【作者】 杨青；

【导师】 高英茂；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2007， 博士

【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。更多还原

【Abstract】 Formation of neural tube is an important event during early embryogenesis and involves formation of neural plate, neural plate folding into neural ridge and neural ridge merging into neural tube. Neural tube is the primordium of central nervous system （CNS）. The genesis and differentiation of neural tube are precondition of normal development of brain and spinal cord. During neural tube development, many environmental teratogens may have adverse effects and thus induce neural tube defects （NTDs）. NTDs are amongst the most prevalent and serious of all human congenital anomalies and place an enormous emotional and economic burden on the population.Since last century, scientists have been working on exploring various environmental teratogens that may induce NTDs. They found that hyperthermia is a prevalent and unavoidable factor. This has motivated extensive research about mechanism of hyperthermia induced NTDs. There is now an extensive literature about the effects of hyperthermia on cell proliferation, apoptosis, differentiation, migration, adhesion and aggregation as well as on tissue induction and organogenesis. Previous studies revealed that many genes participate in the course of hyperthermia induced NTDs. None or insufficent expression of some genes and specific or excessive expression of some other genes may induce NTDs. Hyperthermia induced NTDs are complicated because genes including both upregulated and downregulated are involved. However, there is still no detailed research about the differentially expressed genes in hyperthermia induced NTDs.Recently, several PCR-based techniques for differential genes screening have been established. A technique named suppression subtractive hybridization （SSH）, owing to its high specificity and parity, has been widely applied to many molecular cloning studies for identification of differentially expressed genes in disease, development and different tissues. Here, we constructed subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster in two directions using SSH. Then we identified both upregulated and downregulated genes in hyperthermia induced NTDs from these two subtractive cDNA libraries. Sequence and homology analysis were made to these differentially expressed genes and Northern blot were performed to verify differential expression levels of these genes. Then we selected Npm1, an important gene for embryogenesis as our study target and explored its function in vitro.PART ONE Construction and Validation of Forward and Reverse Subtractive cDNA Libraries of Hyperthermia Induced NTDs of Golden HamsterTo identify differentially expressed genes in hyperthermia induced NTDs of golden hamster, we first constructed subtractive cDNA libraries in two directions. Total RNA were isolated from both control and hyperthermia groups and then were reverse transcripted into double-strand cDNA （ds cDNA） using SMART^TM cDNA synthesis method. We applied method of phenol and chloroform extraction to purify ds cDNA. Then, the purified cDNA were digested by RsaI to generate shorter, blunt-ended ds cDNA fragments and purified again. The final concentration of cDNA were adjusted to 300 ng/μl. Then SSH was carried out in two directions: forward and reverse, with PCR-Select cDNA Subtraction Kit. For the forward SSH, ds cDNA derived from control group was considered the ’driver’ pool, ds cDNA derived from hyperthermia group the ’tester’ pool. From the forward SSH, we could identify upregulated genes in hyperthermia induced NTDs. For the reverse SSH, ds cDNA from hyperthermia group was regarded as the ’driver’ pool, ds cDNA from control group the ’tester’ pool. From the reverse SSH, we could identify downregulated genes in hyperthermia induced NTDs. The housekeeping gene—Gapdh was used as control in SSH to evaluate subtractive efficiency. The significant decrease of Gapdh after SSH suggested high subtractive efficiency in our experiment. Then the purifed subtracted cDNA were ligated with pGEM-T Easy vector and transformed into E. Coli DH5αusing CaCl₂ method. The two subtractive cDNA libraries were separately plated on LB agar plates containing ampicillin （100μg/ml） and IPTG/X-gal. The results of blue-white selection and clony PCR showed that the two subtractive cDNA libraries included differentially expressed gene fragments with length from 150 bp to 1 kb. This suggests that our subtractive cDNA libraries were successfully constructed and suitable for identification of differentially expressed genes in hyperthermia induced NTDs.PART TWO Screening, Sequencing and Verification of Differentially Expressed Genes in Hyperthermia Induced NTDs of Golden HamsterGenerally, a subtractive cDNA library contains so many differentially expressed genes that researchers cannot sequence them all. So we just selected clones from the two subtractive cDNA libraries randomly. Then we sequenced these clones and compared the sequencing results with Genbank database by blastn software. From the reverse subtractive cDNA library, we identified 14 downregulated genes in all and found them all homologous to known genes with high homology. The proteins encoded by these genes included ribosomal proteins, metabolic enzymes, transcription and translation factors and others. Northern blot showed that expression of all these 14 genes decreased significantly in defected neural tubes induced by hyperthermia. However, from the forward subtractive cDNA library, most of the inserts were short and only two meaningful fragments were identified after sequence and homology analysis. The two inserts were of the same sequence and homologous to mouse Pgk1 with homology of 92%. The increased expression of Pgk1 in defected neural tubes induced by hyperthermia was also confirmed by Northern blot. In this part, we verified these differentially expressed genes by Northern blot, showed differential expression of these genes are strongly related to hyperthermia induced NTDs. PART THREE Functional Study of Npm1 in vitro Npm1 was a gene identified from the reverse subtractive cDNA library. Previous studies showed that Npm1 plays an important role in embryogenesis. Npm1^-/- embryos were consistently smaller in size compared with normal ones. The Npm1^-/- mutants also showed distinct developmental abnormalities. Most noticeably, they displayed deficient anterior brain organogenesis, with complete absence of eyes. This suggested that Npm1 is essential for neurogenesis. So we selected Npm1 to explore its role in NTDs induced by hyperthermia. Neural stem cells （NSCs） were used as our study model in vitro. As Npm1 was downregulated in hyperthermia induced NTDs, we suppressed expression of Npm1 in NSCs by RNA interference （RNAi）. After RNAi, Npm1 was downregulated at both mRNA and protein levels. Then we studied cell proliferation, apoptosis and differentiation in NSCs after Npm1 suppression. As a result, suppression of Npm1 decreased proliferation rate of NSCs, increased apoptosis occurrence but did not seem to make any significant difference in cell differentiation. As there was an increase of p53 protein after Npm1 was suppressed, we speculated that apoptosis of NSCs induced by downregulation of Npm1 was strongly related to p53 pathway.In our work, we successfully constructed forward and reverse subtractive cDNA libraries of hyperthermia induced NTDs of golden hamster. And we identified several downregulated and upregulated genes involved in hyperthermia induced NTDs. In addition, we employed RNAi with NSCs in vitro to study the function of Npm1 in hyperthermia induced NTDs. Our work provides important theoretical and technical support for mechanism and prevention of hyperthermia induced NTDs.更多还原