神经细胞中酒精调控基因的鉴定及其功能研究

发布时间：2018-07-06 19:16

本文选题：酒精 + 基因鉴定　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 目的:酒精已被证明参与诱导人类多种疾病,如:动脉硬化、痴呆、糖尿病、以及与锌代谢有关的疾病等。在神经退行性疾病研究中,动物实验表明,酒精可导致大脑与小脑不同区域的损伤,并广泛的触发神经细胞退行性凋亡。酒精诱导的功能改变以及相关的神经病理包括:细胞的外毒性、硫胺素的缺乏、神经胶质细胞的异常、生长因子的改变、细胞的凋亡、氧化压迫、能量代谢异常。发育期间的神经细胞对酒精的伤害尤其敏感:如:1)在发育的大鼠前脑细胞中,酒精一方面通过抑制N-甲基-D-天冬氨酰谷氨酸盐受体,另一方面激活伽马氨基丁酸受体,而触发广泛的由凋亡引起的神经退行性病变。同时酒精还通过改变谷氨酸盐和伽马氨基丁酸的运输,而抑制神经细胞的活性,从而造成数百万神经细胞的死亡;2)在出生后4-6天的大鼠浦肯雅细胞中,酒精可选择性的降低酪氨酸激酶B和C受体的表达,在该过程中酒精通过改变神经营养调控导致浦肯雅细胞的减少;3)人类脑细胞的发育成熟可从妊娠的六个月一直延续到出生后几年,该阶段也是神经突触形成最敏感最脆弱的时期,这种酒精造成的前凋亡效应在人类胚胎发育过程中可导致酒精综合症。长期酗酒诱导的氧化压迫可加速神经系统的退行性病变,神经退行性疾病主要是老年人群,老年痴呆、脑萎缩、帕金森综合征等多种神经系统疾病日益威胁着老年人的健康,当前已成为现代社会老年人的主要致死病之一。在酒精对神经系统损伤的分子机理研究中,酒精调控基因的鉴定是非常重要的内容,对进一步了解疾病的发生与进展具有重要的意义。本文通过抑制性差减杂交技术,在转录水平上筛选酒精调控的相关基因,随后挑选一种基因进行进一步的功能研究。研究内容:通过抑制性差减杂交技术,在神经细胞中进行酒精调控基因的筛选,并分别通过反向点杂交技术以及Northern Blot技术对候选基因进行进一步的证实,序列分析后登陆GenBank数据库进行序列比对。在基因的功能研究中,通过利用Northern Blot技术探索基因表达与酒精处理时间之间的对应关系,探索基因的组织分布特异性;通过利用出生后不同时间的大鼠小脑组织以及不同分化阶段的PC12细胞(神经生长因子诱导),探索细胞分化与基因表达之间的对应关系;通过重组表达技术,对目的基因进行上调与下调,探索基因的过表达与抑制表达对细胞功能的影响,即通过对细胞进行有害处理,观察细胞生存能力的变化;通过利用酵母双杂交技术,筛选与目的基因表达产物相互作用的蛋白分子。方法:建立有效的酒精处理方法;利用抑制性差减杂交技术进行酒精调控基因的筛选,并通过反向点杂交(Reversed dot blot)方法进行验证;利用Northern blot技术探索酒精处理时间与筛选基因表达的对应关系,探索目的基因表达与细胞分化状态之间的关系以及目的基因分布的组织特异性;利用5’RACE技术对目的基因进行全长序列的制备;利用全长序列的重组构建对目的基因进行上调;利用RNAi序列的重组构建对目的基因进行下调;利用MTT方法评价细胞的成活性;利用酵母双杂交技术,筛选与目的基因表达产物相互作用的蛋白分子。结果:1酒精调控基因的筛选:通过差减杂交以及反向点杂交技术筛选出了七个酒精调控的基因,并进一步观察了酒精处理不同时间对这些基因表达的影响。其中酒精上调基因包括:aquaporin 1(NW_047693)、prohibitin(XM_579541)、SCIRP10 ( NW_047402 );酒精下调基因包括: zinc finger protein 341(XM_001074788)、Cab45a(MMU45977)以及两个基因新序列(GenBank数据库没有发现与其高度同源序列)。在这些酒精调控基因中,Cab45和锌脂蛋白341表现出明显的时间依赖性下调,而水孔蛋白1则表现出时间依赖性上调。 2 Cab45基因的特征鉴定及其功能研究:在筛选出的差减杂交克隆中,其中两个序列均与小鼠的Cab45a高度同源,说明该基因在差减杂交过程中出现的频率较高,同时该基因序列在大鼠中未见报道,所以对其进行了进一步的探索。通过5’RACE技术制备了大鼠Cab45基因全长序列,并将推导的氨基酸序列与其在其它物种中的对应体进行了比对,结果显示Cab45具有高度的保守性。象该家族中的其它成员一样, Cab45也具有广泛的组织分布特性。PC12细胞分化模型以及出生不同时间大鼠小脑细胞模型研究表明,Cab45的表达与分化程度呈负相关。对Cab45进行上调与下调的结果表明,上调可显著减弱酒精与紫外线对PC12细胞造成的死亡,而下调则产生相反的结果(紫外线损伤)。另外在通过酵母双杂交方法筛选与其相互作用的蛋白时发现,Cab45具有较强的转录激活功能。结论:本文在神经细胞(B104)中通过差减杂交方法对酒精调控基因进行了筛选,鉴定出了一系列酒精调控的基因(其中两个为新序列),为酒精调控的机理研究提供了重要的数据。随后对其中的钙离子结合蛋白Cab45进行了全长序列的制备,并随后进行了保守性分析、组织分布检测、分化相关性检测以及上调与下调研究等多项指标,系统地阐述了该基因的各种特征,从而将酒精对神经细胞的损伤作用与钙离子结合蛋白建立了重要关系,本研究以下内容均为首次报道:1)酒精短时间处理导致Cab45基因上调,而长时间处理则导致其呈现时间依赖性下调;2)在大鼠中对Cab45基因进行了全序列的测定,并进行了氨基酸序列的保守性分析;3)探索了Cab45组织分布的广谱性;4)探索了Cab45表达与细胞分化状态的负相关性;4)Cab45的过表达可减弱酒精和紫外线对细胞的损伤;5)酵母双杂交显示Cab45具有转录激活功能;6)Cab45基因的调控序列包含多种转录因子的结合位点 Cab45属于弱钙离子结合蛋白家族,定位于高尔基体中,而该家族的其它成员均定位于内质网中,报道表明内质网中钙离子平衡在调控细胞生长、停止与凋亡过程中起着非常重要的作用,推测高尔基体中的中钙离子平衡也有着相似的作用,Cab45可能通过调节钙离子的平衡实现对细胞生长、停止与凋亡的调控。由于该家族中的其他成员被证明与多种疾病有密切的关系,提示Cab45可能在钙离子调控的神经系统疾病中起着重要的作用。该发现对酒精引起的神经损伤与老化的机理研究,提供了新的理论基础,对其进一步研究,将促进其作为药物作用分子靶点的临床应用开发。
[Abstract]:Objective: alcohol has been proved to be involved in inducing a variety of human diseases, such as arteriosclerosis, dementia, diabetes, and diseases related to zinc metabolism. In the study of neurodegenerative diseases, animal experiments show that alcohol can cause damage to different regions of the brain and cerebellum, and widely triggers neurodegenerative apoptosis. Alcohol induced function is widely used. Changes and related neuropathology include: extracellular toxicity of cells, deficiency of thiamine, abnormal glial cells, changes in growth factors, cell apoptosis, oxidative stress, and energy metabolism. The growth of nerve cells is particularly sensitive to alcohol damage: 1) in developing rat forebrain cells, alcohol is suppressed on the one hand. The N- methyl -D- aspartic glutamate receptor, on the other hand, activates the gamma aminobutyric acid receptor and triggers a wide range of neurodegenerative diseases caused by apoptosis. Alcohol also inhibits the activity of nerve cells by altering the transport of glutamate and gamma aminobutyric acid, resulting in the death of millions of nerve cells; 2) In the 4-6 day postnatal pukenya cells, alcohol selectively reduces the expression of tyrosine kinase B and C receptor, in which alcohol leads to a decrease in pukenya cells by changing neurotrophic regulation; 3) human brain cell development can continue from six months of pregnancy to a few years after birth, and this stage is also a synapse. The most sensitive and vulnerable period is that the pre apoptotic effect of this alcohol can lead to alcohol syndrome during human embryonic development. Long-term alcoholism induced oxidative stress can accelerate the degeneration of the nervous system, and neurodegenerative diseases are mainly the old people, Alzheimer's, brain atrophy, Parkinson syndrome and many other nerves. Systemic disease is increasingly threatening the health of the elderly, and it has become one of the main fatal diseases of the old people in modern society. In the study of the molecular mechanism of alcohol on the damage of the nervous system, the identification of the alcohol regulation gene is a very important content. It is of great significance for further understanding the occurrence and progress of the disease.
In this paper, suppression subtractive hybridization (SSH) is used to screen alcohol related genes at transcriptional level, and then select a gene for further functional study.
Research content: through the suppression subtractive hybridization technology, the screening of alcohol regulation genes in the neural cells was screened, and the candidate genes were further confirmed by reverse dot hybridization and Northern Blot technology respectively. The sequence analysis was carried out to the GenBank database after sequence analysis. In the functional study of the gene, the use of Nor Thern Blot explores the relationship between gene expression and alcohol treatment time, exploring the specificity of tissue distribution, and exploring the relationship between cell differentiation and gene expression by using the rat cerebellar tissues at different postnatal time and the PC12 cells of different stages of differentiation (induced by nerve growth factor). The expression technology is up-regulated and down regulated to explore the effect of overexpression and inhibition of gene expression on cell function, that is to observe the changes in cell viability by harmful treatment of cells, and to screen the protein molecules interacting with the target gene surface by using yeast two hybrid technique.
Methods: an effective alcohol treatment method was established, and the alcohol regulation gene was screened by inhibitory subtractive hybridization, and the reverse dot blot (Reversed dot blot) was used to verify the alcohol treatment. The corresponding relationship between the time of alcohol treatment and the expression of the screened gene was explored by Northern blot technology, and the expression of the target gene and the differentiation of the cells were explored. The relationship between the States and the tissue specificity of the target gene distribution; the preparation of the full length sequence of the target gene by 5 'RACE technology; the up regulation of the target gene by the recombination of the full length sequence; the downregulation of the target gene by the recombination of the RNAi sequence; the use of MTT to evaluate the activity of the cells; and the use of yeast double. Hybridization technology to screen protein molecules interacting with target gene expression products.
Results: the screening of 1 alcohol regulation genes: seven alcohol regulated genes were screened by subtractive hybridization and reverse dot blot, and the effects of alcohol treatment at different time on these genes were further observed. The up-regulated alcohol genes included aquaporin 1 (NW_047693), Prohibitin (XM_579541), SCIRP10 (NW_047402); The fine down genes included zinc finger protein 341 (XM_001074788), Cab45a (MMU45977) and two new gene sequences (GenBank database did not find its high homologous sequence). In these alcohol regulation genes, Cab45 and Zinzin 341 showed a significant time dependent downregulation, while the water pore protein 1 showed time dependence. Adjust.
2 Cab45 gene characterization and its function study: in the screened differential hybrids, two of them were highly homologous to the Cab45a of mice, indicating that the frequency of the gene was higher in the process of subtractive hybridization, and the gene sequence was not reported in rats. Therefore, the gene was further explored. Through 5 'RACE, the gene was further explored. The full length sequence of Cab45 gene in rats was prepared by technique, and the deduced amino acid sequence was compared with its counterparts in other species. The results showed that Cab45 had high conservatism. Like other members of the family, Cab45 also had extensive tissue distribution characteristics of.PC12 cell differentiation model and at different times of birth. The study of rat cerebellar cell model showed that the expression of Cab45 was negatively correlated with the degree of differentiation. The up-regulation and down regulation of Cab45 showed that up regulation could significantly weaken the death caused by alcohol and ultraviolet radiation to PC12 cells, and the down regulation produced the opposite result (ultraviolet damage). It is found that Cab45 has a strong transcriptional activation function.
Conclusion: in this paper, the alcohol regulation gene was screened by subtractive hybridization in B104, and a series of alcohol regulated genes were identified (two of them were new sequences), which provided important data for the study of the mechanism of alcohol regulation. Subsequently, the full length sequence of the calcium binding protein Cab45 was prepared. Followed by conservatism analysis, tissue distribution detection, differentiation correlation detection and up regulation and down regulation, the various characteristics of the gene were systematically expounded, and the important relationship between alcohol to nerve cell damage and calcium ion binding protein was established. The following contents were all reported for the first time: 1) alcohol Short time treatment resulted in the up-regulated Cab45 gene, while long time treatment led to a time dependent downregulation; 2) the Cab45 gene was determined in the rat and the conservativeness analysis of the amino acid sequence was carried out; 3) the broad-spectrum distribution of Cab45 was explored; and 4) the negative correlation between the expression of Cab45 and the state of cell differentiation was explored. 4) overexpression of Cab45 can weaken the damage of alcohol and ultraviolet light on cells; 5) yeast two hybrid shows that Cab45 has a transcriptional activation function; 6) the regulatory sequence of the Cab45 gene contains binding sites of a variety of transcription factors.
Cab45 belongs to the weak calcium ion binding protein family, located in the Golgi body, and the other members of the family are located in the endoplasmic reticulum. It is reported that the balance of calcium ions in the endoplasmic reticulum plays a very important role in regulating cell growth and stopping and apoptosis. It is also suggested that the balance of calcium ion in the matrix also has a similar effect. Cab45 may regulate cell growth, stop and apoptosis by regulating the balance of calcium ions. Other members of the family have been shown to have a close relationship with a variety of diseases, suggesting that Cab45 may play an important role in calcium regulated nervous system diseases. Theoretical research provides a new theoretical basis for further research, and will promote its clinical application as a molecular target for drug therapy.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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