GAS7多克隆抗体的制备及雌激素对大鼠海马GAS7表达的影响

发布时间：2019-02-12 10:36

【摘要】： GAS(生长休止蛋白)主要表达于细胞的休止期,调控细胞生长分化。其中GAS7首次发现于血清剥夺的鼠类成纤维细胞,在体内主要表达于神经系统,且特异性地在极度分化的神经细胞大量表达。GAS7对原代培养的小脑神经细胞分化成熟是必需的,而且有研究表明GAS7在PC12细胞的高表达可以促进PC12细胞突起的形成,而应用反义寡核苷酸抑制GAS7在培养的小脑细胞的表达将显著抑制细胞突起的形成,因此GAS7被认为是成熟神经细胞的标志并具有潜在的抗癌功能。近年来GAS7的功能已引起有关学者的重视,但现在尚未有商品化的抗体,而关于GAS7在体内的表达定位及分布等相关资料尚有所欠缺。本研究主要制备抗GAS7抗血清及用免疫组织化学、Western blot和RT-PCR方法观察大鼠神经系统中GAS7的表达定位及分布及雌激素对大鼠海马GAS7表达的影响,以期为研究GAS7在神经系统的功能提供重要的实验资料。目的: 1.利用GST融合蛋白作免疫原制备抗大鼠GAS7多克隆抗血清。 2.观察大鼠神经系统中GAS7不同异构体的表达定位及分布。 3.研究雌激素替代治疗对大鼠海马生长休止蛋白7表达的作用和影响。方法: 1.利用基因工程重组的表达质粒诱导表达的GST融合蛋白作免疫原制备抗大鼠GAS7多克隆抗血清。 2.应用免疫组织化学、Western blot和RT-PCR方法观察大鼠神经系统中GAS7的表达定位及分布。 3.制作卵巢切除雌激素替代模型,以免疫组织化学,Western blot观察雌激素对大鼠海马GAS7表达的作用及影响。结果: 1.成功制备抗大鼠GAS7多克隆抗血清。 2.GAS7广泛表达于大鼠神经系统,在不同的部位有不同的GAS7表达。 3.雌激素上调GAS7b的表达,而下调GAS7a的表达。结论: 1.不同的GAS7蛋白可能参于神经系统不同的功能。 2.雌激素可能通过调节GAS7不同异构体在海马的表达以实现其延缓神经元老化的功能.
[Abstract]:GAS (growth rest protein) is mainly expressed in the resting period of cells and regulates cell growth and differentiation. GAS7 was first found in serum-deprived rat fibroblasts, mainly expressed in the nervous system and specifically expressed in highly differentiated neurons. GAS7 is necessary for the differentiation and maturation of primary cultured cerebellar nerve cells. Moreover, some studies have shown that the high expression of GAS7 in PC12 cells can promote the formation of PC12 cell processes, while the inhibition of GAS7 expression in cultured cerebellar cells by antisense oligonucleotides will significantly inhibit the formation of cell processes. Therefore, GAS7 is considered to be a marker of mature nerve cells and has potential anti-cancer function. In recent years, the function of GAS7 has attracted the attention of the scholars concerned, but there is no commercial antibody, but the expression localization and distribution of GAS7 in vivo and other related data are still lacking. The aim of this study was to prepare antiserum against GAS7 and to observe the localization and distribution of GAS7 in the nervous system of rats by immunohistochemical, Western blot and RT-PCR and the effect of estrogen on the expression of GAS7 in hippocampus of rats. In order to provide important experimental data for studying the function of GAS7 in nervous system. Objective: 1. Anti-rat GAS7 polyclonal antiserum was prepared by using GST fusion protein as immunogen. 2. To observe the expression localization and distribution of different isomers of GAS7 in the nervous system of rats. 3. To study the effect and effect of estrogen replacement therapy on the expression of hippocampal growth reversion protein 7 in rats. Methods: 1. The recombinant GST fusion protein was used as immunogen to prepare anti-rat GAS7 polyclonal antiserum. 2. Immunohistochemical, Western blot and RT-PCR methods were used to observe the localization and distribution of GAS7 expression in rat nervous system. 3. The ovariectomized estrogen replacement model was established and the effects of estrogen on the expression of GAS7 in hippocampus of rats were observed by immunohistochemical, Western blot. Results: 1. Anti-rat GAS7 polyclonal antiserum was successfully prepared. 2.GAS7 is widely expressed in the nervous system of rats and has different GAS7 expression in different sites. 3. Estrogen upregulated the expression of GAS7b and down-regulated the expression of GAS7a. Conclusion: 1. Different GAS7 proteins may be involved in different functions of the nervous system. 2. Estrogen may delay the aging of neurons by regulating the expression of different isomers of GAS7 in the hippocampus.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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