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

【摘要】： 目的:酒精已被證明參與誘導人類多種疾病,如:動脈硬化、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,動物實驗表明,酒精可導致大腦與小腦不同區(qū)域的損傷,并廣泛的觸發(fā)神經(jīng)細胞退行性凋亡。酒精誘導的功能改變以及相關(guān)的神經(jīng)病理包括:細胞的外毒性、硫胺素的缺乏、神經(jīng)膠質(zhì)細胞的異常、生長因子的改變、細胞的凋亡、氧化壓迫、能量代謝異常。發(fā)育期間的神經(jīng)細胞對酒精的傷害尤其敏感:如:1)在發(fā)育的大鼠前腦細胞中,酒精一方面通過抑制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。同時酒精還通過改變谷氨酸鹽和伽馬氨基丁酸的運輸,而抑制神經(jīng)細胞的活性,從而造成數(shù)百萬神經(jīng)細胞的死亡;2)在出生后4-6天的大鼠浦肯雅細胞中,酒精可選擇性的降低酪氨酸激酶B和C受體的表達,在該過程中酒精通過改變神經(jīng)營養(yǎng)調(diào)控導致浦肯雅細胞的減少;3)人類腦細胞的發(fā)育成熟可從妊娠的六個月一直延續(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期,這種酒精造成的前凋亡效應(yīng)在人類胚胎發(fā)育過程中可導致酒精綜合癥。長期酗酒誘導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,當前已成為現(xiàn)代社會老年人的主要致死病之一。在酒精對神經(jīng)系統(tǒng)損傷的分子機理研究中,酒精調(diào)控基因的鑒定是非常重要的內(nèi)容,對進一步了解疾病的發(fā)生與進展具有重要的意義。 本文通過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行進一步的功能研究。 研究內(nèi)容:通過抑制性差減雜交技術(shù),在神經(jīng)細胞中進行酒精調(diào)控基因的篩選,并分別通過反向點雜交技術(shù)以及Northern Blot技術(shù)對候選基因進行進一步的證實,序列分析后登陸GenBank數(shù)據(jù)庫進行序列比對。在基因的功能研究中,通過利用Northern Blot技術(shù)探索基因表達與酒精處理時間之間的對應(yīng)關(guān)系,探索基因的組織分布特異性;通過利用出生后不同時間的大鼠小腦組織以及不同分化階段的PC12細胞(神經(jīng)生長因子誘導),探索細胞分化與基因表達之間的對應(yīng)關(guān)系;通過重組表達技術(shù),對目的基因進行上調(diào)與下調(diào),探索基因的過表達與抑制表達對細胞功能的影響,即通過對細胞進行有害處理,觀察細胞生存能力的變化;通過利用酵母雙雜交技術(shù),篩選與目的基因表達產(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)進行酒精調(diào)控基因的篩選,并通過反向點雜交(Reversed dot blot)方法進行驗證;利用Northern blot技術(shù)探索酒精處理時間與篩選基因表達的對應(yīng)關(guān)系,探索目的基因表達與細胞分化狀態(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)對目的基因進行全長序列的制備;利用全長序列的重組構(gòu)建對目的基因進行上調(diào);利用RNAi序列的重組構(gòu)建對目的基因進行下調(diào);利用MTT方法評價細胞的成活性;利用酵母雙雜交技術(shù),篩選與目的基因表達產(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:通過差減雜交以及反向點雜交技術(shù)篩選出了七個酒精調(diào)控的基因,并進一步觀察了酒精處理不同時間對這些基因表達的影響。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)以及兩個基因新序列(GenBank數(shù)據(jù)庫沒有發(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)出明顯的時間依賴性下調(diào),而水孔蛋白1則表現(xiàn)出時間依賴性上調(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,其中兩個序列均與小鼠的Cab45a高度同源,說明該基因在差減雜交過程中出現(xiàn)的頻率較高,同時該基因序列在大鼠中未見報道,所以對其進行了進一步的探索。通過5’RACE技術(shù)制備了大鼠Cab45基因全長序列,并將推導的氨基酸序列與其在其它物種中的對應(yīng)體進行了比對,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細胞分化模型以及出生不同時間大鼠小腦細胞模型研究表明,Cab45的表達與分化程度呈負相關(guān)。對Cab45進行上調(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PC12細胞造成的死亡,而下調(diào)則產(chǎn)生相反的結(jié)果(紫外線損傷)。另外在通過酵母雙雜交方法篩選與其相互作用的蛋白時發(fā)現(xiàn),Cab45具有較強的轉(zhuǎn)錄激活功能。 結(jié)論:本文在神經(jīng)細胞(B104)中通過差減雜交方法對酒精調(diào)控基因進行了篩選,鑒定出了一系列酒精調(diào)控的基因(其中兩個為新序列),為酒精調(diào)控的機理研究提供了重要的數(shù)據(jù)。隨后對其中的鈣離子結(jié)合蛋白Cab45進行了全長序列的制備,并隨后進行了保守性分析、組織分布檢測、分化相關(guān)性檢測以及上調(diào)與下調(diào)研究等多項指標,系統(tǒng)地闡述了該基因的各種特征,從而將酒精對神經(jīng)細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)容均為首次報道:1)酒精短時間處理導致Cab45基因上調(diào),而長時間處理則導致其呈現(xiàn)時間依賴性下調(diào);2)在大鼠中對Cab45基因進行了全序列的測定,并進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表達與細胞分化狀態(tài)的負相關(guān)性;4)Cab45的過表達可減弱酒精和紫外線對細胞的損傷;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 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,報道表明內(nèi)質(zhì)網(wǎng)中鈣離子平衡在調(diào)控細胞生長、停止與凋亡過程中起著非常重要的作用,推測高爾基體中的中鈣離子平衡也有著相似的作用,Cab45可能通過調(diào)節(jié)鈣離子的平衡實現(xiàn)對細胞生長、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)對酒精引起的神經(jīng)損傷與老化的機理研究,提供了新的理論基礎(chǔ),對其進一步研究,將促進其作為藥物作用分子靶點的臨床應(yīng)用開發(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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R363

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