發(fā)布時(shí)間：2018-03-20 15:25

  本文選題：鼻咽癌　切入點(diǎn)：NP69細(xì)胞系　出處：《南華大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:設(shè)計(jì)靶向STGC3基因的shRNA(miR30-based shRNA),構(gòu)建針對STGC3基因的特異性shRNA真核表達(dá)載體,將pRNAT-U6-shRNA-STGC3瞬時(shí)轉(zhuǎn)染永生化人鼻咽上皮細(xì)胞系NP69,觀察STGC3基因表達(dá)降低對NP69細(xì)胞系的影響,分析STGC3基因在細(xì)胞生長增殖中的作用。 方法:運(yùn)用生物信息學(xué)方法分析與篩選STGC3基因的干擾靶點(diǎn),根據(jù)miRNA30框架結(jié)構(gòu),設(shè)計(jì)靶向STGC3基因的shRNA,構(gòu)建pRNAT-U6-shRNA-STGC3真核表達(dá)載體,用Lipofectamine 2000將重組pRNAT-U6-shRNA-STGC3表達(dá)載體瞬時(shí)轉(zhuǎn)染NP69細(xì)胞系,通過熒光顯微鏡下觀察GFP表達(dá),確定轉(zhuǎn)染效率,以RT-PCR,檢測STGC3基因mRNA表達(dá)水平,評估m(xù)iR30-based shRNA對STGC3基因的干擾效果,擴(kuò)大培養(yǎng)轉(zhuǎn)染細(xì)胞,進(jìn)行細(xì)胞克隆形成實(shí)驗(yàn),觀察pRNAT-U6-shRNA-STGC3- NP69細(xì)胞克隆形成能力,使用流式細(xì)胞儀,分析pRNAT-U6-shRNA-STGC3- NP69細(xì)胞周期的改變和凋亡情況。 結(jié)果構(gòu)建載體,經(jīng)PCR、雙酶切鑒定和測序鑒定,確定成功構(gòu)建pRNAT-U6-shRNA-STGC3真核表達(dá)載體。將pRNAT-U6空質(zhì)粒和pRNAT-U6-shRNA-STGC3重組質(zhì)粒分別轉(zhuǎn)染NP69細(xì)胞系,轉(zhuǎn)染36小時(shí)后,熒光顯微鏡下觀察NP69、pRNAT-U6-NP69和pRNAT-U6-shRNA-STGC3-NP69三組細(xì)胞的GFP表達(dá),pRNAT-U6-NP69和pRNAT-U6-shRNA-STGC3-NP69兩組細(xì)胞可見綠色熒光,而NP69細(xì)胞則無綠色熒光,結(jié)果表明空質(zhì)粒和重組質(zhì)粒均成功轉(zhuǎn)入NP69細(xì)胞系; RT-PCR檢測結(jié)果證實(shí)pRNAT-U6-shRNA-STGC3-NP69細(xì)胞中STGC3基因mRNA的表達(dá)水平明顯低于NP69和pRNAT-U6-NP69細(xì)胞,結(jié)果表明miR30-based shRNA成功地干擾了STGC3在NP69細(xì)胞系中的表達(dá)。細(xì)胞克隆形成實(shí)驗(yàn)結(jié)果顯示,pRNAT-U6-shRNA-STGC3-NP69較對照組克隆形成能力增強(qiáng)(p0.05);流式細(xì)胞儀檢測,pRNAT-U6-shRNA-STGC3-NP69組G_2期和S期的細(xì)胞增加,生長增殖速度增快,凋亡減少,與對照比較差異具有顯著性意義(p㩳0.05)。 結(jié)論 1.成功構(gòu)建了pRNAT-U6-shRNA-STGC3真核表達(dá)載體。 2. miRNA30-based shRNA可下調(diào)STGC3基因在NP69細(xì)胞系中的表達(dá)。 3. STGC3表達(dá)下調(diào),使NP69細(xì)胞系生長增殖速度加快,其機(jī)制可能與影響細(xì)胞周期和減少細(xì)胞凋亡有關(guān)。
[Abstract]:Aim: to design shRNA(miR30-based shRNAs targeting STGC3 gene, construct a specific shRNA eukaryotic expression vector for STGC3 gene, and transfect pRNAT-U6-shRNA-STGC3 into immortalized human nasopharynx epithelial cell line NP69, and observe the effect of decreased STGC3 gene expression on NP69 cell line. To analyze the role of STGC3 gene in cell growth and proliferation. Methods: the interference targets of STGC3 gene were analyzed and screened by bioinformatics. According to the miRNA30 frame structure, the pRNAT-U6-shRNA-STGC3 eukaryotic expression vector was constructed by designing the shRNAs targeting STGC3 gene. The recombinant pRNAT-U6-shRNA-STGC3 expression vector was transiently transfected into NP69 cell line by Lipofectamine 2000. The expression of GFP was observed under fluorescence microscope, the transfection efficiency was determined, the expression level of mRNA of STGC3 gene was detected by RT-PCR, the interference effect of miR30-based shRNA on STGC3 gene was evaluated, the transfected cells were expanded, and the cell clone formation experiment was carried out. The clone forming ability of pRNAT-U6-shRNA-STGC3- NP69 cells was observed. The cell cycle changes and apoptosis of pRNAT-U6-shRNA-STGC3- NP69 cells were analyzed by flow cytometry. Results the eukaryotic expression vector of pRNAT-U6-shRNA-STGC3 was successfully constructed by PCR, restriction endonuclease digestion and sequencing. The empty pRNAT-U6 plasmid and the pRNAT-U6-shRNA-STGC3 recombinant plasmid were transfected into NP69 cell line for 36 hours, respectively. The expression of GFP in three groups of NP69-pRNAT-U6-NP69 and pRNAT-U6-shRNA-STGC3-NP69 cells was observed under fluorescence microscope. Green fluorescence was observed in the cells of pRNAT-U6-NP69 and pRNAT-U6-shRNA-STGC3-NP69, but no green fluorescence was observed in NP69 cells. The results showed that both empty plasmid and recombinant plasmid were successfully transferred into NP69 cell line, and the expression level of STGC3 gene mRNA in pRNAT-U6-shRNA-STGC3-NP69 cells was significantly lower than that in NP69 and pRNAT-U6-NP69 cells. The results showed that miR30-based shRNA successfully interfered with the expression of STGC3 in NP69 cell line. The results of cell clone formation assay showed that pRNAT-U6-shRNA-STGC3-NP69 enhanced the ability of clone formation compared with the control group (p0.05G), and flow cytometry was used to detect the increase of pRNAT-U6-shRNA-STGC3-NP69 cells in G2 and S phases. The rate of growth and proliferation increased and the apoptosis decreased, and the difference was significant compared with the control. 0.05. Conclusion. 1. The eukaryotic expression vector of pRNAT-U6-shRNA-STGC3 was successfully constructed. 2. MiRNA30-based shRNA down-regulated the expression of STGC3 gene in NP69 cell line. 3. The down-regulation of STGC3 expression speeds up the growth and proliferation of NP69 cells, which may be related to the influence of cell cycle and the reduction of apoptosis.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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