one of important brain regions in reward circuits, which is critical for many cognitive functions involving inhibitory control and decision making. As the relationship between a cell's chemistry and structure becomes increasingly apparent, it is not surprised that chronic exposure to drugs of abuse has been shown to alter the morphology of neurons in reward circuits of the brain. Chronic morphine administration decreases the size and caliber of dopamine neurons in ventral tegmental area (VTA). In addition, chronic morphine administration has been shown to decrease the complexity of dendritic branching and the number of spines on medium spiny neurons in the nucleus accumbens (NAc) and PFC. For better understanding the synaptic molecular mechanism involved in addiction, we performed proteomic analysis of PSD fraction of rats' PFC in three phases of morphine induced conditioned place preference (CPP).
The first part of the thesis is to present our proteomic analysis of PSD fraction of rats' PFC in three phases of morphine induced CPP. The effect of morphine dependence is evaluated by CPP model, the procedures of which include nature preference test, conditioned acquisition, conditioned extinction and conditioned reinstatement. Next we analyzed the expression profiling of PSD fraction of rats' PFC in three phases of CPP with two-dimensional gel electrophoresis (2-DE). Furthermore, we identified differential expression proteins with MALDI-TOF-MS and validated the results of proteomics with western blot.
Results:
1. Morphine induced CPP model was established, including acquisition, extinction and reinstatement phases. The rats showed strong preference to morphine-paired chamber in acquisition test after morphine conditioning. The morphine-induced preference is diminished after saline-paired extinction training, and could be re-established after morphine priming injection.
2. Proteomic analysis of PSD fraction of rats' PFC in three phases of morphine induced CPP with 2-DE resulted in 80 differential proteins, of which protein cyclic nucleotide phosphodiesterase 1 (CNPasel) showed significantly differential expression in the three phases of CPP respectively, and the remaining proteins were altered in one or two phases of CPP.
3. The differential proteins were identified with MALDI-TOF-MS spectrum, of which 21 proteins were successfully identified in acquisition phase, 14 in extinction phase and 23 in reinstatement phase. Based on bioinformatics analysis, 58 identified differential proteins are found to involved in many vital biological functions, such as energy metabolism, signal transduction, synaptic transmission, cytoskeleton proteins, molecular chaperones, and synaptic proteins synthetic machine.
4. The expression of representative protein CNPasel was verified for assessment of the accuracy of proteomic results with western blot. The result showed that there were 2.1 fold elevation in the acquisition phase, 1.6 fold decrease in the extinction phase and 1.4 fold increase in the reinstatement phase, which were consistent with the analysis of 2-DE and silver staining. The data indicates that the changes in protein expression identified by proteomic analysis of synaptic fraction of PFC in the three phases of CPP are reliable.
Conclusion:
The proteomic analysis of PSD fraction of rats' PFC in the three phases of morphine induced CPP resulted in identification of many differentially expressed proteins. Based on bioinformatics analysis, these proteins are classified into many important biological functions, such as energy metabolism, signal transduction, synaptic transmission, cytoskeleton proteins, molecular chaperones and synaptic proteins synthetic machine. Therefore, our study, in the first time, revealed the expression profiling of PSD fraction of rats' PFC under morphine induced conditioned dependence including acquisition, extinction and reinstatement, which provides many crucial candidates for further study of molecular synaptic function in morphine addiction.
Epigenetics is to study heritable changes, such as DNA methylation and histone modification that modulate chromatin organization and gene expression in the absence of change in DNA sequence. The most widely studied epigenetic modification in humans is the cytosine methylation of DNA within the dinucleotide CpG. DNA methylation has been shown to involve in many biological processes, such as gene silencing, transposable inhibition, X-chromosome inactivation and cell differentiation. An altered pattern of DNA methylation has been associated with etiology of some severe human diseases such as cancer, mental health and immunological diseases.
A range of approaches are available for assessing DNA methylation, such as sodium bisulfite conversion of genomic DNA combined with sequencing, methylation specific digestion, methylation specific PCR (MSP) and DHPLC. All of the above approaches are limited to study DNA methylation statue on gene-by-gene basis. Obviously, these small-scale approaches can not meet the task to identify epigenetic modification at the genomic level in the complex human diseases. This results in the development of new field, namely epigenomics. Epigenomics is defined as studying epigenetic modifications on genome-scale. DNA methylome analysis is one of important direction of Epigenomics study. Although a few progresses in epigenomics have been achieved, there is still much difficulty to be overcome for developing a reliable and accurate methodology to identify DNA methylome. Therefore, in this project we developed a high through-put 9K CpG island (CGI) array which could be potentially applied into study of underlying DNA methylation modification in a variety of biological processes and common human diseases.
Although 9K CGI array provides an efficient tool for DNA methylation profiling analysis, the bottleneck of DNA methylome study is genome-wide methylated DNA enrichment. Thus, maximal enrichment of methylated DNA is essential in subsequent detection of differential methylation CGI based on high through-put technologies. Most commonly used genome-wide methylated DNA enrichment approaches including methods based on methylation sensitive restriction enzyme digestion -differential methylation hybridization (DMH) and microarray based methylation assessment of single samples (MMASS), as well as ones based on protein affinity purification - methylcytosine binding protein (MBD protein) affinity purification and methylated DNA immunoprecipitation (MeDIP). Previous study has demonstrated that MeDIP is more sensitive than MBD purification for detecting methylated DNA, nevertheless, a systematic comparison of methylation sensitive restriction enzymes digestion based assays including DMH and MMASS, with MeDIP haven't been performed. Therefore, we first optimized several important experimental parameters of these three methods, and then performed evaluation of sensitivity and accuracy among these three methods. Finally, we performed GO analysis of differential CGI related genes from methylation profiling of Ges-1 and MGC-803 cell lines with MMASS and MeDIP assays, and the results showed lots of genes involved in important biological functions and processes.
The second part of the thesis describes our effort in developing human CGI array for DNA methylome analysis as well as optimizing and evaluating methylated DNA enrichment approaches based on the CGI array. We identified CGI clones through sequencing of human CGI library by ourselves and Wellcome Trust Sanger Institute, respectively. In addition, we predicted CGIs from the region of the first exon and 10kb upstream of TSS of some cancer related genes and imprinted genes. All the representative CGI clones and predicted CGIs of target genes were amplified with PCR for CGI array spotting. In addition, we optimized several important parameters of DMH, MMASS and MeDIP, including PCR annealing temperatures and antibody incubation time in immunoprecipitation through methylation profiling of gastric adenocarcinoma cell line MGC-803 with 9K CGI array. Furthermore, we performed a systematic evaluation of sensitivity and accuracy of the three assays through methylation profiling of gastric epithelium cell line Ges-1 and adenocarcinoma cell line MGC-803 with 9K CGI array. Finally, we carried out GO analysis of differential CGI related genes from methylation profiling of Ges-1 and MGC-803 cell lines with MMASS and MeDIP assays.
Results:
1. 18,816 clones (18k) from human CGI library were sequenced. In addition, 12,192 clones identified from the same library bought from Wellcome Trust Sanger Institute for developing high through-put CGI array. After sequences aligning, blasting and clustering, 10,749 representative clones were selected from 18K dataset with the redundancy of 37.02%, as well as 6,445 representative clones from 12K dataset with the redundancy of 37.84%.
2. Based on bioinformatics prediction, 33,116 CGI were identified from whole human genome with CpGi130. 10,749 representative clones selected from 18K dataset and 6,445 representative clones from 12K dataset were matched with predicted CGIs from whole human genome. The overlapped clones were defined as CGI clones, otherwise as non-CGI clones. Totally, 8,967 clones were matched to our bioinformatics prediction and identified as representative CGI clones. In addition, 830 unique CGIs were predicted from the region of the first exon and 10kb upstream of 450 cancer related genes and 90 imprinted genes with CpGi130. We annotated all the representative CGI clones from CGI library and CGIs predicted from target genes including the location on the chromosome, the adjacent genes, the position relationship with genes and the description of genes.
3. 8,967 representative CGI clones from CGI library and 830 CGIs from target genes were amplified, of which 9,223 CGI probes were successfully amplified for 9K CGI array spotting. The evaluation of 9K CGI array manifests that both of the signal and the consistency of hybridization are very high.
4. Several important parameters were optimized in three methylated DNA enrichment assays including DMH, MMASS and MeDIP. The comparison of different PCR annealing temperatures showed that relative high temperature (72℃) for amplification of products, resulted from methylation-sensitive and methylation-dependent digestion, significantly improved the sensitivity of assessment of differential methylation of CGIs. In addition, the comparison of different antibody incubation time showed that the relative long time of the primary antibody incubation (12h) and the suitable time of the secondary antibody incubation (3h around) increased the efficiency of methylated DNA enrichment and, in the meantime, decreased the unspecific binding of unmethylated DNA.
5. A systematic evaluation of the sensitivity and accuracy of the three enrichment methods among DMH, MMASS and MeDIP were performed. The results show that: 1) the three methods identify some common differential methylated CGI probes, however, each method detected unique differential targets that were ranked very low in the other two; 2) MMASS is the highest in sensitivity, MMASS is similar with MeDIP in accuracy and DMH is the lowest either in sensitivity or in accuracy among the three assays.
6. GO analysis of 361 differential CGI related genes from methylation profiling of Ges-1 and MGC-803 cell lines with MMASS and MeDIP assays was performed. The results showed that the genes were involved in many vital biological processes including cellular component, molecular function and biological process. Conclusion:
We developed human 9K CGI array with 9223 CGI probes identified from human CGI library and target genes, which could provide a high through-put tool for DNA methylome analysis on genome-wide. The optimization of several important parameters in DMH, MMASS and MeDIP significantly improves the sensitivity and specificity of the assessment of DNA methylation profiling. Furthermore, the systematic evaluation suggests that MMASS is the highest in sensitivity among these three assays, nevertheless both enzyme digestion based method - MMASS and immunoprecipitation based method - MeDIP are complementary in detection of differential methylated candidates on genome-scale. Therefore, employment combining two or more enrichment approaches could result in better and complete assessment of DNA methylome. In addition, the results of GO analysis suggest the differential CGI related genes which are potentially regulated by differential methylation are involved in lots of vital biological processes and functions. The screening based on 9K CGI array could provide many important candidates for the study of epigenetic mechanism in cancer, and 9K CGI array will be very promising in DNA methylation profiling of human physiological and pathological samples.
蛋白质组学是应用高通量的技术对组织和细胞整体蛋白的组成、表达和相互作用进行研究的学科,在神经科学领域,它越来越多地被应用于各种疾病如帕金森病、阿尔茨海默病、药物成瘾和精神分裂症的蛋白表达谱和分子间相互作用的研究。药物成瘾是精神活性物质反复作用于大脑引发的一种长期适应性改变,并被认为是一种慢性复发性的脑病。目前的研究认为,多巴胺能和谷氨酸能神经的突触可塑性在药物成瘾中起了重要的作用,精神活性物质的强烈作用,使两类受体发生相互调控,神经信号在受体经整合后触发了受体后细胞内的级联反应,从而调控了突触的基因表达和蛋白质合成,长期的基因、蛋白的表达以及分子组成的变化,引起了突触结构和形态发生改变,从而导致神经功能的不可逆变化,这一系列过程在药物依赖的神经适应性过程中起了关键的作用。前额叶皮质(prefrontal cortex,PFC)是奖赏环路的重要组成部分,它对行为的执行起着判定和控制的作用,因而是成瘾相关的重要脑区之一。有研究表明,慢性吗啡作用会引起腹侧背盖核(ventral tegmental area,VTA)多巴胺神经元的体积缩小以及伏隔核(nucleus accumbens,NAc)和PFC树突联系和中间神经原的棘突数量的减少。为了对成瘾相关的突触分子作用机制进行更深入的研究,我们使用双向电泳(two dimensional gel electrophoresis,2-DE)结合质谱的蛋白质组学技术分析了吗啡诱导的条件性位置偏爱(Conditioned Place Preference,CPP)三个阶段大鼠PFC脑区突触后致密区(post synaptic density,PSD)蛋白表达谱的动态变化。
本论文的第一部分描述了吗啡诱导的CPP三个阶段大鼠PFC脑区的PSD蛋白表达谱的分析结果。我们使用CPP模型来评价大鼠的吗啡依赖效应,CPP训练程序包括:自然偏爱测试,条件性偏爱的建立、消退和复燃。基于吗啡诱导的CPP模型,我们使用2-DE技术对CPP三个阶段(建立、消退和复燃)大鼠PFC脑区的PSD蛋白进行分析,筛选了差异表达的蛋白。进而我们使用MALDI-TOF-MS质谱技术成功鉴定了部分差异蛋白,并基于免疫沉淀技术对差异蛋白表达谱的分析结果进行了验证。
结果:
1.建立吗啡诱导的CPP模型,包括条件性获得,消退和复燃三个阶段。吗啡组大鼠在CPP获得阶段表现出对吗啡伴药盒的显著偏爱,在消退阶段对吗啡伴药盒的偏爱消失以及在复燃阶段由于小剂量吗啡的诱导又表现为对伴药盒的显著偏爱。
2.使用2-DE技术对吗啡诱导CPP三个阶段的大鼠PFC脑区的PSD蛋白进行分析,共筛选到80个差异蛋白,其中代表性差异表达的蛋白—CNPasel(cyclicnucleotide phosphodiesterase 1)在CPP三个阶段都发生了显著的变化,其余79个蛋白在CPP的一个或两个阶段有差异表达。
3.使用MALDI-TOF-MS质谱技术对差异表达蛋白进行鉴定,质谱成功鉴定了CPP获得阶段21个差异蛋白,消退阶段14个差异蛋白和复燃阶段23个差异蛋白。通过生物信息学的分析,这些蛋白参与了多种重要的生物学功能如能量代谢,信号传导,突触传递,细胞蛋白骨架,分子伴侣和突触蛋白合成器等。
4.为了评价蛋白质组学分析结果的准确性,我们使用western blot验证了代表性差异蛋白CNPase1。Western blot的结果显示,CNPase1在吗啡诱导CPP的获得阶段上调了2.1倍,消退阶段下调了1.6倍,然而在复燃阶段又上调了1.4倍,该结果与2-DE分析中的变化趋势是一致的。Western blot验证结果表明使用蛋白质组学对吗啡诱导的CPP三个阶段大鼠PFC脑区PSD蛋白表达谱的分析结果是可信的。
结论:
我们对吗啡诱导的CPP三个阶段大鼠PFC脑区PSD蛋白表达谱的分析获得了大量差异表达的蛋白,这些蛋白参与了多种重要的生物学功能如能量代谢,信号传导,突触传递,细胞蛋白骨架,分子伴侣和突触蛋白合成器等。本研究第一次对吗啡诱导的条件性依赖的不同阶段一获得、消退和复燃,基于动物模型分析了成瘾相关重要脑区PFC的PSD蛋白分子的动态变化,从而为吗啡成瘾相关的突触分子作用机制的研究提供了许多重要的靶点。
表观遗传学是指DNA序列没有发生改变,但通过DNA甲基化、组蛋白修饰以及染色质结构的重塑来调控基因表达和功能的可遗传的变化。DNA甲基化是表观遗传学中研究得最为广泛的一种修饰,它参与了哺乳动物的多种生命活动过程,包括基因的沉默、染色体结构的稳定、X染色体的失活、基因印记和细胞分化,因而DNA甲基化的异常改变在人类多种重大疾病如肿瘤、精神性和免疫性等疾病中起了非常重要的作用。目前大多数研究对DNA甲基化修饰的分析都是基于单个候选基因途径,主要采用的检测技术为亚硫酸氢钠转化DNA结合测序、甲基化特异性酶切、甲基化特异性PCR(Methylation Specific PCR,MSP)和变性高效液相色谱(Denaturing High Performance Liquid Chromatography,DHPLC)等。但对于重大、复杂疾病的研究,小规模单基因的研究模式显然不能完整地解释其中表观遗传的调控网络和作用机制,这促使了表观基因组学的发展。表观基因组学则是在全基因组水平上对表观遗传学修饰的研究,基因组水平DNA甲基化修饰谱的研究是表观基因组学的重要组成部分。因而在本研究中,我们开发了高通量的DNA甲基化谱分析芯片—人9K CGI芯片,该芯片可以应用于人类各种生命活动和重大疾病的DNA甲基化修饰谱的分析。虽然9K CGI芯片为基因组DNA甲基化谱分析提供了一个高通量的检测工具,但DNA甲基化谱研究的瓶颈是全基因组甲基化DNA的富集,最大限度地获得纯化的甲基化DNA是后续基于高通量技术进行DNA甲基化分析的关键。目前国际上广泛采用的方法有:(1)基于甲基化限制性酶切技术,代表性的有差异甲基化杂交(Differential Methylation Hybridization,DMH)和基于微阵列芯片的单个样品甲基化评价(Microarray Based Methylation Assessment of Single Sample,MMASS);(2)甲基化结合蛋白(Methylation Binding Protein,MBD Protein)纯化甲基化DNA;(3)甲基化DNA免疫沉淀(Methylated DNA Immunoprecipitation,MeDIP)。已有研究表明MeDIP的灵敏性高于MBD层析柱纯化的方法,但没有研究对基于甲基化限制性酶切和基于免疫沉淀的富集方法进行过系统的评价。因此,在本研究中,我们以胃上皮细胞系Ges-1和胃腺癌细胞系MGC-803为研究对象,基于9K CGI芯片优化了DMH、MMASS和MeDIP三种方法中的一些重要的实验参数,进而对各种优化后的方法进行了灵敏性和准确性的系统评价,从而为9KCGI芯片更好地应用于各种基因组DNA甲基化修饰的表观遗传学研究打下了坚实的基础。同时对MMASS和MeDIP方法分析两种细胞系甲基化谱获得的差异CGI探针相关的基因进行GO分类,结果显示大量的基因参与了多种重要的生物学过程,从而证实了9K CGI芯片对人类的表观遗传学研究具有较大的应用价值。
本论文的第二部分阐述了DNA甲基化谱分析芯片的研制,基于该芯片优化和评价了全基因组甲基化DNA的富集方法,并对甲基化差异的CGI探针相关的基因进行GO分类。首先,我们对CGI文库进行了大规模的克隆测序,合并Wellcome Trust Sanger Institute对同一文库测序获得的克隆,与预测的全基因组CGI比对来鉴定两部分测序获得的CGI克隆。同时我们预测了部分肿瘤相关基因和印记基因第一个外显子和转录起始位点上游10kb范围内的CGI。CGI克隆和目的基因相关CGI进行PCR扩增后的产物作为探针点制成CGI芯片。我们基于CGI芯片,通过对胃腺癌细胞系MGC-803的甲基化谱分析优化了DMH、MMASS和MeDIP三种方法中的一些重要的实验参数,包括PCR退火温度和免疫沉淀反应中的抗体孵育时间。进而对基于胃上皮细胞系Ges-1和胃腺癌细胞系MGC-803的甲基化谱分析比较,系统评价了三种方法的灵敏性和准确性。并对正常和癌症细胞系的甲基化谱分析中筛选到的甲基化差异的CGI探针相关基因进行GO分类。
结果:
1.为了开发高通量的基因组DNA甲基化谱分析芯片,我们对人源性CGI文库进行了18,816个克隆(18K)的测序,序列经拼接、比对及聚类分析后,共获得10,749个代表性克隆,冗余度为37.02%;应用Wellcome Trust Sanger Institute对同一CGI文库进行部分测序获得的12,192个单克隆(12K),同样对12K数据的拼接、比对及聚类分析后,共获得6,445个代表性克隆,冗余度为37.84%。
2.使用CpGi130软件预测到人类全基因组共33,116个CGI。将18K克隆测序获得的10,749个代表性克隆和来自12K克隆的6,445个代表性克隆与全基因组预测得到的CGI进行比对,共鉴定了8,967个特异性CGI克隆。同时从已知的450个肿瘤相关基因和90个印记基因的第一个外显子和转录起始位点上游10K的区域预测了830个特异性CGI。对所有特异性CGI克隆和候选基因的CGI进行了注释,包括在基因组上的位置,邻近的基因,与邻近基因的位置关系以及对基因的描述。
3.为了制备CGI芯片,对8,967个特异性CGI克隆和830个候选基因的CGI进行PCR扩增,成功扩增的9,223个CGI探针用于9K CGI芯片的制备。对9K CGI芯片的质量评价表明芯片杂交的信号强度和重复性都非常高。
4.基于9K CGI芯片,优化了DMH、MMASS和MeDIP三种全基因组甲基化DNA富集方法中的一些重要的参数。对DMH和MMASS方法中不同PCR退火温度的比较表明,使用较高的退火温度(72℃)扩增甲基化和非甲基化酶切产物,大大提高了芯片检测CGI甲基化差异的灵敏性;对MeDIP方法中不同抗体孵育时间的比较表明,较长的一抗孵育时间(12小时)和合适的二抗孵育时间(3小时左右)既提高了甲基化DNA的结合效率,又降低了非甲基化DNA的非特异性结合。
5.基于9K CGI芯片,对优化后的DMH、MMASS和MeDIP三种方法的系统评价结果表明:1)三种方法能检测到共同的甲基化差异的CGI探针,但每种方法也能筛选到特异性的差异CGI探针;2)三种方法中MMASS的敏感性最高,MMASS和MeDIP的准确性类似,但DMH的敏感性和准确性都较低。
6.合并MMASS和MeDIP对Ges-1和MGC-803细胞系甲基化谱分析获得的差异CGI探针,基于探针的基因注释,对361个差异CGI探针相关的基因进行GO分类,包括细胞组分(Cellular Component)、分子功能(Molecular Function)和生物学过程(Biological Process),结果显示大量参与多种重要生物学功能的基因在癌症细胞系中可能发生了甲基化的调控。
结论:
基于对CGI文库的测序及部分目的基因CGI的预测,我们鉴定并扩增了9223个CGI探针,从而开发了9K CGI芯片,该芯片为基因组水平DNA甲基化修饰的研究提供了一个高通量的平台。基于该芯片我们对甲基化DNA的富集方法—DMH、MMASS和MeDIP进行了优化,从而提高了各种方法对基因组CGI甲基化差异评价的灵敏性和特异性。进而对优化后的三种方法的系统评价表明MMASS方法的敏感性最高,但MMASS和MeDIP两种方法的准确性类似,因而基于不同原理的两种方法MMASS和MeDIP对全基因组CGI的甲基化差异的评价是互为补充的。因此在实际的应用中,只有联合不同原理的两种或两种以上的方法,才能对全基因组CGI的甲基化水平进行更全面的分析。同时对差异CGI探针相关基因的GO分类显示,癌症细胞中潜在受甲基化调控的基因参与了多种重要的生物学功能,因而基于9K CGI芯片的甲基化谱分析为癌症的甲基化修饰机制的研究提供了许多重要的靶点,从而也表明9K CGI芯片将在人类生理和疾病的表观遗传学研究中具有广阔的应用前景。
With the completion of Human Genome Project (HGP) the genomic study has moved from genomic sequencing to functional study on genome-scale, emphasizing exploration of the expression and regulatory network of genes on genome-wide. The diversified post-genomic field includes, but not limits, genomic SNP analysis, transcriptomics, proteomics, and newly developed epigenomics.
Proteomics is to study protein expression and interaction of entire proteins in specific tissues and cells, and recently has been intensively used in human neurological diseases such as Parkinson's disease, Alzheimer disease, drug addiction and schizophrenia. Drug abuse is recognized as long-term adaptation in brain which is resulted from repeatedly usage of psychoactive substances. Therefore, drug addiction is also presumed as chronic and relapsing encephalopathy. The numerous lines of evidence have suggested that dopaminergic and glutamatergic neuronal synaptic plasticity play an important role in drug addition. In general, psychoactive substances strongly affect on the neuronal systems through dopamine D-1 and glutamate N-methyl-D-aspartate (NMDA) andα-amino-3-hydroxy-5-methylisoxazole -4-propionic acid (AMPA) receptors, which induce intracellular transcriptional and translational cascades, leading to adaptive changes in gene expression, protein expression and molecular constitute. The long-term adaptation of synaptic plasticity induced from the above molecular alteration, causes the alteration of synaptic morphology and structure which further result in irreversible neural dysfunction.
Prefrontal cortex (PFC) is one of important brain regions in reward circuits, which is critical for many cognitive functions involving inhibitory control and decision making. As the relationship between a cell's chemistry and structure becomes increasingly apparent, it is not surprised that chronic exposure to drugs of abuse has been shown to alter the morphology of neurons in reward circuits of the brain. Chronic morphine administration decreases the size and caliber of dopamine neurons in ventral tegmental area (VTA). In addition, chronic morphine administration has been shown to decrease the complexity of dendritic branching and the number of spines on medium spiny neurons in the nucleus accumbens (NAc) and PFC. For better understanding the synaptic molecular mechanism involved in addiction, we performed proteomic analysis of PSD fraction of rats' PFC in three phases of morphine induced conditioned place preference (CPP).
The first part of the thesis is to present our proteomic analysis of PSD fraction of rats' PFC in three phases of morphine induced CPP. The effect of morphine dependence is evaluated by CPP model, the procedures of which include nature preference test, conditioned acquisition, conditioned extinction and conditioned reinstatement. Next we analyzed the expression profiling of PSD fraction of rats' PFC in three phases of CPP with two-dimensional gel electrophoresis (2-DE). Furthermore, we identified differential expression proteins with MALDI-TOF-MS and validated the results of proteomics with western blot.
Results:
1. Morphine induced CPP model was established, including acquisition, extinction and reinstatement phases. The rats showed strong preference to morphine-paired chamber in acquisition test after morphine conditioning. The morphine-induced preference is diminished after saline-paired extinction training, and could be re-established after morphine priming injection.
2. Proteomic analysis of PSD fraction of rats' PFC in three phases of morphine induced CPP with 2-DE resulted in 80 differential proteins, of which protein cyclic nucleotide phosphodiesterase 1 (CNPasel) showed significantly differential expression in the three phases of CPP respectively, and the remaining proteins were altered in one or two phases of CPP.
3. The differential proteins were identified with MALDI-TOF-MS spectrum, of which 21 proteins were successfully identified in acquisition phase, 14 in extinction phase and 23 in reinstatement phase. Based on bioinformatics analysis, 58 identified differential proteins are found to involved in many vital biological functions, such as energy metabolism, signal transduction, synaptic transmission, cytoskeleton proteins, molecular chaperones, and synaptic proteins synthetic machine.
4. The expression of representative protein CNPasel was verified for assessment of the accuracy of proteomic results with western blot. The result showed that there were 2.1 fold elevation in the acquisition phase, 1.6 fold decrease in the extinction phase and 1.4 fold increase in the reinstatement phase, which were consistent with the analysis of 2-DE and silver staining. The data indicates that the changes in protein expression identified by proteomic analysis of synaptic fraction of PFC in the three phases of CPP are reliable.
Conclusion:
The proteomic analysis of PSD fraction of rats' PFC in the three phases of morphine induced CPP resulted in identification of many differentially expressed proteins. Based on bioinformatics analysis, these proteins are classified into many important biological functions, such as energy metabolism, signal transduction, synaptic transmission, cytoskeleton proteins, molecular chaperones and synaptic proteins synthetic machine. Therefore, our study, in the first time, revealed the expression profiling of PSD fraction of rats' PFC under morphine induced conditioned dependence including acquisition, extinction and reinstatement, which provides many crucial candidates for further study of molecular synaptic function in morphine addiction.
Epigenetics is to study heritable changes, such as DNA methylation and histone modification that modulate chromatin organization and gene expression in the absence of change in DNA sequence. The most widely studied epigenetic modification in humans is the cytosine methylation of DNA within the dinucleotide CpG. DNA methylation has been shown to involve in many biological processes, such as gene silencing, transposable inhibition, X-chromosome inactivation and cell differentiation. An altered pattern of DNA methylation has been associated with etiology of some severe human diseases such as cancer, mental health and immunological diseases.
A range of approaches are available for assessing DNA methylation, such as sodium bisulfite conversion of genomic DNA combined with sequencing, methylation specific digestion, methylation specific PCR (MSP) and DHPLC. All of the above approaches are limited to study DNA methylation statue on gene-by-gene basis. Obviously, these small-scale approaches can not meet the task to identify epigenetic modification at the genomic level in the complex human diseases. This results in the development of new field, namely epigenomics. Epigenomics is defined as studying epigenetic modifications on genome-scale. DNA methylome analysis is one of important direction of Epigenomics study. Although a few progresses in epigenomics have been achieved, there is still much difficulty to be overcome for developing a reliable and accurate methodology to identify DNA methylome. Therefore, in this project we developed a high through-put 9K CpG island (CGI) array which could be potentially applied into study of underlying DNA methylation modification in a variety of biological processes and common human diseases.
Although 9K CGI array provides an efficient tool for DNA methylation profiling analysis, the bottleneck of DNA methylome study is genome-wide methylated DNA enrichment. Thus, maximal enrichment of methylated DNA is essential in subsequent detection of differential methylation CGI based on high through-put technologies. Most commonly used genome-wide methylated DNA enrichment approaches including methods based on methylation sensitive restriction enzyme digestion -differential methylation hybridization (DMH) and microarray based methylation assessment of single samples (MMASS), as well as ones based on protein affinity purification - methylcytosine binding protein (MBD protein) affinity purification and methylated DNA immunoprecipitation (MeDIP). Previous study has demonstrated that MeDIP is more sensitive than MBD purification for detecting methylated DNA, nevertheless, a systematic comparison of methylation sensitive restriction enzymes digestion based assays including DMH and MMASS, with MeDIP haven't been performed. Therefore, we first optimized several important experimental parameters of these three methods, and then performed evaluation of sensitivity and accuracy among these three methods. Finally, we performed GO analysis of differential CGI related genes from methylation profiling of Ges-1 and MGC-803 cell lines with MMASS and MeDIP assays, and the results showed lots of genes involved in important biological functions and processes.
The second part of the thesis describes our effort in developing human CGI array for DNA methylome analysis as well as optimizing and evaluating methylated DNA enrichment approaches based on the CGI array. We identified CGI clones through sequencing of human CGI library by ourselves and Wellcome Trust Sanger Institute, respectively. In addition, we predicted CGIs from the region of the first exon and 10kb upstream of TSS of some cancer related genes and imprinted genes. All the representative CGI clones and predicted CGIs of target genes were amplified with PCR for CGI array spotting. In addition, we optimized several important parameters of DMH, MMASS and MeDIP, including PCR annealing temperatures and antibody incubation time in immunoprecipitation through methylation profiling of gastric adenocarcinoma cell line MGC-803 with 9K CGI array. Furthermore, we performed a systematic evaluation of sensitivity and accuracy of the three assays through methylation profiling of gastric epithelium cell line Ges-1 and adenocarcinoma cell line MGC-803 with 9K CGI array. Finally, we carried out GO analysis of differential CGI related genes from methylation profiling of Ges-1 and MGC-803 cell lines with MMASS and MeDIP assays.
Results:
1. 18,816 clones (18k) from human CGI library were sequenced. In addition, 12,192 clones identified from the same library bought from Wellcome Trust Sanger Institute for developing high through-put CGI array. After sequences aligning, blasting and clustering, 10,749 representative clones were selected from 18K dataset with the redundancy of 37.02%, as well as 6,445 representative clones from 12K dataset with the redundancy of 37.84%.
2. Based on bioinformatics prediction, 33,116 CGI were identified from whole human genome with CpGi130. 10,749 representative clones selected from 18K dataset and 6,445 representative clones from 12K dataset were matched with predicted CGIs from whole human genome. The overlapped clones were defined as CGI clones, otherwise as non-CGI clones. Totally, 8,967 clones were matched to our bioinformatics prediction and identified as representative CGI clones. In addition, 830 unique CGIs were predicted from the region of the first exon and 10kb upstream of 450 cancer related genes and 90 imprinted genes with CpGi130. We annotated all the representative CGI clones from CGI library and CGIs predicted from target genes including the location on the chromosome, the adjacent genes, the position relationship with genes and the description of genes.
3. 8,967 representative CGI clones from CGI library and 830 CGIs from target genes were amplified, of which 9,223 CGI probes were successfully amplified for 9K CGI array spotting. The evaluation of 9K CGI array manifests that both of the signal and the consistency of hybridization are very high.
4. Several important parameters were optimized in three methylated DNA enrichment assays including DMH, MMASS and MeDIP. The comparison of different PCR annealing temperatures showed that relative high temperature (72℃) for amplification of products, resulted from methylation-sensitive and methylation-dependent digestion, significantly improved the sensitivity of assessment of differential methylation of CGIs. In addition, the comparison of different antibody incubation time showed that the relative long time of the primary antibody incubation (12h) and the suitable time of the secondary antibody incubation (3h around) increased the efficiency of methylated DNA enrichment and, in the meantime, decreased the unspecific binding of unmethylated DNA.
5. A systematic evaluation of the sensitivity and accuracy of the three enrichment methods among DMH, MMASS and MeDIP were performed. The results show that: 1) the three methods identify some common differential methylated CGI probes, however, each method detected unique differential targets that were ranked very low in the other two; 2) MMASS is the highest in sensitivity, MMASS is similar with MeDIP in accuracy and DMH is the lowest either in sensitivity or in accuracy among the three assays.
6. GO analysis of 361 differential CGI related genes from methylation profiling of Ges-1 and MGC-803 cell lines with MMASS and MeDIP assays was performed. The results showed that the genes were involved in many vital biological processes including cellular component, molecular function and biological process. Conclusion:
We developed human 9K CGI array with 9223 CGI probes identified from human CGI library and target genes, which could provide a high through-put tool for DNA methylome analysis on genome-wide. The optimization of several important parameters in DMH, MMASS and MeDIP significantly improves the sensitivity and specificity of the assessment of DNA methylation profiling. Furthermore, the systematic evaluation suggests that MMASS is the highest in sensitivity among these three assays, nevertheless both enzyme digestion based method - MMASS and immunoprecipitation based method - MeDIP are complementary in detection of differential methylated candidates on genome-scale. Therefore, employment combining two or more enrichment approaches could result in better and complete assessment of DNA methylome. In addition, the results of GO analysis suggest the differential CGI related genes which are potentially regulated by differential methylation are involved in lots of vital biological processes and functions. The screening based on 9K CGI array could provide many important candidates for the study of epigenetic mechanism in cancer, and 9K CGI array will be very promising in DNA methylation profiling of human physiological and pathological samples.
引文
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