本文選題：GDP-巖藻糖轉運體 + Slc35c1-3-83-KO細胞��； 參考：《大連醫(yī)科大學》2017年碩士論文

【摘要】：GDP-巖藻糖是在細胞質內合成的。而后被GDP-巖藻糖轉運體轉運至高爾基體內,在巖藻糖基轉移酶的催化下,完成巖藻糖基化修飾。巖藻糖基化是蛋白質翻譯后修飾的一種重要方式之一,其對糖蛋白功能發(fā)揮重要調節(jié)作用。在本研究以成熟B細胞受體(BCR)轉基因細胞模型(3-83細胞)作為研究對象,利用CRISPR/Cas9系統(tǒng),設計含有兩個GDP-巖藻糖轉運體基因(Slc35c1)敲除gRNA序列的pGS-U6-gRNA載體,通過凝集素印記等實驗,篩選最佳gRNA干擾序列。利用脂質體轉染技術,把載體導入至3-83細胞后經過G418篩選,挑選單克隆并擴大培養(yǎng),經免疫印跡技術鑒定穩(wěn)定遺傳的Slc35c1基因敲除3-83細胞模型(Slc35c1-3-83-KO)。為建立GDP-巖藻糖轉運體基因恢復細胞模型,將Slc35c1基因導入基因敲除細胞中,并連接至另一種病毒載體(pLHCX),通過梭華試劑將其導入到Slc35c1-3-83-KO細胞中,經過200μg/ml潮霉素篩選,獲得Slc35c1基因恢復模型(Slc35c1-3-83-KO-Re)。利用3-83細胞、Slc35c1-3-83-KO細胞及Slc35c1-3-83-KO-Re細胞,通過MTT及細胞粘附實驗,從細胞整體水平上檢測比較三種細胞的細胞增殖及細胞粘附能力,從而探究GDP-巖藻糖轉運體對成熟B細胞生物學功能的調節(jié)作用。結果顯示,Slc35c1-3-83-KO的整體巖藻糖基化及核心巖藻糖基化水平、細胞增殖、細胞粘附能力顯著低于正常3-83細胞,而在Slc35c1-3-83-KO-Re細胞中其功能得到恢復,提示GDP-巖藻糖轉運體介導的巖藻糖基化修飾對成熟B細胞粘附及增殖具有重要調控作用。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78

【參考文獻】

相關期刊論文 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

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本文編號：1966912