N-乙酰氨基葡萄糖转移酶V高表达细胞株的构建及其对神经生长因子受体TrKA作用影响的研究
发布时间：2018-07-24 19:01
【摘要】： N-糖链在细胞通讯、生长、分化中起重要的作用,文献报道有证据表明N-糖链在神经发育中也起重要作用。目前糖组学(Glycomics)是后后基因组的研究重点。糖基转移酶是广泛存在的一大类酶,参与了聚糖、糖苷和复合糖类中糖部分的生物合成,具有高度的底物专一性。目前国际上关于糖基转移酶的研究较为初步,与其他类型的酶类相比,如参与代谢的酶类等,在酶的表达,受哪些因素调控等方面研究不够透彻,而且其涉及的生物学效应则更为广泛,复杂。糖基转移酶生物功能的完全阐释将揭示庞大的相关生物信息。 N-乙酰氨基葡萄糖基转移酶V(N-acetylglucosaminyltransferase V,GnT-V/Mgat5,EC 2.4.1.155)作为重要的高尔基体糖基转移酶,是糖蛋白N-糖链加工酶之一,具有决定N-糖链类型及复杂型糖链结构的重要作用,催化GlcNAc基团转移至N-糖链核心α1,6臂的α-甘露糖的反应,形成N-糖链的β1,6分支结构,能形成2、3、4天线的N-糖链产物,在蛋白质翻译后修饰中起重要作用,而蛋白糖基化则是生物活性蛋白发挥其生物功能的必要条件之一,其中包括了具有重要功能的生长因子的受体膜蛋白的修饰。N-乙酰氨基葡萄糖基转移酶V是重要的高尔基体糖基转移酶,参与了糖蛋白N-糖链的合成,具有对糖蛋白翻译后糖基化共翻译修饰的重要功能。而且内质网中的蛋白质糖基化共翻译修饰过程能促使蛋白进行正确折叠,同时蛋白质糖基化共翻译修饰也是真核细胞存活必需的。研究发现N-乙酰氨基葡萄糖基转移酶V的缺失能引起受体N-糖链异常,糖基化异常能影响某些类型的细胞表面的受体功能,导致受体信号转导的改变。因此本文构建GnT-V高表达细胞株研究糖蛋白糖链变化对受体功能的影响。 神经生长因子NGF是最早发现的神经生长因子,作为重要的神经营养因子,能调节神经的生长、发育、分化,通过高亲和性的受体TrKA调节营养神经元,不仅在成神经细胞瘤中,NGF受体TrkA表达升高与良好的预后相关,而且在神经退行性病变中,如帕金森和阿尔茨海默氏症(AD),NGF也是治疗药物研究的热点。NGF通过高亲和性的受体引起轴突生长细胞分化,而低亲和性受体引起凋亡,微环境中NGF过低则不能引起轴突生长。PC12细胞是大鼠肾嗜镉瘤细胞,具有神经细胞的特点,是研究NGF神经轴突生长良好的细胞模型,文献报道,在正常状态下50-100 ng/mL左右的NGF刺激几天后能诱导PC12细胞轴突生长。因此选取该细胞作为研究细胞模型,观察低浓度NGF刺激下(降低4-8倍)GnT-V高表达后对细胞的影响,而此低浓度下,NGF单独不足以诱导轴突生长。 人神经生长因子受体(Trk A)含有4个潜在的N-糖链糖基化位点,该位点在神经营养因子受体Trk家族中高度保守,提示糖基化可能有重要的作用,另外还有9个不太保守的N-糖链糖基化位点。研究发现,成熟的Trk A受体在保守和可变的N-糖链糖基化位点均有糖基化。这些位点的糖基化有两种不同的功能,一是Trk A受体糖基化是阻止配体不依赖的激活所必需的,去糖基化的Trk A受体核心蛋白在没有配体激活时也是磷酸化的并且显示组成性激酶活性作用于信号分子Shc和PLC-γ;二是Trk A受体糖基化是受体定位于细胞膜表面所必需的,只有在细胞膜表面的受体能引发Ras/Raf/MAP激酶通路。用激光共聚焦显微镜发现没有糖基化的活性Trk受体陷入在细胞内,不能定位细胞膜,而且没有糖基化的活性Trk A受体不能激活Ras-MAP、MEK和ErK激酶通路,因此,没有糖基化的Trk A受体核心蛋白不能诱导PC12细胞神经元的分化。而且文献报道去糖基化的Trk不能应答其配体NGF,即衣霉素预处理稳定转染的高表达Trk的PC12细胞后,抑制Trk糖基化,改变了Trk蛋白上GM1(ganglioside,神经节苷脂,酸性糖鞘脂)结合位点,失去了与GM1形成复合物的能力。而且文献报道细胞膜N-糖链在神经胚胎发育中有重要作用,值得注意的是GnT-V在神经元细胞分化时期此酶的表达升高2-3倍,而GnT-V高表达引起的糖基化修饰的Trk A受体核心蛋白对其功能的影响和对PC12细胞神经元的分化影响未见报道。 目前N-糖链与肿瘤侵袭转移的关系近年来备受关注,尤其是与GnT-V相关的β1,6分支结构的研究。研究表明在恶性肿瘤中,N-乙酰氨基葡萄糖转移酶V活性增高,其催化产物β1,6分支也增加,肿瘤细胞表面β1,6分支存在能促进其入侵基膜,β1,6分支已成为乳腺癌和结肠癌肿瘤恶化的标志。细胞膜N-糖链在个体发育和肿瘤生成中有显著变化提示N-糖链在细胞分化和细胞增殖转移中有重要作用。而GnT-V在神经中的生物学作用还未见报道。在神经发育中,复合型N-糖链尤其是神经组织胚胎发育必须的。Taguchi et al.报道指出肿瘤的侵袭与神经轴突的应答过程相似的概念,提示在肿瘤转移中起作用的糖基转移酶GnT-V很可能在神经中也起重要作用。 神经轴突的生长与肿瘤的迁移有很多相似之处,包括细胞骨架的变化和参与的信号调节通路。至今关于GnT-V在神经中的作用尚无文献报道,我们主要在此方面进行阐释。 综上所述,本文选NGF为靶点,研究GnT-V转染PC12细胞后GnT-V高表达对NGF及受体诱导的轴突生长通路的变化。考虑到目前GnT-V在神经中的生物学作用未见报道,因此本文首次在此方面进行初步的探索研究,首先成功构建转染稳定表达GnT-V的PC12细胞和mock组细胞,进行了糖链和蛋白鉴定。研究NGF诱导轴突生长的影响,结果表明,GnT-V高表达,能修饰神经生长因子受体,引起NGF低浓度下神经轴突的生长,转染细胞轴突生长明显增强,提示GnT-V表达增加对神经元分化具有调节作用。研究了GnT-V对PC12细胞的生物效应,这种促进PC12细胞突起生长的作用,是促分化作用。为了阐释N-糖链的变化,将NGF受体TrKA免疫沉淀后,用凝集素PHA-L分析GnT-V酶作用产物β1,6分支结构糖链。同时免疫沉淀TrKA受体,测定NGF受体的信号通路的激活,研究了转染稳定表达GnT-V的细胞,mock组细胞TrKA受体磷酸化随时间的变化,TrKA受体用抗TrKA抗体免疫沉淀垂钓分析磷酸化水平。结果表明,转染稳定表达GnT-V的细胞比mock组细胞磷酸化水平明显升高。GnT-V转染PC12细胞对NGF刺激轴突信号转导通路的敏感性增强。基于前期研究发现低浓度的NGF能引起PC12细胞轴突生长的现象,进一步研究GnT-V对TrKA受体作用的机理。 研究发现GnT-V高表达,免疫沉淀神经生长因子细胞膜受体TrKA糖链β1,6分支结构增加,说明此酶GnT-V能直接糖基化修饰神经生长因子高亲和性的受体TrKA,同时发现糖基化修饰的高亲和性的受体TrKA的磷酸化增强。为进一步解释其机制,我们研究了不同时间点TrKA受体的内吞,NGF-β孵育不同的时间点,测定转染稳定表达GnT-V的细胞,mock组细胞NGF-β与受体TrKA结合内吞的情况。结果发现转染稳定表达GnT-V的PC12细胞与mock组相比,TrKA受体内吞延迟,提示GnT-V高表达引起的N-糖链变化,使得NGF受体TrKA内吞延迟,进而增强了受体信号转导通路,导致神经轴突的生长。这些结果表明,GnT-V高表达,修饰神经生长因子受体,增强其功能,能引起低浓度NGF刺激下神经轴突的生长, GnT-V在神经中可能通过修饰糖蛋白受体调节NGF诱导的信号通路,可能在与神经生长因子受体相关的神经退行性病变中起作用。本文研究发现N-乙酰氨基葡萄糖基转移酶V糖基化修饰神经生长因子受体TrKA,能调节TrKA受体的功能,激活TrKA受体介导的轴突生长的信号通路。 此论文的研究意义在于首次阐释了N-乙酰氨基葡萄糖基转移酶V对神经生长因子受体的糖基化修饰作用对TrKA受体功能的影响,其研究结果为研究N-糖链在神经中的作用提供了深层次的理论依据,拓展了对N-乙酰氨基葡萄糖基转移酶V在神经中作用的认识。发现N-乙酰氨基葡萄糖基转移酶V具有促神经分化作用,这种对神经生长因子受体TrKA的调节作用,可能与神经生长因子相关的神经退行性病症的药物设计提供了新的靶点,拓展了糖基转移酶研究的领域。
[Abstract]:N- sugar chains play an important role in cell communication, growth and differentiation. There is evidence that N- sugar chains play an important role in neural development. Glycosylation (Glycomics) is the focus of post genome research. Glycosyltransferase is a widely distributed enzyme that participates in the biosynthesis of sugar parts in chitosan, glycosides and carbohydrates. There is a high degree of substrate specificity. The international research on glycosyltransferases is relatively preliminary. Compared with other types of enzymes, such as the enzymes involved in metabolism, the expression of enzymes and what factors are regulated, the biological effects of glycosyltransferase are more extensive and complex. The full interpretation of energy will reveal a large number of related biological information.
N- acetylglucosaminosyltransferase V (N-acetylglucosaminyltransferase V, GnT-V/Mgat5, EC 2.4.1.155), as an important Golgi body glycosyltransferase, is one of the glycoprotein N- sugar chain processing enzymes. It plays an important role in determining the type of N- chain and the complex structure of sugar chain. It catalyzes the transfer of the GlcNAc group to the alpha glycosaminophone of the alpha 1,6 arm of the N- sugar chain. The reaction of sugar and the formation of the beta 1,6 branching structure of N- sugar chain can form the N- sugar chain product of the 2,3,4 antenna, which plays an important role in the post-translational modification of the protein, and the protein glycosylation is one of the necessary conditions for the biological activity protein to play its biological function, including the modified.N- of the receptor membrane protein with the heavy function of the growth factor. Acetylglucosaminosyltransferase V is an important Golgi body glycosyltransferase, which participates in the synthesis of glycoprotein N- sugar chain, and has an important function for the co translation of glycosylation after glycoprotein translation. Translation modification is also necessary for the survival of eukaryotic cells. The study found that the deletion of N- acetylglucosaminosyltransferase V can cause the abnormal glucose chain of the receptor N-, and the abnormal glycosylation can affect the receptor function of certain types of cell surface and lead to the change of receptor signal transduction. Therefore, this paper constructs a GnT-V high expression cell line to study glycoprotein carbohydrate chain change. The effect of chemistry on the function of the receptor.
Nerve growth factor NGF is the earliest discovered nerve growth factor. As an important neurotrophic factor, it can regulate nerve growth, development, differentiation, and regulate nutrient neurons through a highly compatible receptor TrKA. Not only in the neurocytoma, the elevated expression of NGF receptor TrkA is associated with a good prognosis, but also in neurodegenerative diseases. For example, Parkinson and Alzheimer's disease (AD), NGF is also a hot spot in the research of therapeutic drugs,.NGF can cause differentiation of axon growth cells through high affinity receptor, while low affinity receptor causes apoptosis, and low NGF in microenvironment can not cause axonal growth.PC12 cells are rat kidney eosinophil cells, and have the characteristics of neural cells, which is a study of NG. F neurite growth good cell model, the literature reported that the NGF stimulation of about 50-100 ng/mL in normal state can induce the growth of PC12 cell axon in a few days. Therefore, the cell model was selected as the study cell model, and the effect of the high expression of GnT-V on the cell was observed under the low concentration of NGF stimulation (reduced by 4-8 times), and at this low concentration, NGF was not alone. Enough to induce axon growth.
The human nerve growth factor receptor (Trk A) contains 4 potential N- glycosylation sites, which are highly conserved in the neurotrophic factor receptor Trk family, suggesting that glycosylation may play an important role. There are also 9 less conservative N- glycosylation sites. The study found that the mature Trk A receptor is conservative and variable in the sugar chain sugar chain sugar. The glycosylation sites are glycosylated. Glycosylation of these sites has two different functions. One is that the glycosylation of Trk A receptor is necessary to prevent the activation of ligands without dependence. The deglycosylated Trk A receptor core protein is phosphorylated without ligand activation and shows the activity of the constituent kinase activity to the signal molecule Shc and PLC- gamma; two It is the Trk A receptor glycosylation is necessary for the receptor to locate on the surface of the cell membrane. Only the receptor on the surface of the cell membrane can trigger the Ras/Raf/MAP kinase pathway. The laser confocal microscope shows that the non glycosylated active Trk receptor is trapped in the cell and can not locate the cell membrane, and the non glycosylated active Trk A receptor does not activate the Ras- MAP, MEK and ErK kinase pathways, therefore, the non glycosylated Trk A receptor core protein can not induce the differentiation of PC12 cell neurons. Moreover, it is reported that the glycosylated Trk can not respond to its ligand NGF, that is, after preconditioning the stable transfected PC12 cells with high expression of Trk, the Trk glycosylation is suppressed and the Trk protein is changed. The binding site of ganglioside, acid sphingolipid) loses the ability to form complex with GM1. Moreover, it is reported that the N- sugar chain of cell membrane plays an important role in the development of neural embryos. It is worth noting that the expression of this enzyme increased by 2-3 times of GnT-V during the differentiation period of neuron cells, and the glycosylated Trk A receptor nucleus caused by the high expression of GnT-V The effect of cardiac protein on its function and the differentiation of neurons in PC12 cells has not been reported.
The relationship between N- sugar chain and tumor invasion and metastasis has attracted much attention in recent years, especially the study of the GnT-V related beta 1,6 branch structure. The study shows that in malignant tumors, N- acetylglucosaminosamine transferase V activity increases, its catalytic product beta 1,6 branch is also increased, the tumor cell surface beta 1,6 branch exists to promote its invasion of the basement membrane, beta 1,6 The branching has become a sign of the deterioration of breast and colon cancer. The significant changes in the cell membrane N- sugar chain in ontogenesis and tumor formation suggest that the N- sugar chain plays an important role in cell differentiation and cell proliferation and metastasis. The biological role of GnT-V in the nerve has not been reported. In the development of nerve, the complex N- sugar chain is especially the nerve. .Taguchi et al., which is necessary for tissue development, indicates that the invasion of the tumor is similar to that of the neurite response process, suggesting that the glycosyltransferase GnT-V, which plays a role in tumor metastasis, is likely to play an important role in the nerve.
There are many similarities between the growth of the axon and the migration of the tumor, including the changes in the cytoskeleton and the signaling pathways involved. There has been no literature on the role of GnT-V in the nerve, and we are mainly explaining it in this respect.
To sum up, NGF is selected as the target to study the change of GnT-V high expression of NGF and receptor induced axon growth pathway after the transfection of GnT-V into PC12 cells. Considering that the biological role of GnT-V in the nerve is not reported, the first exploration and research in this paper is the first time to construct a PC1 that transfected the stable expression of GnT-V. 2 cells and mock cells were identified. The effects of NGF on the growth of axon were studied. The results showed that the high expression of GnT-V could modify the nerve growth factor receptor, cause the growth of the axon under the low concentration of NGF, and the growth of the axon in the transfected cells was obviously enhanced, suggesting that the expression of GnT-V has a regulatory effect on the differentiation of neurons. The biological effect of GnT-V on PC12 cells, which promotes the growth of PC12 cells, is the role of promoting differentiation. In order to explain the changes in the N- sugar chain, the NGF receptor TrKA is immune to the precipitation of the NGF receptor, and the agglutinin PHA-L is used to analyze the sugar chain of the beta 1,6 branching structure of the product of the GnT-V enzyme. At the same time, the NGF receptor is immune to the TrKA receptor and the signal pathway of the NGF receptor is determined. The cells transfected stably expressing GnT-V, the phosphorylation of TrKA receptor in mock group was changed with time, and the phosphorylation level of TrKA receptor was analyzed by anti TrKA antibody immunoprecipitation. The results showed that the transfected cells with stable expression of GnT-V significantly increased the phosphorylation level of.GnT-V transfected PC12 cells to NGF stimulated axon signal to NGF. The sensitivity of the transduction pathway is enhanced. Based on previous studies, a low concentration of NGF can cause the growth of PC12 cells, and the mechanism of the effect of GnT-V on the TrKA receptor is further studied.
The high expression of GnT-V and the increase of the branching structure of TrKA sugar chain beta 1,6 in the immunoprecipitation neural growth factor cell membrane receptor indicate that the enzyme GnT-V can directly glycosylate the highly compatible receptor TrKA of the nerve growth factor, and the phosphorylation of the glycosylated high affinity receptor TrKA is also found. The endocytosis of TrKA receptor at different time points was studied. NGF- beta incubated at different time points, the transfection of cells with stable expression of GnT-V and the endocytosis of NGF- beta and receptor TrKA in group mock were found. The results showed that the PC12 cells that transfected the stable expression of GnT-V were delayed in the TrKA receptor endocytosis compared with the mock group, suggesting the change of the N- sugar chain caused by the GnT-V high expression. The NGF receptor TrKA endocytosis is delayed and the receptor signal transduction pathway is enhanced, resulting in the growth of the axon. These results suggest that GnT-V is highly expressed, modifies the NGF receptor, enhances its function, and causes the growth of the axon under the low concentration of NGF stimulation, and GnT-V may regulate the NGF induced by modifying the glycoprotein receptor in the nerve. The signaling pathway may play a role in neurodegenerative diseases associated with NGF receptor. This study found that the N- acetylglucosaminyltransferase V glycosylated NGF receptor TrKA, which regulates the function of the TrKA receptor, and activates the signal pathway of the axon growth mediated by TrKA receptor.
The significance of this paper is for the first time to explain the effect of the glycosylation of N- acetylglucosaminotransferase V on the function of TrKA receptor, which provides a deep theoretical basis for the study of the role of N- sugar chain in the nerve and extends the N- acetylglucosaminosyltransferase (V). It is found that N- acetylglucosaminosyltransferase V has the role of promoting nerve differentiation, and this regulation of NGF receptor TrKA may provide new targets for the drug design of neurodegenerative diseases related to NGF, and expand the field of glycosyltransferase research.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341
本文编号：2142338
[Abstract]:N- sugar chains play an important role in cell communication, growth and differentiation. There is evidence that N- sugar chains play an important role in neural development. Glycosylation (Glycomics) is the focus of post genome research. Glycosyltransferase is a widely distributed enzyme that participates in the biosynthesis of sugar parts in chitosan, glycosides and carbohydrates. There is a high degree of substrate specificity. The international research on glycosyltransferases is relatively preliminary. Compared with other types of enzymes, such as the enzymes involved in metabolism, the expression of enzymes and what factors are regulated, the biological effects of glycosyltransferase are more extensive and complex. The full interpretation of energy will reveal a large number of related biological information.
N- acetylglucosaminosyltransferase V (N-acetylglucosaminyltransferase V, GnT-V/Mgat5, EC 2.4.1.155), as an important Golgi body glycosyltransferase, is one of the glycoprotein N- sugar chain processing enzymes. It plays an important role in determining the type of N- chain and the complex structure of sugar chain. It catalyzes the transfer of the GlcNAc group to the alpha glycosaminophone of the alpha 1,6 arm of the N- sugar chain. The reaction of sugar and the formation of the beta 1,6 branching structure of N- sugar chain can form the N- sugar chain product of the 2,3,4 antenna, which plays an important role in the post-translational modification of the protein, and the protein glycosylation is one of the necessary conditions for the biological activity protein to play its biological function, including the modified.N- of the receptor membrane protein with the heavy function of the growth factor. Acetylglucosaminosyltransferase V is an important Golgi body glycosyltransferase, which participates in the synthesis of glycoprotein N- sugar chain, and has an important function for the co translation of glycosylation after glycoprotein translation. Translation modification is also necessary for the survival of eukaryotic cells. The study found that the deletion of N- acetylglucosaminosyltransferase V can cause the abnormal glucose chain of the receptor N-, and the abnormal glycosylation can affect the receptor function of certain types of cell surface and lead to the change of receptor signal transduction. Therefore, this paper constructs a GnT-V high expression cell line to study glycoprotein carbohydrate chain change. The effect of chemistry on the function of the receptor.
Nerve growth factor NGF is the earliest discovered nerve growth factor. As an important neurotrophic factor, it can regulate nerve growth, development, differentiation, and regulate nutrient neurons through a highly compatible receptor TrKA. Not only in the neurocytoma, the elevated expression of NGF receptor TrkA is associated with a good prognosis, but also in neurodegenerative diseases. For example, Parkinson and Alzheimer's disease (AD), NGF is also a hot spot in the research of therapeutic drugs,.NGF can cause differentiation of axon growth cells through high affinity receptor, while low affinity receptor causes apoptosis, and low NGF in microenvironment can not cause axonal growth.PC12 cells are rat kidney eosinophil cells, and have the characteristics of neural cells, which is a study of NG. F neurite growth good cell model, the literature reported that the NGF stimulation of about 50-100 ng/mL in normal state can induce the growth of PC12 cell axon in a few days. Therefore, the cell model was selected as the study cell model, and the effect of the high expression of GnT-V on the cell was observed under the low concentration of NGF stimulation (reduced by 4-8 times), and at this low concentration, NGF was not alone. Enough to induce axon growth.
The human nerve growth factor receptor (Trk A) contains 4 potential N- glycosylation sites, which are highly conserved in the neurotrophic factor receptor Trk family, suggesting that glycosylation may play an important role. There are also 9 less conservative N- glycosylation sites. The study found that the mature Trk A receptor is conservative and variable in the sugar chain sugar chain sugar. The glycosylation sites are glycosylated. Glycosylation of these sites has two different functions. One is that the glycosylation of Trk A receptor is necessary to prevent the activation of ligands without dependence. The deglycosylated Trk A receptor core protein is phosphorylated without ligand activation and shows the activity of the constituent kinase activity to the signal molecule Shc and PLC- gamma; two It is the Trk A receptor glycosylation is necessary for the receptor to locate on the surface of the cell membrane. Only the receptor on the surface of the cell membrane can trigger the Ras/Raf/MAP kinase pathway. The laser confocal microscope shows that the non glycosylated active Trk receptor is trapped in the cell and can not locate the cell membrane, and the non glycosylated active Trk A receptor does not activate the Ras- MAP, MEK and ErK kinase pathways, therefore, the non glycosylated Trk A receptor core protein can not induce the differentiation of PC12 cell neurons. Moreover, it is reported that the glycosylated Trk can not respond to its ligand NGF, that is, after preconditioning the stable transfected PC12 cells with high expression of Trk, the Trk glycosylation is suppressed and the Trk protein is changed. The binding site of ganglioside, acid sphingolipid) loses the ability to form complex with GM1. Moreover, it is reported that the N- sugar chain of cell membrane plays an important role in the development of neural embryos. It is worth noting that the expression of this enzyme increased by 2-3 times of GnT-V during the differentiation period of neuron cells, and the glycosylated Trk A receptor nucleus caused by the high expression of GnT-V The effect of cardiac protein on its function and the differentiation of neurons in PC12 cells has not been reported.
The relationship between N- sugar chain and tumor invasion and metastasis has attracted much attention in recent years, especially the study of the GnT-V related beta 1,6 branch structure. The study shows that in malignant tumors, N- acetylglucosaminosamine transferase V activity increases, its catalytic product beta 1,6 branch is also increased, the tumor cell surface beta 1,6 branch exists to promote its invasion of the basement membrane, beta 1,6 The branching has become a sign of the deterioration of breast and colon cancer. The significant changes in the cell membrane N- sugar chain in ontogenesis and tumor formation suggest that the N- sugar chain plays an important role in cell differentiation and cell proliferation and metastasis. The biological role of GnT-V in the nerve has not been reported. In the development of nerve, the complex N- sugar chain is especially the nerve. .Taguchi et al., which is necessary for tissue development, indicates that the invasion of the tumor is similar to that of the neurite response process, suggesting that the glycosyltransferase GnT-V, which plays a role in tumor metastasis, is likely to play an important role in the nerve.
There are many similarities between the growth of the axon and the migration of the tumor, including the changes in the cytoskeleton and the signaling pathways involved. There has been no literature on the role of GnT-V in the nerve, and we are mainly explaining it in this respect.
To sum up, NGF is selected as the target to study the change of GnT-V high expression of NGF and receptor induced axon growth pathway after the transfection of GnT-V into PC12 cells. Considering that the biological role of GnT-V in the nerve is not reported, the first exploration and research in this paper is the first time to construct a PC1 that transfected the stable expression of GnT-V. 2 cells and mock cells were identified. The effects of NGF on the growth of axon were studied. The results showed that the high expression of GnT-V could modify the nerve growth factor receptor, cause the growth of the axon under the low concentration of NGF, and the growth of the axon in the transfected cells was obviously enhanced, suggesting that the expression of GnT-V has a regulatory effect on the differentiation of neurons. The biological effect of GnT-V on PC12 cells, which promotes the growth of PC12 cells, is the role of promoting differentiation. In order to explain the changes in the N- sugar chain, the NGF receptor TrKA is immune to the precipitation of the NGF receptor, and the agglutinin PHA-L is used to analyze the sugar chain of the beta 1,6 branching structure of the product of the GnT-V enzyme. At the same time, the NGF receptor is immune to the TrKA receptor and the signal pathway of the NGF receptor is determined. The cells transfected stably expressing GnT-V, the phosphorylation of TrKA receptor in mock group was changed with time, and the phosphorylation level of TrKA receptor was analyzed by anti TrKA antibody immunoprecipitation. The results showed that the transfected cells with stable expression of GnT-V significantly increased the phosphorylation level of.GnT-V transfected PC12 cells to NGF stimulated axon signal to NGF. The sensitivity of the transduction pathway is enhanced. Based on previous studies, a low concentration of NGF can cause the growth of PC12 cells, and the mechanism of the effect of GnT-V on the TrKA receptor is further studied.
The high expression of GnT-V and the increase of the branching structure of TrKA sugar chain beta 1,6 in the immunoprecipitation neural growth factor cell membrane receptor indicate that the enzyme GnT-V can directly glycosylate the highly compatible receptor TrKA of the nerve growth factor, and the phosphorylation of the glycosylated high affinity receptor TrKA is also found. The endocytosis of TrKA receptor at different time points was studied. NGF- beta incubated at different time points, the transfection of cells with stable expression of GnT-V and the endocytosis of NGF- beta and receptor TrKA in group mock were found. The results showed that the PC12 cells that transfected the stable expression of GnT-V were delayed in the TrKA receptor endocytosis compared with the mock group, suggesting the change of the N- sugar chain caused by the GnT-V high expression. The NGF receptor TrKA endocytosis is delayed and the receptor signal transduction pathway is enhanced, resulting in the growth of the axon. These results suggest that GnT-V is highly expressed, modifies the NGF receptor, enhances its function, and causes the growth of the axon under the low concentration of NGF stimulation, and GnT-V may regulate the NGF induced by modifying the glycoprotein receptor in the nerve. The signaling pathway may play a role in neurodegenerative diseases associated with NGF receptor. This study found that the N- acetylglucosaminyltransferase V glycosylated NGF receptor TrKA, which regulates the function of the TrKA receptor, and activates the signal pathway of the axon growth mediated by TrKA receptor.
The significance of this paper is for the first time to explain the effect of the glycosylation of N- acetylglucosaminotransferase V on the function of TrKA receptor, which provides a deep theoretical basis for the study of the role of N- sugar chain in the nerve and extends the N- acetylglucosaminosyltransferase (V). It is found that N- acetylglucosaminosyltransferase V has the role of promoting nerve differentiation, and this regulation of NGF receptor TrKA may provide new targets for the drug design of neurodegenerative diseases related to NGF, and expand the field of glycosyltransferase research.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341
【相似文献】
相关期刊论文 前10条
1 何学元;张有成;;神经生长因子及其受体与肿瘤相关性研究进展[J];国际消化病杂志;2010年04期
2 甄红英,孙素元,于肇英,李劲松;脑损伤后神经生长因子受体的变化[J];中国病理生理杂志;1991年01期
3 张雪宝,陆天才;神经生长因子及其受体与肿瘤的关系[J];临床与实验病理学杂志;2002年05期
4 宓宝杰;王相利;;神经生长因子与其受体的信号转导[J];中国煤炭工业医学杂志;2007年06期
5 徐鹏;舒畅;赵子义;张伟;;免疫磁珠法分离纯化骨髓间充质干细胞及向神经细胞定向分化[J];中国组织工程研究与临床康复;2007年20期
6 徐娟;刘洪臣;袁维秀;黄旭明;;实验性牙移动三叉神经节内神经生长因子受体mRNA的改变[J];口腔医学研究;2006年02期
7 涂建锋;杨悦;张跃明;;银杏叶复方对急性全脑缺血再灌注损伤大鼠下丘脑NGFR表达的影响[J];中华中医药学刊;2010年01期
8 甄红英,于肇英,孙素元,李劲松;大鼠脑中脑神经生长因子受体[J];中国病理生理杂志;1991年01期
9 刘浩;神经生长因子受体在先天性巨结肠表达的意义[J];中华小儿外科杂志;1999年01期
10 高红,张志波,王维林,黄英;先天性巨结肠神经生长因子及神经生长因子受体基因表达[J];中华小儿外科杂志;2005年03期
相关博士学位论文 前4条
1 杨晓云;N-乙酰氨基葡萄糖转移酶V高表达细胞株的构建及其对神经生长因子受体TrKA作用影响的研究[D];中国海洋大学;2007年
2 李姣;抑制GnT-V表达诱导SMMC-7721细胞发生内质网应激的机制探讨—GLUT1结构和功能异常[D];复旦大学;2007年
3 王海涛;HCMV感染神经瘤细胞对其神经生长因子和受体表达影响的研究[D];青岛大学;2009年
4 王伟岸;中枢神经系统对肠易激综合征内脏敏感性的调节[D];中国协和医科大学;2000年
相关硕士学位论文 前2条
1 杨以恒;先天性胆总管囊肿肠神经分布及发病机制研究[D];河北医科大学;2009年
2 赏金标;神经细胞粘附分子（NCAM）、神经生长因子受体（NGFR/P75NRT）在涎腺腺样囊性癌中的表达及临床意义[D];浙江大学;2007年
，本文编号：2142338
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2142338.html
最近更新
教材专著
