【摘要】： 最近幾年,miRNA已成為生命科學領域研究的熱點,它的發(fā)現不僅為生物學開辟了新的領域,更為其生物學原理應用于疾病治療的轉譯研究提供了新的武器,曾一度被認為是“垃圾”RNA的小分子近十年先后7次被美國《SCIENCE》評選為“年度全球十大科技突破”。到目前為止,人們已經發(fā)現了三千余種miRNAs,被認為是最大的一類基因表達調控因子,它通過與靶基因的結合調節(jié)細胞分裂、增殖、分化和多種生物學信號通路,也可以起到腫瘤抑制基因和癌基因的作用,以及應用在疾病診斷和基因功能研究中。 多項研究結果表明miR-122是肝臟特異的miRNA,調節(jié)脂肪酸、膽固醇的代謝和肝臟的分化,上調嗜肝病毒HCV的復制。為了研究miR-122與HBV的復制關系,我們利用生物信息學預測在HBV上1689nt-1711nt區(qū)域存在miR-122的可能靶序列。首先利用miRNA的生物學原理,將miR-122的編碼基因hcr構建在包含綠色熒光蛋白的載體上,將此重組質粒轉染到細胞內,hcr基因在細胞內表達,繼而在宿主細胞內發(fā)展為成熟的miR-122,實驗中利用RT-PCR、T-A克隆技術對于細胞內的miR-122的表達進行檢測,證實獲得能穩(wěn)定表達miR-122的真核表達細胞系。為了驗證miR-122作用HBV的靶點,首先,從HepG2215基因組DNA擴增miR-15靶序列—BCL2,插入熒光素酶報告基因(luciferase)下游的Xba1酶切位點,構建了陽性的miR-15熒光素酶報告基因系統(tǒng),用于驗證miR-122靶點的陽性對照系統(tǒng)。已有研究證明HBV在1689nt-1711nt位置有四種轉錄本,根據miRNA的生物學原理推測miR-122可能對這四種mRNA都有作用,本實驗選用其中之一的HBx來驗證1689nt-1711nt序列與miR-122之間是否為靶向的關系。本研究構建了包含HBx序列的熒光素酶報告基因系統(tǒng)及HBx蛋白的表達系統(tǒng),與miR-122共轉染到自身低表達miR-122的細胞系中,在過表達mirR-122、鎖定miR-122和突變miR-122三個層面上,用熒光素酶報告基因的檢測及western blot實驗方法,證實miR-122通過作用靶序列HBx對HBV復制進行負向調控,并在翻譯水平抑制HBX蛋白的表達。然后將表達HBV1.1倍體的質粒和miR-122共轉染細胞,提取細胞內的乙肝病毒復制中間體DNA,做southern blot檢測,結果發(fā)現與miR-122共轉染之后HBV復制中間體DNA的含量明顯減少,確定miR-122與HBV的復制的降低有關。
[Abstract]:In recent years, miRNA has become a hot spot in the field of life sciences. Its discovery has not only opened up a new field for biology, but also provided a new weapon for the translation of its biological principles in the study of disease therapy. Small molecules once known as "garbage" RNA have been named "Top Ten technological breakthroughs of the year" by the United States'< SCIENCE > for seven times in the past decade. So far, more than 3, 000 species of miRNAs, have been found to be the largest class of gene expression regulators, which regulate cell division, proliferation, differentiation and a variety of biological signaling pathways by binding to target genes. It can also play the role of tumor suppressor gene and oncogene, and be used in disease diagnosis and gene function research. Many studies have shown that miR-122 is a liver-specific miRNA, that regulates fatty acids, cholesterol metabolism and liver differentiation, and up-regulates the replication of hepatoviral HCV. In order to study the replication relationship between miR-122 and HBV, we used bioinformatics to predict the possible target sequences of miR-122 in the 1689nt-1711nt region on HBV. Firstly, based on the biological principle of miRNA, the encoding gene hcr of miR-122 was constructed on the vector containing green fluorescent protein. The recombinant plasmid was transfected into the cells and the hcr gene was expressed in the cells. Then the expression of miR-122 in host cells was detected by RT-PCR,T-A cloning in the mature miR-122, experiment, and the eukaryotic expression cell lines which could stably express miR-122 were obtained. In order to verify the target of miR-122 acting on HBV, firstly, the miR-15 target sequence-BCL2, was amplified from HepG2215 genome DNA and inserted into the downstream Xba1 cleavage site of luciferase reporter gene (luciferase), and a positive miR-15 luciferase reporter gene system was constructed. A positive control system used to validate miR-122 targets. Studies have shown that HBV has four transcripts at the 1689nt-1711nt site, and it is speculated that miR-122 may play a role in all four mRNA based on the biological principles of miRNA. In this study, one of the HBx was selected to verify the relationship between 1689nt-1711nt sequence and miR-122. In this study, the luciferase reporter gene system containing HBx sequence and the expression system of HBx protein were constructed. The system co-transfected with miR-122 into a cell line with low expression of miR-122 and overexpressed mirR-122,. By using luciferase reporter gene detection and western blot assay, it was confirmed that miR-122 negatively regulated HBV replication by acting on target sequence HBx, and inhibited the expression of HBX protein at the translation level on the three levels of locking miR-122 and mutated miR-122, and the expression of HBX protein was inhibited at the translation level by luciferase reporter gene detection and western blot assay. Then the plasmid expressing HBV1.1 was co-transfected with miR-122 to extract the DNA, of HBV replication intermediates in the cells for southern blot detection. The results showed that the content of DNA, the intermediate of HBV replication, was significantly decreased after co-transfection with miR-122. Determine that miR-122 is associated with reduced replication of HBV.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346

【相似文獻】

相關期刊論文 前10條

1 韓卿;宋方洲;易發(fā)平;卜友泉;李長燕;楊曉明;;JNK3抑制NF-κB轉錄活性的研究[J];醫(yī)學分子生物學雜志;2008年04期

2 劉東遠,全碩,王娟,方福德;大鼠谷胱甘肽S-轉移酶P1基因順式調控元件的搜尋[J];中國醫(yī)學科學院學報;1998年05期

3 賈連群;王啟明;柳春;田麗;傅明德;;高密度脂蛋白亞類抗脂多糖作用的研究[J];細胞與分子免疫學雜志;2007年07期

4 張振明;李暉;董欽;劉宇;李鳳蘭;馬寧;李金波;劉端陽;舒暢;逄淑超;;SCN5A基因啟動區(qū)+495bp～+584bp片段的克隆與功能分析[J];哈爾濱醫(yī)科大學學報;2007年06期

5 高曉寧;于力;林季;;KIR3DL1基因啟動子亞克隆及表達調控載體的構建[J];標記免疫分析與臨床;2007年04期

6 鐘波;葉楓;王濤;姜帆;梁后杰;龐學利;鄒仲敏;;不同活性的survivin基因啟動子片段的克隆及鑒定[J];第三軍醫(yī)大學學報;2011年11期

7 崔麗艷;楊碩;張捷;;LCN2基因啟動子區(qū)熒光素酶報告基因載體的構建及鑒定[J];中國誤診學雜志;2011年21期

8 倪才飛;趙帆;鄭子瑞;黃蓓;馬紅芳;李力;宋婷;魏從文;何湘;張艷紅;鐘輝;;線粒體抗病毒信號蛋白MAVS真核表達質粒的構建及表達[J];軍事醫(yī)學科學院院刊;2008年06期

9 劉貝娜;何英;王爽;;PLUNC基因啟動子區(qū)熒光素酶報告載體的構建與鑒定[J];熱帶醫(yī)學雜志;2009年02期

10 隋江東;李華;吳玉章;;NLRP1基因啟動子區(qū)熒光素酶報告載體的構建與鑒定[J];免疫學雜志;2011年04期

相關會議論文 前4條

1 龔鳳英;鄧潔英;史軼蘩;;干擾素γ對垂體GH_3細胞中人生長激素基因啟動子活性的影響及其機制[A];中華醫(yī)學會第六次全國內分泌學術會議論文匯編[C];2001年

2 鄧潔英;欒好江;史軼蘩;;垂體特異性轉錄因子Pit-1與IM-9細胞內生長激素表達的關系[A];中華醫(yī)學會第六次全國內分泌學術會議論文匯編[C];2001年

3 魏旭東;范瑞華;E.W Gelfand;;有絲分裂原激酶(MEKK_2)調節(jié)白細胞介素2生成的研究[A];第九屆全國實驗血液學會議論文摘要匯編[C];2003年

4 王壘;李園園;陸長德;;人增殖細胞核抗原啟動子的順式作用元件以及DNA損傷試劑作用后p53蛋白對該啟動子的轉錄激活[A];中國生物化學與分子生物學會第八屆會員代表大會暨全國學術會議論文摘要集[C];2001年

相關博士學位論文 前3條

1 張艷;USF1下調結直腸癌中FHL2基因轉錄調控的機制研究[D];南方醫(yī)科大學;2009年

2 權金星;前列腺素E_2對大鼠成骨細胞OCIL基因表達的調節(jié)及其分子機制的研究[D];天津醫(yī)科大學;2007年

3 畢楊;Wnt拮抗因子對胚胎肝干細胞分化的影響研究[D];重慶醫(yī)科大學;2009年

相關碩士學位論文 前6條

1 郝美君;miR-122對HBV調節(jié)作用的研究[D];重慶醫(yī)科大學;2009年

2 聶棟;α1-B糖蛋白前體基因的原核表達及功能鑒定與CYP4F2基因的腎臟靶向表達[D];中國醫(yī)科大學;2005年

3 夏浩;嚴重創(chuàng)傷患者外周血清對核因子-κB的體外調節(jié)作用及其與臟器功能不全關系的研究[D];第三軍醫(yī)大學;2006年

4 周廣舉;人內皮細胞高表達脂多糖相關因子1（EOLA1）的啟動子克隆及初步鑒定[D];第三軍醫(yī)大學;2008年

5 胡潔淼;神經系統(tǒng)特異表達基因Human Neurotrimin對膠質瘤抑制作用的研究[D];中國協(xié)和醫(yī)科大學;2007年

6 洪志霞;小干擾RNA對小鼠Znf313基因的沉默作用及人ZNF313基因的轉錄調控研究[D];四川大學;2006年

，

本文編號：2428525