本文關(guān)鍵詞：應(yīng)用siRNA特異沉默EBV潛伏期基因EBNA2表達(dá)的初步研究　出處：《青島大學(xué)》2007年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 小干涉RNA EB病毒核抗2 GT38 pSUPER逆轉(zhuǎn)錄病毒載體 小發(fā)夾RNA PA317 Epsetin-Barr病毒

【摘要】： EBNA2是EBV感染宿主細(xì)胞后最先表達(dá)的病毒基因產(chǎn)物之一，由BYRF1基因編碼，與細(xì)胞內(nèi)蛋白無(wú)任何明顯的同源序列。EBNA2是一種病毒轉(zhuǎn)錄因子，不能直接與DNA結(jié)合，但可通過(guò)與細(xì)胞抑制重組信號(hào)結(jié)合蛋白R(shí)BP-Jκ及其它一些胞內(nèi)DNA結(jié)合蛋白的結(jié)合間接作用于EBNA2反應(yīng)啟動(dòng)子。EBNA2具有核定位序列、DNA結(jié)合部位及轉(zhuǎn)錄激活部位等功能區(qū)域，是首個(gè)被確定與病毒細(xì)胞轉(zhuǎn)化功能有密切關(guān)系的病毒蛋白。EBNA2缺失的EBV毒株P(guān)3-HR1失去轉(zhuǎn)化B細(xì)胞的能力，提示我們可以通過(guò)干擾EBNA2的表達(dá)影響其轉(zhuǎn)化細(xì)胞的能力。同時(shí)作為EBV BYRF1基因特異性表達(dá)的產(chǎn)物，EBNA2可以反映EBV的存在情況以及在細(xì)胞內(nèi)的拷貝數(shù)。 RNAi是由雙鏈RNA介導(dǎo)的遺傳干擾現(xiàn)象，能夠特異、有效地降解mRNA，引起轉(zhuǎn)錄后水平的基因沉默；研究表明，21-23nt的小干擾RNA(small interferenceRNA，siRNA)可介導(dǎo)哺乳動(dòng)物細(xì)胞特定基因的沉默。RNAi具有高效性和高度特異性，可能成為封閉基因的新技術(shù)而在基因功能研究和疾病基因治療中發(fā)揮重要作用。大量研究結(jié)果證實(shí)，siRNA可以有效抑制所有內(nèi)源性基因的mRNA從而敲除該基因的功能，目前siRNA已經(jīng)成為敲除特定基因在哺乳動(dòng)物細(xì)胞系中表達(dá)的強(qiáng)大工具。 本研究中我們化學(xué)合成了靶向EBNA2編碼基因的特異性siRNA，采用脂質(zhì)體法轉(zhuǎn)染EBVⅢ型潛伏的胃癌上皮細(xì)胞GT38，觀(guān)察分析siRNA對(duì)EBNA2基因表達(dá)的抑制作用以及靶基因沉默對(duì)GT38細(xì)胞的影響。同時(shí)構(gòu)建能轉(zhuǎn)錄產(chǎn)生靶向EBNA2編碼基因的siRNA的pSUPER逆轉(zhuǎn)錄病毒載體，進(jìn)而篩選出穩(wěn)定產(chǎn)毒的細(xì)胞克隆，以觀(guān)察比較兩種干涉方式對(duì)目的基因表達(dá)的抑制效果以及對(duì)靶細(xì)胞的影響。 第一部分化學(xué)合成siRNA對(duì)GT38細(xì)胞EBNA2基因表達(dá)的抑制作用 目的采用化學(xué)合成法合成針對(duì)EBNA2編碼基因的siRNA，分別轉(zhuǎn)染EBV陽(yáng)性腫瘤細(xì)胞GT38細(xì)胞，觀(guān)察人工合成siRNA對(duì)EBNA2的特異沉默效果以及EBNA2特異性沉默對(duì)EBV陽(yáng)性腫瘤細(xì)胞的影響。 方法①化學(xué)合成3組靶向EBNA2編碼基因的siRNA，以EBVⅢ型潛伏的胃癌上皮細(xì)胞GT38為靶細(xì)胞，采用脂質(zhì)體法分別將3組siRNA轉(zhuǎn)染GT38細(xì)胞，同時(shí)設(shè)脂質(zhì)體對(duì)照和細(xì)胞對(duì)照。②采用RT-PCR檢測(cè)靶基因EBNA2 mRNA的轉(zhuǎn)錄表達(dá)水平，并篩選出最佳轉(zhuǎn)染濃度及抑制效果最為理想的siRNA鏈。③將抑制效果最好的siRNA鏈以最佳轉(zhuǎn)染濃度轉(zhuǎn)染靶細(xì)胞，采用Hoechst 33258、透射電鏡和流式細(xì)胞術(shù)檢測(cè)GT38細(xì)胞周期和凋亡的變化。 結(jié)果①RT-PCR檢測(cè)結(jié)果表明，與未轉(zhuǎn)染siRNA的細(xì)胞對(duì)照相比，siRNA789、siRNA764和siRNA257對(duì)GT38細(xì)胞EBNA2的表達(dá)均具有明顯的抑制作用(P＜0.01)，其中以siRNA789的抑制作用更強(qiáng)；②與未轉(zhuǎn)染的細(xì)胞對(duì)照比較，轉(zhuǎn)染siRNA789后48h和72h GT38細(xì)胞中EBNA2 mRNA表達(dá)水平明顯下降，兩兩間比較均有顯著性差異(P＜0.01)，而siRNA789轉(zhuǎn)染后24h抑制效果不明顯(P＞0.05)；③在0～50nM濃度范圍內(nèi)siRNA789轉(zhuǎn)染72h后EBNA2 mRNA轉(zhuǎn)錄表達(dá)水平隨濃度增加而逐漸降低，差別均有統(tǒng)計(jì)學(xué)意義(P＜0.05)；當(dāng)siRNA作用濃度為50nM～100nM siRNA各組間差別不明顯(P＞0.05)，轉(zhuǎn)染非特異siRNA細(xì)胞組與細(xì)胞對(duì)照比較無(wú)顯著性差異(P＞0.05)；④Hoechst 33258染色和透射電鏡結(jié)果顯示，與對(duì)照組比較，，實(shí)驗(yàn)組GT38細(xì)胞未見(jiàn)胞核濃縮，也未觀(guān)察到凋亡小體；流式細(xì)胞分析結(jié)果表明，與對(duì)照組比較，實(shí)驗(yàn)組細(xì)胞未出現(xiàn)凋亡，但其S其細(xì)胞數(shù)量明顯減少。 結(jié)論化學(xué)合成siRNA能有效抑制GT38細(xì)胞中EBNA2編碼基因的表達(dá)，其抑制作用具有時(shí)間依賴(lài)性，在一定濃度范圍內(nèi)呈明顯的量效關(guān)系，且對(duì)靶基因的特定序列具有較強(qiáng)的選擇偏向性�；瘜W(xué)合成siRNA誘導(dǎo)EBNA2編碼基因沉默后，不能引起靶細(xì)胞明顯的凋亡，但可引起靶細(xì)胞S期數(shù)量減少。 第二部分特異性沉默BV潛伏期基因EBNA2 pSUPER retro RNAi系統(tǒng)的構(gòu)建 目的構(gòu)建能轉(zhuǎn)錄產(chǎn)生靶向EBV核抗原EBNA2編碼基因siRNA的pSUPER逆轉(zhuǎn)錄病毒載體，并篩選出穩(wěn)定產(chǎn)毒的細(xì)胞克隆。 方法采用DNA重組技術(shù)將60nt能轉(zhuǎn)錄產(chǎn)生靶向EBNA2小發(fā)夾RNA(small hairpin RNA，shRNA)的寡核苷酸序列定向克隆入逆轉(zhuǎn)錄病毒載體pSUPER.retro.neo gfp，并用限制性?xún)?nèi)切酶酶切鑒定以及測(cè)序鑒定；脂質(zhì)體法將重組逆轉(zhuǎn)錄病毒載體轉(zhuǎn)染包裝細(xì)胞系PA317，G418篩選獲得穩(wěn)定產(chǎn)生逆轉(zhuǎn)錄病毒的細(xì)胞克隆。 結(jié)果酶切鑒定及測(cè)序鑒定結(jié)果表明插入片段的序列和方向完全正確；重組載體轉(zhuǎn)染包裝細(xì)胞后可表達(dá)綠色熒光蛋白，經(jīng)G418篩選獲得可穩(wěn)定產(chǎn)生逆轉(zhuǎn)錄病毒的抗性細(xì)胞克�。徊《镜味葹�2.5×10~5 CFU／ml。 結(jié)論成功構(gòu)建了能轉(zhuǎn)錄產(chǎn)生靶向EBV核抗原EBNA2編碼基因siRNA的pSUPER逆轉(zhuǎn)錄病毒載體，經(jīng)PA317細(xì)胞包裝篩選出穩(wěn)定產(chǎn)毒的細(xì)胞克隆，為進(jìn)一步探討EBNA2在EBV相關(guān)腫瘤發(fā)生發(fā)展的生物學(xué)意義提供了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:EBNA2 is one of the viral gene product EBV infection of host cells after the first expression, encoding by BYRF1 gene, no obvious homology.EBNA2 is a viral transcription factor and intracellular protein, not directly combined with DNA, but the RBP-J kappa binding protein and other intracellular DNA binding protein with an indirect effect on the reaction of EBNA2.EBNA2 promoter with nuclear localization sequence and cell inhibition of recombinant signal, DNA binding site and transcription activation site and other functional areas, EBV is the first P3-HR1 strain of virus protein was identified and transformed virus cell function have close relationship to the deletion of the.EBNA2 lost the ability to transform B cells, suggesting that we can influence the ability of transformed cells by the expression of EBNA2 interference. At the same time as the product of EBV BYRF1 gene specific expression, EBNA2 can reflect the existence of EBV and the copy number within the cell.
RNAi is the phenomenon of genetic interference mediated by double stranded RNA can specifically and efficiently degrade mRNA, cause post transcriptional gene silencing; studies show that small interfering RNA 21-23nt (small interferenceRNA siRNA) can mediate specific gene silencing in mammalian cells.RNAi with high efficiency and high specificity, may be the new technology of closed gene and gene in the gene function study and treatment of diseases play an important role. A large number of research results confirmed that siRNA can effectively inhibit all endogenous gene mRNA to knockdown the gene function, the siRNA has become a powerful tool for knockout specific gene expression in mammalian cell lines.
In this study we chemically synthesized specific siRNA EBNA2 encoding gene, gastric epithelial cells GT38 transfected with EBV type III latent by liposome method, observation and analysis of the inhibition effect of siRNA on the expression of EBNA2 gene and the target gene silencing on GT38 cells. At the same time to construct encoded targeting pSUPER retroviral vector siRNA the EBNA2 encoding gene, and then screened stable cell clones producing toxin, to observe the inhibitory effect of two kinds of interference on the expression of target gene and effect on target cells.
The inhibitory effect of chemical synthesis of siRNA on the expression of EBNA2 gene in GT38 cells
Objective to synthesize siRNA targeting EBNA2 coding gene by chemical synthesis, and transfect EBV positive tumor cells into GT38 cells. Observe the effect of synthetic siRNA on EBNA2 and the effect of EBNA2 specific silence on EBV positive tumor cells.
The siRNA method of chemical synthesis of 3 groups of target gene encoding EBNA2, gastric epithelial cells GT38 with EBV type III latent target cells by liposome method. The 3 groups of siRNA were transfected into GT38 cells, and liposome control and control cells. The expression level of the transcription of RT-PCR target gene EBNA2 and mRNA. Select the most ideal siRNA chain for the best transfection concentration and inhibition effect. The best inhibitory effect of siRNA chain with the best transfection concentration of transfected target cells by Hoechst 33258, the change of GT38 cell cycle and apoptosis was detected by electron microscopy and flow cytometry.
Results the results of RT-PCR showed that siRNA789 and siRNA transfected cells compared to the control, the expression of siRNA764 and siRNA257 on GT38 EBNA2 cells were significantly inhibited (P < 0.01), the stronger the inhibition of siRNA789; and the non transfected cells compared with EBNA2 48h and 72h GT38 cells the expression level of mRNA was significantly decreased after transfection of siRNA789, there were significant differences between the 22 (P < 0.01), and siRNA789 24h after transfection had no obvious inhibition effects (P > 0.05); in 0 ~ 50nM in the concentration range of siRNA789 after transfection of 72h EBNA2 mRNA mRNA expression level decreased gradually with the increase of concentration difference statistical significance (P < 0.05); when the concentration of siRNA 50nM ~ 100nM siRNA no significant differences between groups (P > 0.05), non transfection of specific siRNA cells and cell control group showed no significant difference (P > 0.05); Hoechst 33258 The results of staining and transmission electron microscopy showed that compared with the control group, the GT38 cells in the experimental group did not concentrate on the nucleus and no apoptotic bodies were observed. Flow cytometry analysis showed that compared with the control group, the cells in the experimental group did not show apoptosis, but the number of S cells in the experimental group was significantly reduced.
Conclusion the chemical synthesis of siRNA can effectively inhibit the expression of EBNA2 encoding gene in GT38 cells, the inhibition is time dependent, was in a dose-dependent manner within a certain concentration range, and the specific sequence of target gene with strong selection bias. The chemical synthesis of siRNA induced by EBNA2 encoding gene silencing, can induce apoptosis of target cells, but can cause the number of target cells in S phase decreased.
Construction of the second part specific silent BV latency gene EBNA2 pSUPER retro RNAi system
Objective to construct a pSUPER retrovirus vector capable of transcriptional production of the target EBV nuclear antigen EBNA2 encoding gene siRNA, and to screen out the stable and toxic cell clones.
Methods the 60nt encoded targeting EBNA2 small hairpin RNA using recombinant DNA Technology (small hairpin RNA, shRNA) oligonucleotide sequences cloned into the retroviral vector pSUPER.retro.neo GFP and restriction enzyme digestion and sequencing; Lipofectamine recombinant retroviral vector was transfected into packaging cell line PA317 cell clone G418 stably produce retrovirus.
The results of enzyme digestion and sequencing showed that the sequence and direction of insert completely correct; green fluorescent protein expression recombinant vector was transfected into packaging cell, can obtain clones stably produce retrovirus by G418 screening; virus titer was 2.5 * 10~5 / ml. CFU
Conclusion we have successfully constructed the encoded targeting pSUPER retroviral vector EBNA2 encoding gene siRNA EBV nuclear antigen, PA317 cells were screened by packaging cell clones stably produce poison, it provides the experimental basis for further study of the biological significance of EBNA2 in EBV is related to the occurrence and development of tumor.

【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】：2007
【分類(lèi)號(hào)】：R373

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張麗;Galectin-3、CD44V6、LMP-1、EBNA-2在濾泡上皮起源甲狀腺腫瘤中的表達(dá)及意義[D];新疆醫(yī)科大學(xué);2009年

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