發(fā)布時(shí)間：2018-05-26 13:56

  本文選題：EB病毒 + 潛伏膜蛋白1��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:探討LMP1特異性表達(dá)沉默對(duì)EBV陽(yáng)性鼻咽癌細(xì)胞系C666-1細(xì)胞增殖、侵襲轉(zhuǎn)移能力的影響;以及EBV編碼基因LMP1對(duì)lncRNA H19基因啟動(dòng)子區(qū)甲基化和表達(dá)的影響。方法:①采用慢病毒感染法分別以MOI值為1,10,100綠色熒光標(biāo)記的陰性對(duì)照病毒sgRNA(U6-SV40-EGFP)接種至C666-1細(xì)胞,并分別將4種感染條件加至3種MOI值中,感染72h后采用倒置熒光顯微鏡觀察感染效率。②體外培養(yǎng)鼻咽癌C666-1細(xì)胞,加入梯度濃度的嘌呤霉素溶液篩選,確定最佳篩選濃度。③設(shè)計(jì)并合成2種針對(duì)LMP1基因的sgRNA(LV-LMP1-413,LV-LMP1-414)及Cas9嘌呤霉素抗性的慢病毒顆粒。在預(yù)實(shí)驗(yàn)確認(rèn)的MOI值和最佳感染條件下,將攜帶嘌呤霉素標(biāo)記的Cas9慢病毒顆粒接種至C666-1細(xì)胞中,采用預(yù)實(shí)驗(yàn)確認(rèn)的嘌呤霉素濃度篩選出穩(wěn)定表達(dá)Cas9的細(xì)胞株,然后將攜帶綠色熒光蛋白與sgRNA(LV-LMP1-413,LV-LMP1-414)的慢病毒分別感染至Cas9蛋白穩(wěn)轉(zhuǎn)C666-1細(xì)胞,同時(shí)設(shè)病毒對(duì)照和細(xì)胞對(duì)照;采用有限稀釋法篩選出同時(shí)攜帶綠色熒光和嘌呤霉素抗性的單克隆細(xì)胞,擴(kuò)大培養(yǎng)并建立穩(wěn)定細(xì)胞系。④采用Western blotting檢測(cè)靶基因LMP1沉默前后蛋白水平的變化;CCK8細(xì)胞增殖實(shí)驗(yàn)和transwell小室檢測(cè)LMP1基因沉默前后對(duì)C666-1細(xì)胞增殖及侵襲轉(zhuǎn)移能力的影響;亞硫酸鹽基因組測(cè)序法(bisulfate genomic sequencing,BGS)檢測(cè) LMP1 沉默前后 C666-1 細(xì)胞系中 lncRNA H19 動(dòng)子區(qū)甲基化程度;實(shí)時(shí)熒光定量 PCR(Real time fluorescent quantitative PCR,real time qPCR)檢測(cè)LMP1沉默前后C666-1細(xì)胞系中l(wèi)ncRNAH19的轉(zhuǎn)錄表達(dá)。結(jié)果:①對(duì)照病毒轉(zhuǎn)染C666-1細(xì)胞后,72h在倒置熒光顯微鏡下觀察到細(xì)胞內(nèi)有點(diǎn)狀綠色熒光出現(xiàn),感染效率高于80%,且細(xì)胞生長(zhǎng)良好,表明轉(zhuǎn)染成功。本實(shí)驗(yàn)選用MOI值為100和最佳感染條件(polybrene和ENi.S.液)進(jìn)行Cas9蛋白和sgRNA轉(zhuǎn)染C666-1細(xì)胞。②經(jīng)篩選確認(rèn)嘌呤霉素最佳使用濃度為1μg/ml。③成功篩選出同時(shí)攜帶綠色熒光和嘌呤霉素抗性的C666-1細(xì)胞克隆。Western blotting結(jié)果顯示,與陰性病毒對(duì)照及細(xì)胞對(duì)照相比,LMP1沉默后C666-1細(xì)胞中LMP1表達(dá)明顯減弱。④與陰性對(duì)照及細(xì)胞對(duì)照比較,LMP1基因沉默后C666-1細(xì)胞增殖、侵襲、遷移能力明顯減弱(P0.05)。⑤BGS結(jié)果顯示LMP1沉默后C666-1細(xì)胞系平均甲基化率(Cas9-EGFP-LMP1-413-C666-1,89.9%,Cas9-EGFP-LMP1-414-C666-1,87.4%);陰性病毒對(duì)照及細(xì)胞對(duì)照平均甲基化率分別為(81.8%,84.8%),統(tǒng)計(jì)學(xué)分析顯示LMP1沉默前后C666-1細(xì)胞系中H19基因啟動(dòng)子甲基化水平無(wú)顯著性差異(P0.05)。⑥LMP1沉默前后C666-1細(xì)胞系lncRNA H19的轉(zhuǎn)錄表達(dá)無(wú)顯著性差異(P0.05)。結(jié)論:①成功構(gòu)建靶向敲減EBV潛伏期基因LMP1的CRISPR/Cas9雙載體慢病毒,并篩選出穩(wěn)定感染的細(xì)胞克隆,初步實(shí)驗(yàn)結(jié)果表明慢病毒轉(zhuǎn)染能有效沉默靶基因LMP1的表達(dá)。②EBV潛伏期基因LMP1表達(dá)沉默能抑制EBV陽(yáng)性鼻咽癌C666-1細(xì)胞增殖、侵襲遷移能力。③LMP1表達(dá)沉默對(duì)lncRNA H19啟動(dòng)子區(qū)甲基化及轉(zhuǎn)錄表達(dá)無(wú)明顯影響,提示尚有其他因素參與EBV對(duì)lncRNA H19甲基化和表達(dá)的調(diào)控作用。
[Abstract]:Objective: To investigate the effect of LMP1 specific expression silencing on the proliferation, invasion and metastasis of EBV positive nasopharyngeal carcinoma cell line C666-1 cells, and the effect of EBV encoding gene LMP1 on the methylation and expression of lncRNA H19 promoter region. GRNA (U6-SV40-EGFP) was inoculated to C666-1 cells, and 4 kinds of infection conditions were added to 3 MOI values. Infection efficiency was observed by inverted fluorescence microscope after 72h infection. (2) C666-1 cells of nasopharyngeal carcinoma were cultured in vitro, and the optimum screening concentration was selected by adding a gradient concentration of purinomycin solution. (3) 2 kinds of sgRN for LMP1 gene were designed and synthesized. A (LV-LMP1-413, LV-LMP1-414) and Cas9 purinomycin resistant lentivirus particles. Under the pre confirmed MOI value and the optimal infection condition, the Cas9 lentivirus particles labeled with purinomycin were inoculated into C666-1 cells, and the cell lines that stably expressed Cas9 were screened by the preconfirmed concentration of purinomycin, and then the Cas9 was carried green. The lentivirus of fluorescent protein and sgRNA (LV-LMP1-413, LV-LMP1-414) infected with Cas9 protein to stabilize C666-1 cells, and also set the virus control and cell control. The monoclonal cells carrying both green fluorescence and purinamycin resistance were screened by the finite dilution method, and Kuo Dapei cultured and established the stable cell line. (4) the Western blotting examination was used. The changes in the protein level of the target gene LMP1 before and after the silencing of the target gene, the effect of the proliferation and invasion and metastasis of C666-1 cells before and after the LMP1 gene silencing by the Transwell cell test and the CCK8 cell proliferation test, and the assay of bisulfate genomic sequencing, BGS to detect the lncRNA zone in the C666-1 cell line of LMP1 before and after the silencing of LMP1 Real-time fluorescence quantitative PCR (Real time fluorescent quantitative PCR, real time qPCR) was used to detect the transcriptional expression of lncRNAH19 in C666-1 cell lines before and after the silencing of LMP1. Results: (1) after transfection of the virus to the cells, the appearance of a dot like green fluorescence in the cell was observed under the inverted fluorescence microscope, and the infection efficiency was higher than 80% The cell growth was good and the transfection was successful. The experiment selected the MOI value of 100 and the best infection condition (Polybrene and ENi.S. liquid) for Cas9 protein and sgRNA transfected C666-1 cells. 2. The optimal use concentration of purinamycin was 1 mu g/ml. (3), and a successful screening of C666-1 cell clone carrying green fluorescence and purinamycin resistance was successfully screened. The results of.Western blotting showed that the LMP1 expression in C666-1 cells was significantly weakened after LMP1 silencing with negative viruses and cell pairs. 4. Compared with negative control and cell control, the proliferation, invasion, and migration ability of C666-1 cells decreased significantly after LMP1 gene silencing (P0.05). 5. BGS results showed that C666-1 cell line methylated by LMP1 after the LMP1 was silent. The rate of Cas9-EGFP-LMP1-413-C666-1,89.9% (Cas9-EGFP-LMP1-414-C666-1,87.4%) and the average methylation rate of negative virus control and cell control were respectively (81.8%, 84.8%). Statistical analysis showed that there was no significant difference in the level of H19 gene promoter methylation in C666-1 cell lines before and after LMP1 silence (P0.05). 6. C666-1 cell line lncRN before and after LMP1 silencing. There was no significant difference in the transcriptional expression of A H19 (P0.05). Conclusion: (1) a successful construction of a CRISPR/Cas9 double vector lentivirus targeting the target EBV latency gene LMP1 was successfully constructed and a stable infection cell clone was screened. The preliminary experimental results showed that the gene expression of the target gene LMP1 could be silenced effectively by the transfection of the lentivirus. (2) the LMP1 expression silencing of the EBV latency gene could inhibit E. BV positive nasopharyngeal carcinoma C666-1 cell proliferation and invasion and migration. LMP1 expression silencing has no obvious effect on the methylation and transcriptional expression of lncRNA H19 promoter region, suggesting that there are other factors involved in the regulatory role of EBV on the methylation and expression of lncRNA H19.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373

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本文編號(hào)：1937473