【摘要】： EB病毒(Epstein-Barr virus,EBV)屬皰疹病毒γ亞科,是重要的DNA腫瘤病毒,與鼻咽癌(NPC)、Burkitt淋巴瘤(BL)、胃癌(GC)等多種腫瘤的發(fā)生有關(guān)。在EBV編碼基因中,潛伏膜蛋白1(Latent membrane protein 1,LMP1)編碼基因已被確認(rèn)具有癌基因的功能。研究表明,LMP1具有介導(dǎo)細(xì)胞增殖、抑制細(xì)胞凋亡和細(xì)胞分化,以及促進(jìn)腫瘤的侵襲和轉(zhuǎn)移等多種生物學(xué)活性。 siRNA是一種短片段雙鏈RNA分子,能以同源互補(bǔ)序列的mRNA為靶目標(biāo)降解特定的mRNA,從而高效、特異地阻斷體內(nèi)特定基因的表達(dá),誘使細(xì)胞表現(xiàn)出特定基因缺失表型。因此采用siRNA技術(shù)深入探討LMP1的生物活性對(duì)于揭示EBV致癌機(jī)制以及開展EBV相關(guān)腫瘤的特異性治療具有實(shí)際意義。 本研究選擇EBV陽性胃上皮細(xì)胞GT38作為靶細(xì)胞,采用人工合成的siRNA特異性阻斷LMP1的表達(dá),檢測(cè)LMP1沉默對(duì)GT38增殖和凋亡的影響,探討LMP1在EBV相關(guān)腫瘤發(fā)生發(fā)展中的分子機(jī)制,為EBV相關(guān)腫瘤的生物治療提供實(shí)驗(yàn)依據(jù)。 目的探討化學(xué)合成小干涉RNA(small interfering RNA,siRNA)對(duì)EBV陽性胃上皮細(xì)胞(GT38)LMP1編碼基因表達(dá)的抑制作用以及對(duì)細(xì)胞增殖和凋亡的影響。 方法①采用脂質(zhì)體法分別以20、30、50、80和100nM終濃度熒光標(biāo)記的陰性對(duì)照siRNA(FAM-siRNA)轉(zhuǎn)染GT38細(xì)胞,轉(zhuǎn)染12h內(nèi)用倒置熒光顯微鏡檢測(cè)轉(zhuǎn)染效率。②化學(xué)合成靶向LMP1 mRNA 649、979和1348位點(diǎn)的3對(duì)siRNA,以EBVⅢ型潛伏的胃上皮細(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)非特異性對(duì)照和細(xì)胞對(duì)照。采用RT-PCR和Western blotting檢測(cè)靶基因LMP1的轉(zhuǎn)錄表達(dá)水平,篩選出抑制效果最為理想的siRNA鏈并檢測(cè)對(duì)LMP1表達(dá)影響的時(shí)效關(guān)系。③選擇抑制效果最好的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)胞凋亡和周期的變化;Western blotting檢測(cè)LMP1沉默對(duì)GT38細(xì)胞、細(xì)胞漿和細(xì)胞核內(nèi)NFκB基因表達(dá)的影響;RT-PCR檢測(cè)對(duì)Bcl-2、Bax、MMP9、ICAM-1轉(zhuǎn)錄表達(dá)的影響。 結(jié)果①FAM-siRNA轉(zhuǎn)染GT38細(xì)胞后12h各濃度組在熒光顯微鏡下均可觀察到細(xì)胞內(nèi)有點(diǎn)狀綠色熒光出現(xiàn),表明轉(zhuǎn)染成功。siRNA終濃度為50,80和100nM時(shí)轉(zhuǎn)染效率較高,本實(shí)驗(yàn)選用50nM濃度siRNA轉(zhuǎn)染GT38細(xì)胞。②RT-PCR檢測(cè)結(jié)果表明,與轉(zhuǎn)染非特異性siRNA的細(xì)胞相比,siRNA649、siRNA979和siRNA1348對(duì)靶細(xì)胞LMP1的轉(zhuǎn)錄表達(dá)均具有抑制作用,差異均有統(tǒng)計(jì)學(xué)意義(F=235.99,P＜0.05);其中以siRNA649對(duì)LMP1轉(zhuǎn)錄表達(dá)的抑制作用最為明顯,轉(zhuǎn)染后24,48和72h時(shí)LMP1mRNA的轉(zhuǎn)錄水平均明顯低于細(xì)胞對(duì)照組(F=89.93,P＜0.05),而轉(zhuǎn)染后48和72h時(shí)LMP1mRNA的轉(zhuǎn)錄表達(dá)水平明顯低于轉(zhuǎn)染后24h時(shí)LMP1mRNA的轉(zhuǎn)錄表達(dá)水平。Western blotting結(jié)果顯示,與細(xì)胞對(duì)照比較,轉(zhuǎn)染siRNA649后48h未檢測(cè)到LMP1的表達(dá),轉(zhuǎn)染后72和96h時(shí)LMP1的表達(dá)明顯減弱。③Hochest 33258染色可見siRNA649作用后的GT38細(xì)胞發(fā)生凋亡,透射電鏡觀察到GT38細(xì)胞線粒體空泡化,染色質(zhì)固縮;而轉(zhuǎn)染非特異性對(duì)照siRNA的GT38細(xì)胞未觀察到上述改變。流式細(xì)胞分析顯示,與非特異性對(duì)照組和細(xì)胞對(duì)照組比較,siRNA649作用24,72以及120h后的細(xì)胞其細(xì)胞周期無明顯改變。④Western blotting結(jié)果顯示與細(xì)胞對(duì)照比較,siRNA649轉(zhuǎn)染后GT38細(xì)胞總蛋白和核蛋白中NFκB的表達(dá)水平降低,細(xì)胞漿蛋白中NFκB表達(dá)水平增高。⑤RT-PCR檢測(cè)Bcl-2、Bax、MMP9和ICAM-1 mRNA的轉(zhuǎn)錄表達(dá),電泳結(jié)果顯示與細(xì)胞對(duì)照比較,轉(zhuǎn)染后24,48和72h GT38細(xì)胞Bcl-2(F=39.83,P＜0.05)、MMP9(F=177.47,P＜0.05)、ICAM-1(F=467.69,P＜0.05)的轉(zhuǎn)錄表達(dá)明顯下調(diào),差別有統(tǒng)計(jì)學(xué)意義,對(duì)Bax的轉(zhuǎn)錄表達(dá)無明顯影響(F=0.75,P＞0.05),Bcl-2/Bax比值降低,差別有統(tǒng)計(jì)學(xué)意義(F=5.45,P＜0.05)。 結(jié)論化學(xué)合成siRNA能有效抑制GT38細(xì)胞中LMP1編碼基因的表達(dá),其抑制作用具有時(shí)間依賴性,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。LMP1特異性沉默可抑制NFκB的表達(dá)和核轉(zhuǎn)移,進(jìn)而下調(diào)Bcl-2/Bax、MMP9、ICAM-1等下游基因的表達(dá),最終導(dǎo)致靶細(xì)胞的凋亡。GT38細(xì)胞可用作研究LMP1生物活性及其在EBV相關(guān)腫瘤發(fā)生中所起作用的理想的靶細(xì)胞。
[Abstract]:Epstein-Barr virus (EBV) belongs to the herpesvirus gamma subfamily. It is an important DNA oncovirus. It is associated with nasopharyngeal carcinoma (NPC), Burkitt lymphoma (BL), gastric cancer (GC) and many other tumors. In the EBV coding gene, the latent membrane protein 1 (LMP1) coding gene has been confirmed to have the function of oncogene. It has many biological activities, such as mediating cell proliferation, inhibiting cell apoptosis and differentiation, and promoting tumor invasion and metastasis.
SiRNA is a short-fragment double-stranded RNA molecule, which can degrade specific mRNA with the target of homologous complementary sequences, thus effectively and specifically block the expression of specific genes in vivo and induce cells to show specific gene deletion phenotypes. Therefore, the biological activity of LMP1 is studied by siRNA technology to reveal the carcinogenic mechanism of EBV and to carry out EBV carcinogenesis. The specific treatment of V related tumors is of practical significance.
In this study, we selected EBV-positive gastric epithelial cells GT38 as target cells, and used synthetic siRNA to specifically block the expression of LMP1. We examined the effects of LMP1 silencing on the proliferation and apoptosis of GT38, and explored the molecular mechanism of LMP1 in the development of EBV-related tumors.
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of LMP1 gene in EBV-positive gastric epithelial cells (GT38) and its effect on cell proliferation and apoptosis.
Methods (1) GT38 cells were transfected with 20,30,50,80 and 100 nM fluorescent-labeled negative control siRNA (FAM-siRNA) by liposome method, and the transfection efficiency was detected by inverted fluorescence microscopy within 12 hours. 2) Three pairs of siRNA targeting at sites 649,979 and 1348 of LMP1 mRNA were synthesized by chemosynthesis, and GT38 cells were taken as target cells. Three groups of siRNA were transfected into GT38 cells by liposome method, and non-specific control and cell control were set up. The target gene LMP1 was detected by RT-PCR and Western blotting. The siRNA chains with the best inhibitory effect were screened and the time-dependent relationship of LMP1 expression was detected. 3. The siRNA chains with the best inhibitory effect were selected. Hoechst 33258 was used to detect the apoptosis and cell cycle of GT38 cells; Western blotting was used to detect the effect of LMP1 silencing on the expression of NF-kappa B gene in GT38 cells, cytoplasm and nucleus; RT-PCR was used to detect the effect of Bcl-2, Bax, MMP9, ICAM-1 transcription and expression.
Results: (1) The dot-like green fluorescence was observed in all concentration groups 12 hours after transfection of FAM-siRNA into GT38 cells, indicating that the transfection was successful. The transfection efficiency was high when the final concentration of siRNA was 50,80 and 100 nM. The 50 nM concentration of siRNA was selected to transfect GT38 cells in this experiment. The results of RT-PCR showed that the transfection was successful. Compared with the control cells, siRNA 649, siRNA 979 and siRNA 1348 all inhibited the transcriptional expression of LMP1, and the difference was statistically significant (F = 235.99, P < 0.05); among them, siRNA 649 inhibited the transcriptional expression of LMP1 most significantly, and the transcriptional level of LMP1 mRNA at 24, 48 and 72 hours after transfection was significantly lower than that of the control group (F = 89.93, P < 0.05). The expression of LMP1 mRNA at 48 and 72 hours after transfection was significantly lower than that at 24 hours after transfection. Western blotting showed that LMP1 expression was not detected at 48 hours after transfection, but decreased significantly at 72 and 96 hours after transfection. Mitochondrial vacuolation and chromatin condensation were observed in GT38 cells after apoptosis, but not in GT38 cells transfected with non-specific siRNA. Flow cytometry analysis showed that the cell cycle of GT38 cells treated with siRNA 649 at 24, 72 and 120 h was not observed in non-specific control group and cell control group. Western blotting showed that the expression of NF-kappa B in the total protein and nucleoprotein of GT38 cells decreased and the expression of NF-kappa B in cytoplasm protein increased after transfection with siRNA 649. _The transcriptional expression of Bcl-2, Bax, MMP9 and ICAM-1 mRNA was detected by RT-PCR, and the electrophoresis results showed that compared with the cell control, the expression of NF-kappa B in the transfected GT38 cells decreased and the expression of NF-kappa B in the cytoplasm protein increased. Bcl-2 (F = 39.83, P < 0.05), MMP-9 (F = 177.47, P < 0.05), ICAM-1 (F = 467.69, P < 0.05) were significantly down-regulated, and the difference was statistically significant. There was no significant effect on the transcriptional expression of Bax (F = 0.75, P > 0.05), and the ratio of Bcl-2 to Bax was decreased (F = 5.45, P < 0.05).
Conclusion Chemically synthesized siRNA can effectively inhibit the expression of LMP1-encoded gene in GT38 cells. The inhibitory effect is time-dependent and dose-dependent in a certain concentration range. Specific silencing of LMP1 can inhibit the expression and nuclear transfer of NF-kappa B and then down-regulate Bcl-2/Ba. The expression of downstream genes such as x, MMP9 and ICAM-1 eventually leads to the apoptosis of target cells. GT38 cells can be used to study the biological activity of LMP1 and its role in EBV-related tumorigenesis.
【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】：2008
【分類號(hào)】：R373

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