本文選題：乙肝病毒　切入點：肝細胞癌　出處：《第二軍醫(yī)大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究目的構(gòu)建穩(wěn)定表達人AR的Hep G2細胞系,分別轉(zhuǎn)染空質(zhì)粒、HBx野生型質(zhì)粒和變異型質(zhì)粒至Hep G2細胞,通過檢測細胞的遷移力、增殖周期和細胞凋亡等,探究HCC相關(guān)HBx變異與AR的交互作用對肝癌惡性程度的影響。研究方法1.構(gòu)建攜帶人AR外顯子基因、綠色熒光蛋白基因(GFP)及嘌呤霉素基因(PM)的重組表達質(zhì)粒(p CDH-CMV-MCS-EF1-GFP-Puro);2.重組表達質(zhì)粒包裝感染Hep G2細胞及嘌呤霉素加壓篩選,構(gòu)建能穩(wěn)定表達人AR的Hep G2細胞系;3.分別以慢病毒空載體、攜帶HBx野生型全長基因的慢病毒載體以及攜帶A1727G+A1762T/G1764組合突變或3’端缺失突變的HBx基因的慢病毒載體,瞬時轉(zhuǎn)染Hep G2-GFP-AR細胞,通過細胞遷移實驗和流式細胞技術(shù)檢測細胞周期和細胞凋亡,觀察不同HBx與AR在乙肝相關(guān)性肝癌發(fā)生發(fā)展中的作用。結(jié)果1.成功構(gòu)建了攜帶人AR基因的慢病毒表達載體p CDH-CMV-MCSEF1-GFP-Puro-AR。2.通過攜帶AR基因的慢病毒感染Hep G2細胞,篩選出穩(wěn)定高表達人AR的Hep G2細胞株,經(jīng)流式細胞術(shù)驗證其GFP的表達量,傳至第10代后,該細胞株GFP的表達量為97.7%;Western-blot實驗鑒定單克隆細胞株傳至第14代時AR的表達量,2號克隆AR的表達量較高。3.選取AR表達量較高的2號克隆用于后續(xù)功能實驗,Transwell實驗結(jié)果顯示:HBx空轉(zhuǎn)染組在加入DHT后,下室中細胞OD值(0.45±0.02)顯著高于未加DHT組(0.33±0.03),p0.001;與未轉(zhuǎn)染HBx基因的Hep G2-GFP-AR細胞(空-組)(0.33±0.03)比較,轉(zhuǎn)染攜帶HBx A1727G+A1762T/G1764組合突變(167-組)(0.43±0.01,p=0.001)或3’端缺失突變(CT-組)(0.45±0.03,p0.001)的質(zhì)粒后,下室中細胞OD值顯著增高;Hep G2-GFP-AR細胞在分別轉(zhuǎn)染HBx A1727G+A1762T/G1764組合突變(167-組)或3’端缺失突變(CT-組)的質(zhì)粒后,加入DHT時,下室中細胞OD值顯著降低(167-組vs 167+組,0.43±0.01 vs 0.36±0.02,p=0.009;CT-組vs CT+組,0.45±0.03 vs 0.35±0.04,p=0.001)。細胞周期實驗結(jié)果顯示:各組間G2+S期細胞總占比無顯著統(tǒng)計學差異。細胞凋亡實驗結(jié)果顯示:各組間細胞總凋亡率無顯著統(tǒng)計學差異。結(jié)論1.本實驗成功構(gòu)建了攜帶人AR外顯子基因的慢病毒表達載體p CDH-CMVMCS-EF1-GFP-Puro-AR;并利用該載體,慢病毒感染Hep G2細胞,成功構(gòu)建了穩(wěn)定表達AR的Hep G2細胞系Hep G2-GFP-AR。2.通過AR高表達細胞系Hep G2-GFP-AR進一步功能實驗,發(fā)現(xiàn)在高表達AR的Hep G2-GFP-AR細胞中,轉(zhuǎn)染空載體組加入DHT后細胞遷移能力顯著增強,而轉(zhuǎn)染HBx組合突變或3’端缺失突變的HBx后亦能夠增強細胞的遷移能力;但加入DHT后則對HBx A1727G+A1762T/G1764組合突變或3’端缺失突變提高腫瘤細胞遷移能力的效應可能具有抑制作用;本研究未發(fā)現(xiàn)HBx相關(guān)突變株以及AR對Hep G2細胞的增殖和凋亡有顯著影響。
[Abstract]:Objective to construct a stable Hep G2 cell line expressing human AR and transfect empty plasmid HBX wild-type plasmid and variant plasmid into Hep G2 cell line. To explore the effect of HCC related HBx variation and AR interaction on the malignancy of hepatocellular carcinoma. 1. Construct the gene carrying human AR exon. The recombinant expression plasmids of green fluorescent protein (GFP) and purine mycin gene (PMM) were constructed by pCDH-CMV-MCS-EF1-GFP-Puromo-2.The recombinant expression plasmid was packaged to infect Hep G2 cells and purine mycin to construct Hep G2 cell lines which could stably express human AR. The lentivirus vector carrying HBx wild-type full-length gene and the lentivirus vector carrying A1727G A1762T / G1764 combination mutation or HBx gene with 3 'deletion mutation were transiently transfected into Hep G2-GFP-AR cells. Cell cycle and apoptosis were detected by cell migration assay and flow cytometry. To observe the role of different HBx and AR in the occurrence and development of Hepatitis B related liver cancer. Results 1. The lentivirus expression vector pCDH-CMV-MCSEF1-GFP-Puro-AR.2 was successfully constructed. Hep G2 cells were infected by the lentivirus carrying AR gene. Hep G2 cell lines with stable and high expression of human AR were screened. The expression of GFP was confirmed by flow cytometry and passed on to the 10th passage. The GFP expression of the cell line was 97.7 and the expression of AR was identified by Western-blot. The expression of AR in clone 2 was higher than that in clone 2 at the 14th passage. Clone 2 with high AR expression was selected for further functional experiment. After adding DHT to the empty transfection group, Compared with Hep G2-GFP-AR cells without transfection of HBx gene (0.33 鹵0.03p0.001), the cells were transfected with HBx A1727G A1762T / G1762T- G177- group (0.43 鹵0.01p0.001) or 3'terminal deletion mutation CT-group (0.45 鹵0.03p0.001), compared with Hep G2-GFP-AR cells without HBx gene transfection (0.33 鹵0.03p0.001). After transfection of HBx A1727G / A1762T / G1764 combination mutagenesis (167- group) or 3'deletion mutation (CT- group), Hep G2-GFP-AR cells were added to DHT. The OD value of the cells in the lower chamber decreased significantly in 167- group vs 167 group (0.43 鹵0.01) vs 0.36 鹵0.02p0.009 CT- group vs CT group (0.45 鹵0.03 vs 0.35 鹵0.04 p0. 001). The results of cell cycle test showed that there was no significant difference in the total proportion of cells in G 2 S phase between the three groups. Conclusion: 1. The lentivirus expression vector pCDH-CMVMCS-EF1-GFP-Puro-ARA was successfully constructed in this study. The stable Hep G2 cell line Hep G2-GFP-AR.2 was successfully constructed by lentivirus infection with AR. Through further functional experiments of AR overexpression cell line Hep G2-GFP-AR, Hep G2-GFP-AR cell line with high AR expression was found. After transfection of empty vector with DHT, the cell migration ability was significantly enhanced, and the migration ability was also enhanced after transfection of HBx combination mutation or HBx with 3 'deletion mutation. However, the addition of DHT may inhibit the effect of HBx A1727G A1762T / G1764 combination mutation or 3'deletion mutation on the ability of tumor cell migration, and no significant effect of HBx related mutant and AR on the proliferation and apoptosis of Hep G2 cells was found in this study.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

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本文編號：1635279