GDP-岩藻糖转运体生物学功能的基础研究

发布时间：2018-06-02 02:26

本文选题：GDP-岩藻糖转运体 + Slc35c1-3-83-KO细胞　；参考：《大连医科大学》2017年硕士论文

【摘要】：GDP-岩藻糖是在细胞质内合成的。而后被GDP-岩藻糖转运体转运至高尔基体内,在岩藻糖基转移酶的催化下,完成岩藻糖基化修饰。岩藻糖基化是蛋白质翻译后修饰的一种重要方式之一,其对糖蛋白功能发挥重要调节作用。在本研究以成熟B细胞受体(BCR)转基因细胞模型(3-83细胞)作为研究对象,利用CRISPR/Cas9系统,设计含有两个GDP-岩藻糖转运体基因(Slc35c1)敲除gRNA序列的pGS-U6-gRNA载体,通过凝集素印记等实验,筛选最佳gRNA干扰序列。利用脂质体转染技术,把载体导入至3-83细胞后经过G418筛选,挑选单克隆并扩大培养,经免疫印迹技术鉴定稳定遗传的Slc35c1基因敲除3-83细胞模型(Slc35c1-3-83-KO)。为建立GDP-岩藻糖转运体基因恢复细胞模型,将Slc35c1基因导入基因敲除细胞中,并连接至另一种病毒载体(pLHCX),通过梭华试剂将其导入到Slc35c1-3-83-KO细胞中,经过200μg/ml潮霉素筛选,获得Slc35c1基因恢复模型(Slc35c1-3-83-KO-Re)。利用3-83细胞、Slc35c1-3-83-KO细胞及Slc35c1-3-83-KO-Re细胞,通过MTT及细胞粘附实验,从细胞整体水平上检测比较三种细胞的细胞增殖及细胞粘附能力,从而探究GDP-岩藻糖转运体对成熟B细胞生物学功能的调节作用。结果显示,Slc35c1-3-83-KO的整体岩藻糖基化及核心岩藻糖基化水平、细胞增殖、细胞粘附能力显著低于正常3-83细胞,而在Slc35c1-3-83-KO-Re细胞中其功能得到恢复,提示GDP-岩藻糖转运体介导的岩藻糖基化修饰对成熟B细胞粘附及增殖具有重要调控作用。
[Abstract]:GDP- fucose is synthesized in cytoplasm. Then it was transported to Golgi by GDP-fucose transporter and modified by fucosyltransferase. Fucose glycosylation is one of the important ways of protein post-translational modification, which plays an important role in regulating the function of glycoprotein. In this study, a mature B cell receptor (B cell receptor) transgenic cell model, Guan3-83 cell, was used to design a pGS-U6-gRNA vector containing two GDP-fucose transporter genes (Slc35c1) and knockout the gRNA sequence by lectin imprinting. Screening the best gRNA interference sequence. Using liposome transfection technique, the vector was introduced into 3-83 cells and screened by G418. The monoclonal was selected and cultured. The stable inherited Slc35c1 gene knockout 3-83 cell model Slc35c1-3-83-KOA was identified by Western blotting. In order to establish a GDP- fucose transporter gene recovery cell model, the Slc35c1 gene was introduced into knockout cells and ligated to another virus vector, pLHCXN, which was transfected into Slc35c1-3-83-KO cells by Sovar reagent and screened by 200 渭 g/ml hygromycin. Slc35c1 gene restoration model Slc35c1-3-83-KO-Rea was obtained. The cell proliferation and cell adhesion ability of three kinds of cells were measured and compared by MTT and cell adhesion assay using Slc35c1-3-83-KO and Slc35c1-3-83-KO-Re cells. To explore the regulation of GDP- fucose transporter on the biological function of mature B cells. The results showed that the integral fucosylation and core fucosylation of Slc35c1-3-83-KO, cell proliferation and cell adhesion were significantly lower than those of normal 3-83 cells, but the function of SLAC35c1-3-83-KO was recovered in Slc35c1-3-83-KO-Re cells. These results suggest that GDP- fucose transporter mediated fucosylation modification plays an important role in the regulation of adhesion and proliferation of mature B cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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