本文選題：GDP-巖藻糖轉(zhuǎn)運(yùn)體 + Slc35c1-3-83-KO細(xì)胞　； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：GDP-巖藻糖是在細(xì)胞質(zhì)內(nèi)合成的。而后被GDP-巖藻糖轉(zhuǎn)運(yùn)體轉(zhuǎn)運(yùn)至高爾基體內(nèi),在巖藻糖基轉(zhuǎn)移酶的催化下,完成巖藻糖基化修飾。巖藻糖基化是蛋白質(zhì)翻譯后修飾的一種重要方式之一,其對(duì)糖蛋白功能發(fā)揮重要調(diào)節(jié)作用。在本研究以成熟B細(xì)胞受體(BCR)轉(zhuǎn)基因細(xì)胞模型(3-83細(xì)胞)作為研究對(duì)象,利用CRISPR/Cas9系統(tǒng),設(shè)計(jì)含有兩個(gè)GDP-巖藻糖轉(zhuǎn)運(yùn)體基因(Slc35c1)敲除gRNA序列的pGS-U6-gRNA載體,通過(guò)凝集素印記等實(shí)驗(yàn),篩選最佳gRNA干擾序列。利用脂質(zhì)體轉(zhuǎn)染技術(shù),把載體導(dǎo)入至3-83細(xì)胞后經(jīng)過(guò)G418篩選,挑選單克隆并擴(kuò)大培養(yǎng),經(jīng)免疫印跡技術(shù)鑒定穩(wěn)定遺傳的Slc35c1基因敲除3-83細(xì)胞模型(Slc35c1-3-83-KO)。為建立GDP-巖藻糖轉(zhuǎn)運(yùn)體基因恢復(fù)細(xì)胞模型,將Slc35c1基因?qū)牖蚯贸?xì)胞中,并連接至另一種病毒載體(pLHCX),通過(guò)梭華試劑將其導(dǎo)入到Slc35c1-3-83-KO細(xì)胞中,經(jīng)過(guò)200μg/ml潮霉素篩選,獲得Slc35c1基因恢復(fù)模型(Slc35c1-3-83-KO-Re)。利用3-83細(xì)胞、Slc35c1-3-83-KO細(xì)胞及Slc35c1-3-83-KO-Re細(xì)胞,通過(guò)MTT及細(xì)胞粘附實(shí)驗(yàn),從細(xì)胞整體水平上檢測(cè)比較三種細(xì)胞的細(xì)胞增殖及細(xì)胞粘附能力,從而探究GDP-巖藻糖轉(zhuǎn)運(yùn)體對(duì)成熟B細(xì)胞生物學(xué)功能的調(diào)節(jié)作用。結(jié)果顯示,Slc35c1-3-83-KO的整體巖藻糖基化及核心巖藻糖基化水平、細(xì)胞增殖、細(xì)胞粘附能力顯著低于正常3-83細(xì)胞,而在Slc35c1-3-83-KO-Re細(xì)胞中其功能得到恢復(fù),提示GDP-巖藻糖轉(zhuǎn)運(yùn)體介導(dǎo)的巖藻糖基化修飾對(duì)成熟B細(xì)胞粘附及增殖具有重要調(diào)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q78

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Ruby Yanru Chen-Tsai;Ruhong Jiang;Luping Zhuang;Junfeng Wu;Lingsong Li;Jiarui Wu;;Genome editing and animal models[J];Chinese Science Bulletin;2014年01期

，

本文編號(hào)：1966912