發(fā)布時間：2018-06-17 07:00

  本文選題：高溫 + 神經(jīng)管畸形��； 參考：《山東大學(xué)》2007年博士論文

【摘要】： 神經(jīng)管形成是胚胎早期發(fā)育中的重要事件，是指從神經(jīng)板出現(xiàn)、卷折形成神經(jīng)褶到左右神經(jīng)褶在背側(cè)中線靠攏愈合形成神經(jīng)管的連續(xù)生物學(xué)過程。神經(jīng)管是中樞神經(jīng)系統(tǒng)發(fā)生的原基，神經(jīng)管的發(fā)生和分化是腦和脊髓正常發(fā)育的前提。在神經(jīng)管發(fā)育過程中，多種環(huán)境致畸因素可對其產(chǎn)生影響，，引發(fā)神經(jīng)管畸形(neural tube defects，NTDs)。神經(jīng)管畸形是發(fā)生率最高、危害也最為嚴(yán)重的一類先天畸形，不僅危及患兒的生命和健康，而且給社會和家庭造成沉重的經(jīng)濟(jì)負(fù)擔(dān)。 從上個世紀(jì)開始，科學(xué)家開始檢測誘發(fā)神經(jīng)管畸形的各種致畸因子，其中高溫是較常見且難以預(yù)防和避免的一種環(huán)境致畸因素。因此，探討高溫致神經(jīng)管畸形的發(fā)生機(jī)理成為實驗畸形學(xué)中的一個研究熱點(diǎn)。近年來，人們從細(xì)胞、亞細(xì)胞和分子水平對高溫致神經(jīng)管畸形的機(jī)理進(jìn)行了大量的動物實驗和細(xì)胞培養(yǎng)研究，觀察了高溫對細(xì)胞增殖、凋亡、分化、粘著、類聚、組織誘導(dǎo)和器官發(fā)生等方面的影響。研究發(fā)現(xiàn)，在神經(jīng)管形成和分化過程中，某一或某些相關(guān)基因的特異表達(dá)或特異不表達(dá)、上調(diào)表達(dá)或下調(diào)表達(dá)都會導(dǎo)致神經(jīng)管發(fā)育異常，引發(fā)神經(jīng)管畸形。高溫致神經(jīng)管畸形的發(fā)生是一個多基因參與的復(fù)雜過程，這其中有些基因上調(diào)表達(dá)，有些基因下調(diào)表達(dá)，而關(guān)于這些差異表達(dá)基因的系統(tǒng)研究至今仍未見報道。 抑制性消減雜交(suppression subtractive hybridization，SSH)是一種尋找差異表達(dá)基因的技術(shù)，可以克隆出與驅(qū)動子相比在檢測子中特異表達(dá)和上調(diào)表達(dá)的基因。應(yīng)用抑制性消減雜交方法構(gòu)建的消減cDNA文庫具有特異性和均等化兩大突出優(yōu)點(diǎn)。在本項實驗中，我們應(yīng)用抑制性消減雜交技術(shù)構(gòu)建了高溫致金黃地鼠神經(jīng)管畸形的雙向消減cDNA文庫，從中篩選到了高溫致神經(jīng)管畸形過程中上調(diào)表達(dá)和下調(diào)表達(dá)的基因。通過序列測定和同源性比較對這些差異表達(dá)基因進(jìn)行了分析，然后通過Northern雜交方法進(jìn)一步證實這些基因在高溫致神經(jīng)管畸形過程中的差異表達(dá)情況。隨后從這些差異表達(dá)基因中，挑選在胚胎發(fā)育中起重要作用的下調(diào)表達(dá)基因——Npm1，對其功能進(jìn)行了體外研究。 第一部分高溫致金黃地鼠神經(jīng)管畸形雙向消減cDNA文庫的構(gòu)建及鑒定 為篩選高溫致神經(jīng)管畸形過程中的差異表達(dá)基因，我們首先構(gòu)建了高溫致金黃地鼠神經(jīng)管畸形雙向消減cDNA文庫。實驗中，我們分別提取高溫組和對照組金黃地鼠胚胎神經(jīng)管組織總RNA，通過SMART~(TM) cDNA合成的方法反轉(zhuǎn)錄得到雙鏈cDNA(ds cDNA)。將酚氯仿純化后的ds cDNA用RsaI消化，消化后的ds cDNA再次經(jīng)酚氯仿純化，并用雙蒸水調(diào)整濃度至300ng/μl。取純化后的ds cDNA用于抑制性消減雜交。按PCR-select~(TM) cDNA subtraction kit說明進(jìn)行正反兩個方向的消減雜交，以高溫組ds cDNA做檢測子、對照組ds cDNA做驅(qū)動子進(jìn)行消減雜交，構(gòu)建高溫致金黃地鼠神經(jīng)管畸形正向消減cDNA文庫，可從中篩選高溫致神經(jīng)管畸形過程中上調(diào)表達(dá)的基因。反之，以對照組ds cDNA做檢測子、高溫組ds cDNA做驅(qū)動子進(jìn)行消減雜交，構(gòu)建高溫致金黃地鼠神經(jīng)管畸形反向消減cDNA文庫，可從中篩選高溫致神經(jīng)管畸形過程中下調(diào)表達(dá)的基因。將看家基因Gapdh設(shè)立為消減雜交實驗的對照以檢測消減效率。結(jié)果，Gapdh的表達(dá)豐度在消減雜交結(jié)束后明顯降低，表明我們的消減效率很高。將消減雜交得到的產(chǎn)物純化，然后通過T-A克隆的方法將其與T載體連接，轉(zhuǎn)化感受態(tài)大腸桿菌DH5α，鋪含氨芐青霉素的LB/X-gal/IPTG平板，構(gòu)建消減cDNA文庫。經(jīng)藍(lán)白斑初步篩選后，再利用菌落PCR方法進(jìn)一步鑒定。結(jié)果顯示，構(gòu)建的消減cDNA文庫中包含150 bp-1 kb的長短不一的差異表達(dá)基因片段。這說明我們構(gòu)建的高溫致金黃地鼠神經(jīng)管畸形雙向消減cDNA文庫是成功的，可用于篩選差異表達(dá)基因。 第二部分高溫致金黃地鼠神經(jīng)管畸形差異表達(dá)基因的篩選、序列分析及鑒定 由于消減cDNA文庫中包含的基因信息較多，一般采用隨機(jī)法挑選文庫中的克隆進(jìn)行測序。本實驗中，我們選取經(jīng)菌落PCR證實插入片段較長的克隆進(jìn)行測序，并將測序結(jié)果通過blastn軟件與Genbank數(shù)據(jù)庫中的已知基因進(jìn)行序列比對和同源性分析。在高溫致金黃地鼠神經(jīng)管畸形反向消減cDNA文庫中，我們共篩選到14個下調(diào)表達(dá)基因，經(jīng)分析均與已知基因有較高的同源性。這些基因編碼的蛋白有核糖體蛋白，參與代謝的酶，翻譯及轉(zhuǎn)錄因子和其他。隨后通過Northern雜交證實了這些基因在高溫致畸胚胎神經(jīng)管中表達(dá)水平均明顯降低。而在高溫致金黃地鼠神經(jīng)管畸形正向消減cDNA文庫中，由于擴(kuò)增得到的片段普遍較短，測序和同源性分析后僅發(fā)現(xiàn)了一個有意義的差異表達(dá)基因片段。此片段與小鼠磷酸甘油酸酯激酶(Pgk1)同源且同源性高達(dá)92％。Northern雜交證實該基因在高溫致畸胚胎神經(jīng)管中表達(dá)水平明顯升高。從高溫致金黃地鼠神經(jīng)管畸形雙向消減cDNA文庫中篩選得到的基因，它們的差異表達(dá)情況均得到Northern雜交驗證，證實這些基因在高溫致神經(jīng)管畸形過程中的表達(dá)發(fā)生了異常變化，同時也說明這些基因的差異表達(dá)與高溫致神經(jīng)管畸形的發(fā)生密切相關(guān)。 第三部分Npm1基因功能的體外研究 Npm1(nucleophosmin)是我們從高溫致金黃地鼠神經(jīng)管畸形反向消減cDNA文庫中篩選到的一個基因。以往的研究顯示，Npm1在胚胎發(fā)育過程中扮演著重要的角色。Npm1~(-/-)的小鼠胚胎較正常胚胎發(fā)育遲緩，前腦發(fā)育缺陷，眼睛缺失。說明Npm1對神經(jīng)系統(tǒng)的正常發(fā)育是必需的。因此我們對Npm1在高溫致畸中的作用機(jī)制進(jìn)行了研究。由于在高溫致神經(jīng)管畸形過程中Npm1基因呈下調(diào)表達(dá)，我們在神經(jīng)干細(xì)胞(neural stem cells，NSCs)的體外培養(yǎng)中，應(yīng)用RNA干擾(RNA interference，RNAi)技術(shù)，對該基因的功能進(jìn)行了比較深入的研究。實驗結(jié)果顯示，RNAi技術(shù)成功地在mRNA和蛋白水平上降低了Npm1的表達(dá)，干擾效果得到了RT-PCR和Western blot證實。隨后我們從細(xì)胞增殖、凋亡和分化三個方面著手，研究Npm1基因表達(dá)降低對神經(jīng)干細(xì)胞的影響。結(jié)果表明，Npm1基因表達(dá)降低后，神經(jīng)干細(xì)胞的增殖速度明顯減慢，凋亡的發(fā)生率明顯增加，而神經(jīng)干細(xì)胞向神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞方向分化的比例未受到影響。此外，Npm1基因表達(dá)降低后，p53蛋白表達(dá)升高，說明Npm1基因干擾后引發(fā)的神經(jīng)干細(xì)胞凋亡與p53信號通路密切相關(guān)。 本項研究成功地構(gòu)建了高溫致金黃地鼠神經(jīng)管畸形雙向消減cDNA文庫，并從中篩選到了與高溫致神經(jīng)管畸形密切相關(guān)的下調(diào)表達(dá)基因和上調(diào)表達(dá)基因；還以體外培養(yǎng)的神經(jīng)干細(xì)胞為研究模型，用RNAi技術(shù)，對在胚胎發(fā)育中起重要作用的下調(diào)表達(dá)基因Npm1在高溫致神經(jīng)管畸形中的作用機(jī)制進(jìn)行了比較深入的研究，從而為高溫致神經(jīng)管畸形在基因水平上的機(jī)理和防治研究提供了重要的理論和技術(shù)支持。
[Abstract]:Neural tube formation is an important event in the early development of the embryo. It refers to the continuous biological process from the appearance of the nerve plate to the formation of the nerve folds to the right of the left and right nerve folds in the dorsal middle line to form the neural tube. The neural tube is the primordial base of the central nervous system. The birth and differentiation of the nerve canal is the prerequisite for the normal development of the brain and spinal cord. In the process of neural tube development, a variety of environmental teratogenicity factors can affect it, causing neural tube defects (NTDs). Neural tube malformation is the highest incidence and the most serious type of congenital malformation, which not only endangers the life and health of the children, but also causes a heavy economic burden to the society and the family.
From the beginning of the last century, scientists began to detect all kinds of teratogenic factors inducing neural tube malformation. High temperature is a common and difficult environmental teratogenic factor that is difficult to prevent and avoid. Therefore, the study of the mechanism of high temperature induced neural tube malformation has become a research hotspot in experimental malformation. In recent years, people have been from cells, subcells and A large number of animal experiments and cell culture studies on the mechanism of high temperature induced neural tube malformation have been carried out at molecular level. The effects of high temperature on cell proliferation, apoptosis, differentiation, adhesion, cohesion, tissue induction and organogenesis are observed. The specific expression of some or some related genes in the formation and differentiation of neural tube is found. The up-regulation or down regulation of expression or down regulation can lead to abnormal development of neural tube and cause neural tube malformation. The occurrence of neural tube malformation caused by high temperature is a complex process of multi gene participation, some of which are up-regulated, some genes are downregulated, and the systematic study of these differentially expressed genes has not yet been studied. Report.
Suppression subtractive hybridization (SSH) is a technique for finding differentially expressed genes, which can clone the genes that express and express the expression specifically in the detector compared with the driver. The subtractive cDNA library constructed by the suppression subtractive hybridization method has two prominent advantages of specificity and equalization. In this experiment, we constructed a bi-directional subtractive cDNA Library of neural tube malformation in golden hamster with inhibitory subtractive hybridization. We screened genes for expression and down regulation during the process of high temperature induced neural tube malformation. The differential expression of these genes in the process of high temperature induced neural tube malformation was further confirmed by Northern hybridization. Then, from these differentially expressed genes, the down regulated expression gene, Npm1, which plays an important role in the development of embryo, was selected, and the function of these genes was studied in vitro.
Part I construction and identification of bidirectional reduced cDNA Library of hamster neural tube defects induced by hyperthermia
In order to screen the differentially expressed genes in the process of high temperature induced neural tube malformation, we first constructed a bi-directional subtractive cDNA Library of the nervous tube malformation in golden hamster. In the experiment, we extracted the total RNA of the embryonic neural tube tissue of the high temperature group and the control group of golden hamster, and reverse transcribed the double chain cDNA (DS C) through the method of SMART~ (TM) cDNA synthesis. DNA). The purified DS cDNA after phenol chloroform was digested with RsaI, and the DS cDNA after digestion was purified again by phenol chloroform, and the concentration to 300ng/ Mu L. was adjusted to the purified DS cDNA for the suppression subtractive hybridization. On the other hand, the control group DS cDNA as the driver for subtractive hybridization and the construction of the positive subtractive cDNA Library of the hyperthermia induced neural tube malformation in golden hamster, which can be screened from the high temperature induced neural tube malformation. On the contrary, the control group DS cDNA was used as the detector and the high temperature group DS cDNA was used as the driver for subtractive hybridization and the high temperature induced golden hamster was constructed. The cDNA Library of neural tube malformation can be used to reduce the down-regulation of genes in the process of high temperature induced neural tube malformation. The family gene Gapdh was set up as a control of subtractive hybridization to detect subtractive efficiency. The results showed that the expression abundance of Gapdh decreased significantly after the end of subtractive hybridization, indicating that our subtractive efficiency was very high. The obtained product was purified and then connected to the T vector by T-A cloning, transforming the receptive Escherichia coli DH5 alpha into a LB/X-gal/IPTG tablet containing ampicillin and constructing a subtractive cDNA library. After preliminary screening by blue leukoplakia, it was further identified by the colony PCR method. The results showed that the constructed subtracted cDNA library contains 150 bp-1. The differentially expressed gene fragment of KB shows that the bi-directional subtractive cDNA Library of the hyperthermia induced neural tube malformation in golden hamster is successful and can be used to screen differentially expressed genes.
The second part is screening, sequence analysis and identification of differentially expressed genes in hyperthermia induced hamster neural tube defects.
In order to reduce the gene information contained in the cDNA library, the clones in the random selected library are usually sequenced. In this experiment, we selected a long clone by colony PCR to confirm the sequence of the inserted fragment, and the sequence results were compared with the known genes in the Genbank database by BLASTN software and homology. In the reverse subtractive cDNA Library of the nervous tube malformation in golden hamster, we screened 14 down-regulated genes, which had high homology with the known genes. The proteins encoded by these genes were ribosome proteins, enzymes involved in metabolism, translation and transcriptional genes and others. Subsequently, this protein was confirmed by Northern hybridization. The expression level of some genes in the neural tube of high temperature teratogenic embryo was significantly reduced, and the amplified fragment was generally short in the cDNA Library of the nervous tube malformation in the golden hamster, and only a meaningful differential expression base fragment was found after sequencing and homology analysis. Enzyme (Pgk1) homology and homology up to 92%.Northern hybridization confirmed that the gene expression level in the high temperature teratogenic embryo neural tube increased obviously. The differential expression of the genes obtained from the bidirectional subtractive cDNA Library of the golden hamster's neural tube malformation was verified by Northern hybridization and confirmed that these genes were induced at high temperature. Abnormal expression of neural tube defects was observed during the course of neural tube defects. Meanwhile, the differential expression of these genes was closely related to the occurrence of neural tube defects induced by hyperthermia.
In vitro study on the function of the third part of Npm1 gene
Npm1 (nucleophosmin) is a gene that we screened from the reverse cDNA Library of the neural tube malformation in golden hamsters. Previous studies showed that Npm1 was playing an important role in the embryonic development of.Npm1~ (- / -) mouse embryos more slowly than normal embryos, hypoplasia of the forebrain, and absence of the eyes. It explained Npm1 to the nervous system. The normal development of the Npm1 is necessary. Therefore, we have studied the mechanism of the action in high temperature teratogenicity. Due to the downregulation of the Npm1 gene in the process of high temperature induced neural tube malformation, we used the RNA interference (RNA interference, RNAi) technique in the culture of neural stem cells (neural stem cells, NSCs), and the work of this gene. The experimental results show that RNAi technology has successfully reduced the expression of Npm1 on the level of mRNA and protein, and the effect of interference is confirmed by RT-PCR and Western blot. Then we start from three aspects of cell proliferation, apoptosis and differentiation, and study the effect of Npm1 gene expression reduction on neural stem cells. The results show that the effect of Npm1 gene expression on neural stem cells. After the decrease of Npm1 gene expression, the proliferation rate of neural stem cells significantly slowed, the incidence of apoptosis increased obviously, while the proportion of neural stem cells to neuron and glial cells was not affected. In addition, the expression of p53 protein increased after the decrease of Npm1 gene expression, indicating the apoptosis of neural stem cells caused by Npm1 gene interference. It is closely related to the p53 signaling pathway.
This study successfully constructed a bi-directional subtractive cDNA Library of neural tube malformation in golden hamster, and screened the down regulated gene and up-regulated gene closely related to the high temperature induced neural tube malformation. The neural stem cells cultured in vitro were used as the research model and the RNAi technique was used to play an important role in the development of the embryo. The mechanism of the down-regulation of expression gene Npm1 in the hyperthermia induced neural tube malformation has been deeply studied, which provides important theoretical and technical support for the mechanism and prevention and control of the high temperature induced neural tube malformation at the gene level.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 呂鋒，高英茂，管英俊;高溫致神經(jīng)管畸形動物模型的建立及其形態(tài)發(fā)生的研究[J];解剖學(xué)報;1996年03期

本文編號：2030158