【摘要】：EB病毒(Epstein-Barr virus,EBV)是皰疹病毒屬γ皰疹病毒亞科成員,是重要的DNA腫瘤病毒。EBV感染與部分胃癌的關(guān)系已經(jīng)得到確認(rèn),但EBV相關(guān)胃癌(EBV-associated gastric carcinoma,EBVa GC)的發(fā)病機(jī)制尚未完全明了。近來研究表明EBV可能通過誘導(dǎo)某些抑癌基因啟動(dòng)子區(qū)Cp G島高甲基化而使該基因功能失活參與胃癌的發(fā)生發(fā)展。目的:明確EBV陽性和陰性胃癌細(xì)胞系以及EBVaGC和EBV陰性胃癌(EBV-negatived gastric carcinoma,EBVn GC)組織中RASSF1A基因啟動(dòng)子區(qū)甲基化狀態(tài)以及對(duì)其表達(dá)的影響,為進(jìn)一步明確Ras相關(guān)結(jié)構(gòu)域家族基因1A(ras associated domain family gene1A,RASSF1A)基因在EBVaGC發(fā)生中的作用及作用機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。方法:采用甲基化特異性PCR(Methylation-Specific PCR,MSP)和基于焦亞硫酸氫鹽修飾的基因組測(cè)序法(Bisulfite genomic sequencing,BGS)檢測(cè)5-氮雜-2'-脫氧胞苷(5-Aza-2’deoxycytidine,5-Aza-dc)處理前后8種胃癌細(xì)胞系以及胃癌組織中RASSF1A基因啟動(dòng)子區(qū)甲基化狀態(tài);實(shí)時(shí)熒光定量PCR(Real time quantitative PCR,real time q PCR)檢測(cè)EBV陰性及陽性細(xì)胞系中RASSF1Am RNA的轉(zhuǎn)錄表達(dá);免疫組化技術(shù)檢測(cè)EBVa GC和EBVn GC組織中RASSF1A蛋白表達(dá)。結(jié)果:(1)MSP檢測(cè)結(jié)果顯示4種EBV陽性胃癌細(xì)胞系(GT38,GT39,SNU719,AGS-EBV)RASSF1A基因啟動(dòng)子區(qū)呈未甲基化狀態(tài),電泳結(jié)果為U型,4種EBV陰性胃癌細(xì)胞系(HGC-27,MKN-45,SGC7901,AGS)RASSF1A基因啟動(dòng)子區(qū)呈高甲基化狀態(tài),電泳結(jié)果為M型。BGS檢測(cè)RASSF1A基因啟動(dòng)子區(qū)的86個(gè)Cp G位點(diǎn)甲基化狀態(tài),結(jié)果顯示:4種EBV陽性胃癌細(xì)胞系除少量位點(diǎn)發(fā)生甲基化外,其余位點(diǎn)均未發(fā)生甲基化,甲基化率均小于10%;而4種EBV陰性胃癌細(xì)胞系則表現(xiàn)為幾乎所有位點(diǎn)的甲基化,甲基化率均達(dá)90%以上。(2)MSP檢測(cè)結(jié)果顯示:36例EBVa GC組織中有28例為M型(28/36,77.8%),M+U型為8例(8/36,22.2%);41例EBVn GC組織中U型有33例(33/41,80.5%),M+U型為8例(8/41,19.5%)。BGS結(jié)果顯示:36例EBVa GC平均甲基化率達(dá)86%以上;41例EBVn GC平均甲基化率為22.3%。(3)經(jīng)去甲基化試劑5-Aza-dc處理后,4種EBV陰性細(xì)胞系MSP電泳結(jié)果均發(fā)生變化,由原來的M型轉(zhuǎn)變成M+U型;而4種EBV陽性細(xì)胞系MSP電泳結(jié)果仍為U型。(4)EBV陽性胃癌細(xì)胞系RASSF1A基因的轉(zhuǎn)錄表達(dá)水平均高于陰性胃癌細(xì)胞系。5-Aza-dc作用后,EBV陰性胃癌細(xì)胞RASSF1A基因的轉(zhuǎn)錄表達(dá)水平明顯升高,EBV陽性胃癌細(xì)胞系則無明顯變化。(5)EBVa GC和EBVn GC組織中RASSF1A蛋白表達(dá)在EBVa GC和EBVn GC組織中無顯著差異(P0.05)。結(jié)論:(1)EBV陽性和EBV陰性胃癌細(xì)胞系中RASSF1A基因啟動(dòng)子區(qū)甲基化狀態(tài)不同,提示EBV感染可導(dǎo)致RASSF1A基因啟動(dòng)子區(qū)甲基化狀態(tài)發(fā)生改變。(2)RASSF1A基因啟動(dòng)子區(qū)的高甲基化可抑制該基因的轉(zhuǎn)錄表達(dá);去甲基化試劑5-Aza-dc處理可使RASSF1A基因啟動(dòng)子區(qū)去甲基化,使得該基因表達(dá)水平升高。(3)EBVa GC和EBVn GC中組織中RASSF1A基因啟動(dòng)子區(qū)甲基化狀態(tài)不同,提示EBV感染可能是導(dǎo)致RASSF1A基因啟動(dòng)子區(qū)域甲基化的原因;但其與EBV陽性細(xì)胞系RASSF1A基因啟動(dòng)子區(qū)低甲基化現(xiàn)象相左的結(jié)果需要我們進(jìn)一步深入研究加以證實(shí)。
[Abstract]:EB virus (Epstein-Barr virus, EBV) is a member of the subfamily of herpes simplex virus (HSV), an important DNA tumor virus.EBV infection and some gastric cancer, but the pathogenesis of EBV related gastric cancer (EBV-associated gastric carcinoma, EBVa GC) has not been fully understood. The methylation of the Cp G island of the promoter region of the oncogene makes the gene function inactivation to participate in the development of gastric cancer. Objective: to clarify the methylation status of the promoter region of the EBV positive and negative gastric cancer cell lines and the EBVaGC and EBV negative gastric carcinoma (EBV-negatived gastric carcinoma, EBVn GC) and the effect on its expression. The Ras related domain family gene 1A (RAS associated domain family gene1A, RASSF1A) gene provides an experimental basis for the role and mechanism of the gene in the occurrence of EBVaGC. G, BGS) detected the methylation status of the RASSF1A gene promoter region of 8 gastric cancer cell lines and gastric cancer tissues before and after the treatment of 5- heterozygous -2'- deoxycytidine (5-Aza-2 'deoxycytidine, 5-Aza-dc), and the real-time fluorescent quantitative PCR (Real time quantitative) was used to detect the transcription of the negative and positive cell lines. The expression of RASSF1A protein in the tissues of EBVa GC and EBVn GC was detected by histochemical technique. Results: (1) the results of MSP detection showed that the promoter region of the 4 EBV positive gastric cancer cell lines (GT38, GT39, SNU719, AGS-EBV) was not methylation state, and the electrophoresis results were type, 4 kinds of negative gastric cancer cell lines were in the promoter region. The methylation status of the 86 Cp G loci in the promoter region of the RASSF1A gene was detected by M type.BGS. The results showed that the 4 EBV positive gastric cancer cell lines had no methylation and the methylation rate was less than 10% except for a small number of loci methylation, while the 4 EBV negative gastric cancer cell lines were almost all sites. Methylation and methylation rates were above 90%. (2) MSP detection results showed that 28 of the 36 EBVa GC tissues were M (28/36,77.8%), M+U in 8 (8/36,22.2%), 33 in EBVn GC tissue in 41 cases, and 8 for M+U type. After the rate of 22.3%. (3) was treated with the demethylation reagent 5-Aza-dc, the results of the MSP electrophoresis of the 4 EBV negative cell lines were all changed, from the original M type to the M+U type, while the MSP electrophoresis results of the 4 EBV positive cell lines were still U type. (4) the RASSF1A gene expression level of the EBV positive gastric cancer cell lines was higher than that of the negative gastric cancer cell line.5-Aza-dc. After use, the transcriptional expression level of RASSF1A gene in EBV negative gastric cancer cells was significantly increased, and there was no significant change in EBV positive gastric cancer cell lines. (5) there was no significant difference in the expression of RASSF1A protein in EBVa GC and EBVn GC tissues in EBVa GC and EBVn GC tissues. (1) the promoter region of the gene promoter region was methylated in the positive and negative gastric cancer cell lines. It is suggested that EBV infection can lead to a change in the methylation status of the promoter region of the RASSF1A gene. (2) the hypermethylation of the promoter region of the RASSF1A gene inhibits the transcriptional expression of the gene, and the demethylation agent 5-Aza-dc treatment can demethmethylation of the promoter region of the RASSF1A gene, making the gene expression level higher. (3) EBVa GC and EBVn G The methylation status of the promoter region of the RASSF1A gene in the tissue of C is different, suggesting that EBV infection may be the cause of the methylation of the promoter region of the RASSF1A gene, but the results from the dismethylation of the RASSF1A gene promoter region of the EBV positive cell line need to be further confirmed by our further study.
【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)】：R735.2

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本文編號(hào)：2142518