高温致金黄地鼠神经管畸形差异表达基因的筛

发布时间：2018-06-17 07:00

本文选题：高温 + 神经管畸形　；参考：《山东大学》2007年博士论文

【摘要】： 神经管形成是胚胎早期发育中的重要事件，是指从神经板出现、卷折形成神经褶到左右神经褶在背侧中线靠拢愈合形成神经管的连续生物学过程。神经管是中枢神经系统发生的原基，神经管的发生和分化是脑和脊髓正常发育的前提。在神经管发育过程中，多种环境致畸因素可对其产生影响，引发神经管畸形(neural tube defects，NTDs)。神经管畸形是发生率最高、危害也最为严重的一类先天畸形，不仅危及患儿的生命和健康，而且给社会和家庭造成沉重的经济负担。从上个世纪开始，科学家开始检测诱发神经管畸形的各种致畸因子，其中高温是较常见且难以预防和避免的一种环境致畸因素。因此，探讨高温致神经管畸形的发生机理成为实验畸形学中的一个研究热点。近年来，人们从细胞、亚细胞和分子水平对高温致神经管畸形的机理进行了大量的动物实验和细胞培养研究，观察了高温对细胞增殖、凋亡、分化、粘着、类聚、组织诱导和器官发生等方面的影响。研究发现，在神经管形成和分化过程中，某一或某些相关基因的特异表达或特异不表达、上调表达或下调表达都会导致神经管发育异常，引发神经管畸形。高温致神经管畸形的发生是一个多基因参与的复杂过程，这其中有些基因上调表达，有些基因下调表达，而关于这些差异表达基因的系统研究至今仍未见报道。抑制性消减杂交(suppression subtractive hybridization，SSH)是一种寻找差异表达基因的技术，可以克隆出与驱动子相比在检测子中特异表达和上调表达的基因。应用抑制性消减杂交方法构建的消减cDNA文库具有特异性和均等化两大突出优点。在本项实验中，我们应用抑制性消减杂交技术构建了高温致金黄地鼠神经管畸形的双向消减cDNA文库，从中筛选到了高温致神经管畸形过程中上调表达和下调表达的基因。通过序列测定和同源性比较对这些差异表达基因进行了分析，然后通过Northern杂交方法进一步证实这些基因在高温致神经管畸形过程中的差异表达情况。随后从这些差异表达基因中，挑选在胚胎发育中起重要作用的下调表达基因——Npm1，对其功能进行了体外研究。第一部分高温致金黄地鼠神经管畸形双向消减cDNA文库的构建及鉴定为筛选高温致神经管畸形过程中的差异表达基因，我们首先构建了高温致金黄地鼠神经管畸形双向消减cDNA文库。实验中，我们分别提取高温组和对照组金黄地鼠胚胎神经管组织总RNA，通过SMART~(TM) cDNA合成的方法反转录得到双链cDNA(ds cDNA)。将酚氯仿纯化后的ds cDNA用RsaI消化，消化后的ds cDNA再次经酚氯仿纯化，并用双蒸水调整浓度至300ng/μl。取纯化后的ds cDNA用于抑制性消减杂交。按PCR-select~(TM) cDNA subtraction kit说明进行正反两个方向的消减杂交，，以高温组ds cDNA做检测子、对照组ds cDNA做驱动子进行消减杂交，构建高温致金黄地鼠神经管畸形正向消减cDNA文库，可从中筛选高温致神经管畸形过程中上调表达的基因。反之，以对照组ds cDNA做检测子、高温组ds cDNA做驱动子进行消减杂交，构建高温致金黄地鼠神经管畸形反向消减cDNA文库，可从中筛选高温致神经管畸形过程中下调表达的基因。将看家基因Gapdh设立为消减杂交实验的对照以检测消减效率。结果，Gapdh的表达丰度在消减杂交结束后明显降低，表明我们的消减效率很高。将消减杂交得到的产物纯化，然后通过T-A克隆的方法将其与T载体连接，转化感受态大肠杆菌DH5α，铺含氨苄青霉素的LB/X-gal/IPTG平板，构建消减cDNA文库。经蓝白斑初步筛选后，再利用菌落PCR方法进一步鉴定。结果显示，构建的消减cDNA文库中包含150 bp-1 kb的长短不一的差异表达基因片段。这说明我们构建的高温致金黄地鼠神经管畸形双向消减cDNA文库是成功的，可用于筛选差异表达基因。第二部分高温致金黄地鼠神经管畸形差异表达基因的筛选、序列分析及鉴定由于消减cDNA文库中包含的基因信息较多，一般采用随机法挑选文库中的克隆进行测序。本实验中，我们选取经菌落PCR证实插入片段较长的克隆进行测序，并将测序结果通过blastn软件与Genbank数据库中的已知基因进行序列比对和同源性分析。在高温致金黄地鼠神经管畸形反向消减cDNA文库中，我们共筛选到14个下调表达基因，经分析均与已知基因有较高的同源性。这些基因编码的蛋白有核糖体蛋白，参与代谢的酶，翻译及转录因子和其他。随后通过Northern杂交证实了这些基因在高温致畸胚胎神经管中表达水平均明显降低。而在高温致金黄地鼠神经管畸形正向消减cDNA文库中，由于扩增得到的片段普遍较短，测序和同源性分析后仅发现了一个有意义的差异表达基因片段。此片段与小鼠磷酸甘油酸酯激酶(Pgk1)同源且同源性高达92％。Northern杂交证实该基因在高温致畸胚胎神经管中表达水平明显升高。从高温致金黄地鼠神经管畸形双向消减cDNA文库中筛选得到的基因，它们的差异表达情况均得到Northern杂交验证，证实这些基因在高温致神经管畸形过程中的表达发生了异常变化，同时也说明这些基因的差异表达与高温致神经管畸形的发生密切相关。第三部分Npm1基因功能的体外研究 Npm1(nucleophosmin)是我们从高温致金黄地鼠神经管畸形反向消减cDNA文库中筛选到的一个基因。以往的研究显示，Npm1在胚胎发育过程中扮演着重要的角色。Npm1~(-/-)的小鼠胚胎较正常胚胎发育迟缓，前脑发育缺陷，眼睛缺失。说明Npm1对神经系统的正常发育是必需的。因此我们对Npm1在高温致畸中的作用机制进行了研究。由于在高温致神经管畸形过程中Npm1基因呈下调表达，我们在神经干细胞(neural stem cells，NSCs)的体外培养中，应用RNA干扰(RNA interference，RNAi)技术，对该基因的功能进行了比较深入的研究。实验结果显示，RNAi技术成功地在mRNA和蛋白水平上降低了Npm1的表达，干扰效果得到了RT-PCR和Western blot证实。随后我们从细胞增殖、凋亡和分化三个方面着手，研究Npm1基因表达降低对神经干细胞的影响。结果表明，Npm1基因表达降低后，神经干细胞的增殖速度明显减慢，凋亡的发生率明显增加，而神经干细胞向神经元和神经胶质细胞方向分化的比例未受到影响。此外，Npm1基因表达降低后，p53蛋白表达升高，说明Npm1基因干扰后引发的神经干细胞凋亡与p53信号通路密切相关。本项研究成功地构建了高温致金黄地鼠神经管畸形双向消减cDNA文库，并从中筛选到了与高温致神经管畸形密切相关的下调表达基因和上调表达基因；还以体外培养的神经干细胞为研究模型，用RNAi技术，对在胚胎发育中起重要作用的下调表达基因Npm1在高温致神经管畸形中的作用机制进行了比较深入的研究，从而为高温致神经管畸形在基因水平上的机理和防治研究提供了重要的理论和技术支持。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

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