本文選題：酒精 + 基因鑒定��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年博士論文

【摘要】： 目的:酒精已被證明參與誘導(dǎo)人類(lèi)多種疾病,如:動(dòng)脈硬化、癡呆、糖尿病、以及與鋅代謝有關(guān)的疾病等。在神經(jīng)退行性疾病研究中,動(dòng)物實(shí)驗(yàn)表明,酒精可導(dǎo)致大腦與小腦不同區(qū)域的損傷,并廣泛的觸發(fā)神經(jīng)細(xì)胞退行性凋亡。酒精誘導(dǎo)的功能改變以及相關(guān)的神經(jīng)病理包括:細(xì)胞的外毒性、硫胺素的缺乏、神經(jīng)膠質(zhì)細(xì)胞的異常、生長(zhǎng)因子的改變、細(xì)胞的凋亡、氧化壓迫、能量代謝異常。發(fā)育期間的神經(jīng)細(xì)胞對(duì)酒精的傷害尤其敏感:如:1)在發(fā)育的大鼠前腦細(xì)胞中,酒精一方面通過(guò)抑制N-甲基-D-天冬氨酰谷氨酸鹽受體,另一方面激活伽馬氨基丁酸受體,而觸發(fā)廣泛的由凋亡引起的神經(jīng)退行性病變。同時(shí)酒精還通過(guò)改變谷氨酸鹽和伽馬氨基丁酸的運(yùn)輸,而抑制神經(jīng)細(xì)胞的活性,從而造成數(shù)百萬(wàn)神經(jīng)細(xì)胞的死亡;2)在出生后4-6天的大鼠浦肯雅細(xì)胞中,酒精可選擇性的降低酪氨酸激酶B和C受體的表達(dá),在該過(guò)程中酒精通過(guò)改變神經(jīng)營(yíng)養(yǎng)調(diào)控導(dǎo)致浦肯雅細(xì)胞的減少;3)人類(lèi)腦細(xì)胞的發(fā)育成熟可從妊娠的六個(gè)月一直延續(xù)到出生后幾年,該階段也是神經(jīng)突觸形成最敏感最脆弱的時(shí)期,這種酒精造成的前凋亡效應(yīng)在人類(lèi)胚胎發(fā)育過(guò)程中可導(dǎo)致酒精綜合癥。長(zhǎng)期酗酒誘導(dǎo)的氧化壓迫可加速神經(jīng)系統(tǒng)的退行性病變,神經(jīng)退行性疾病主要是老年人群,老年癡呆、腦萎縮、帕金森綜合征等多種神經(jīng)系統(tǒng)疾病日益威脅著老年人的健康,當(dāng)前已成為現(xiàn)代社會(huì)老年人的主要致死病之一。在酒精對(duì)神經(jīng)系統(tǒng)損傷的分子機(jī)理研究中,酒精調(diào)控基因的鑒定是非常重要的內(nèi)容,對(duì)進(jìn)一步了解疾病的發(fā)生與進(jìn)展具有重要的意義。 本文通過(guò)抑制性差減雜交技術(shù),在轉(zhuǎn)錄水平上篩選酒精調(diào)控的相關(guān)基因,隨后挑選一種基因進(jìn)行進(jìn)一步的功能研究。 研究?jī)?nèi)容:通過(guò)抑制性差減雜交技術(shù),在神經(jīng)細(xì)胞中進(jìn)行酒精調(diào)控基因的篩選,并分別通過(guò)反向點(diǎn)雜交技術(shù)以及Northern Blot技術(shù)對(duì)候選基因進(jìn)行進(jìn)一步的證實(shí),序列分析后登陸GenBank數(shù)據(jù)庫(kù)進(jìn)行序列比對(duì)。在基因的功能研究中,通過(guò)利用Northern Blot技術(shù)探索基因表達(dá)與酒精處理時(shí)間之間的對(duì)應(yīng)關(guān)系,探索基因的組織分布特異性;通過(guò)利用出生后不同時(shí)間的大鼠小腦組織以及不同分化階段的PC12細(xì)胞(神經(jīng)生長(zhǎng)因子誘導(dǎo)),探索細(xì)胞分化與基因表達(dá)之間的對(duì)應(yīng)關(guān)系;通過(guò)重組表達(dá)技術(shù),對(duì)目的基因進(jìn)行上調(diào)與下調(diào),探索基因的過(guò)表達(dá)與抑制表達(dá)對(duì)細(xì)胞功能的影響,即通過(guò)對(duì)細(xì)胞進(jìn)行有害處理,觀察細(xì)胞生存能力的變化;通過(guò)利用酵母雙雜交技術(shù),篩選與目的基因表達(dá)產(chǎn)物相互作用的蛋白分子。 方法:建立有效的酒精處理方法;利用抑制性差減雜交技術(shù)進(jìn)行酒精調(diào)控基因的篩選,并通過(guò)反向點(diǎn)雜交(Reversed dot blot)方法進(jìn)行驗(yàn)證;利用Northern blot技術(shù)探索酒精處理時(shí)間與篩選基因表達(dá)的對(duì)應(yīng)關(guān)系,探索目的基因表達(dá)與細(xì)胞分化狀態(tài)之間的關(guān)系以及目的基因分布的組織特異性;利用5’RACE技術(shù)對(duì)目的基因進(jìn)行全長(zhǎng)序列的制備;利用全長(zhǎng)序列的重組構(gòu)建對(duì)目的基因進(jìn)行上調(diào);利用RNAi序列的重組構(gòu)建對(duì)目的基因進(jìn)行下調(diào);利用MTT方法評(píng)價(jià)細(xì)胞的成活性;利用酵母雙雜交技術(shù),篩選與目的基因表達(dá)產(chǎn)物相互作用的蛋白分子。 結(jié)果:1酒精調(diào)控基因的篩選:通過(guò)差減雜交以及反向點(diǎn)雜交技術(shù)篩選出了七個(gè)酒精調(diào)控的基因,并進(jìn)一步觀察了酒精處理不同時(shí)間對(duì)這些基因表達(dá)的影響。其中酒精上調(diào)基因包括:aquaporin 1(NW_047693)、prohibitin(XM_579541)、SCIRP10 ( NW_047402 );酒精下調(diào)基因包括: zinc finger protein 341(XM_001074788)、Cab45a(MMU45977)以及兩個(gè)基因新序列(GenBank數(shù)據(jù)庫(kù)沒(méi)有發(fā)現(xiàn)與其高度同源序列)。在這些酒精調(diào)控基因中,Cab45和鋅脂蛋白341表現(xiàn)出明顯的時(shí)間依賴(lài)性下調(diào),而水孔蛋白1則表現(xiàn)出時(shí)間依賴(lài)性上調(diào)。 2 Cab45基因的特征鑒定及其功能研究:在篩選出的差減雜交克隆中,其中兩個(gè)序列均與小鼠的Cab45a高度同源,說(shuō)明該基因在差減雜交過(guò)程中出現(xiàn)的頻率較高,同時(shí)該基因序列在大鼠中未見(jiàn)報(bào)道,所以對(duì)其進(jìn)行了進(jìn)一步的探索。通過(guò)5’RACE技術(shù)制備了大鼠Cab45基因全長(zhǎng)序列,并將推導(dǎo)的氨基酸序列與其在其它物種中的對(duì)應(yīng)體進(jìn)行了比對(duì),結(jié)果顯示Cab45具有高度的保守性。象該家族中的其它成員一樣, Cab45也具有廣泛的組織分布特性。PC12細(xì)胞分化模型以及出生不同時(shí)間大鼠小腦細(xì)胞模型研究表明,Cab45的表達(dá)與分化程度呈負(fù)相關(guān)。對(duì)Cab45進(jìn)行上調(diào)與下調(diào)的結(jié)果表明,上調(diào)可顯著減弱酒精與紫外線對(duì)PC12細(xì)胞造成的死亡,而下調(diào)則產(chǎn)生相反的結(jié)果(紫外線損傷)。另外在通過(guò)酵母雙雜交方法篩選與其相互作用的蛋白時(shí)發(fā)現(xiàn),Cab45具有較強(qiáng)的轉(zhuǎn)錄激活功能。 結(jié)論:本文在神經(jīng)細(xì)胞(B104)中通過(guò)差減雜交方法對(duì)酒精調(diào)控基因進(jìn)行了篩選,鑒定出了一系列酒精調(diào)控的基因(其中兩個(gè)為新序列),為酒精調(diào)控的機(jī)理研究提供了重要的數(shù)據(jù)。隨后對(duì)其中的鈣離子結(jié)合蛋白Cab45進(jìn)行了全長(zhǎng)序列的制備,并隨后進(jìn)行了保守性分析、組織分布檢測(cè)、分化相關(guān)性檢測(cè)以及上調(diào)與下調(diào)研究等多項(xiàng)指標(biāo),系統(tǒng)地闡述了該基因的各種特征,從而將酒精對(duì)神經(jīng)細(xì)胞的損傷作用與鈣離子結(jié)合蛋白建立了重要關(guān)系,本研究以下內(nèi)容均為首次報(bào)道:1)酒精短時(shí)間處理導(dǎo)致Cab45基因上調(diào),而長(zhǎng)時(shí)間處理則導(dǎo)致其呈現(xiàn)時(shí)間依賴(lài)性下調(diào);2)在大鼠中對(duì)Cab45基因進(jìn)行了全序列的測(cè)定,并進(jìn)行了氨基酸序列的保守性分析;3)探索了Cab45組織分布的廣譜性;4)探索了Cab45表達(dá)與細(xì)胞分化狀態(tài)的負(fù)相關(guān)性;4)Cab45的過(guò)表達(dá)可減弱酒精和紫外線對(duì)細(xì)胞的損傷;5)酵母雙雜交顯示Cab45具有轉(zhuǎn)錄激活功能;6)Cab45基因的調(diào)控序列包含多種轉(zhuǎn)錄因子的結(jié)合位點(diǎn) Cab45屬于弱鈣離子結(jié)合蛋白家族,定位于高爾基體中,而該家族的其它成員均定位于內(nèi)質(zhì)網(wǎng)中,報(bào)道表明內(nèi)質(zhì)網(wǎng)中鈣離子平衡在調(diào)控細(xì)胞生長(zhǎng)、停止與凋亡過(guò)程中起著非常重要的作用,推測(cè)高爾基體中的中鈣離子平衡也有著相似的作用,Cab45可能通過(guò)調(diào)節(jié)鈣離子的平衡實(shí)現(xiàn)對(duì)細(xì)胞生長(zhǎng)、停止與凋亡的調(diào)控。由于該家族中的其他成員被證明與多種疾病有密切的關(guān)系,提示Cab45可能在鈣離子調(diào)控的神經(jīng)系統(tǒng)疾病中起著重要的作用。該發(fā)現(xiàn)對(duì)酒精引起的神經(jīng)損傷與老化的機(jī)理研究,提供了新的理論基礎(chǔ),對(duì)其進(jìn)一步研究,將促進(jìn)其作為藥物作用分子靶點(diǎn)的臨床應(yīng)用開(kāi)發(fā)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R363

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