本文選題：肝細胞肝癌　切入點：SET8　出處：《河北醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:SET8是一種賴氨酸甲基轉(zhuǎn)移酶,主要參與基因轉(zhuǎn)錄,DNA合成與修復(fù),異染色質(zhì)形成,基因組的穩(wěn)定性,細胞周期進程等方面的調(diào)控;另有多項研究顯示SET8可能與多種腫瘤的發(fā)生和發(fā)展密切相關(guān)。我們的前期研究發(fā)現(xiàn)mi R-502可調(diào)控SET8基因的表達,并且發(fā)現(xiàn)SET8的表達量與肝細胞肝癌預(yù)后相關(guān);隨后在體外實驗中,利用小干擾RNA(siRNA)將SMMC-7721細胞中的SET8基因表達沉默,發(fā)現(xiàn)SET8基因沉默可顯著抑制肝細胞肝癌細胞在體外的遷移、增殖和侵襲能力,因此,本實驗同樣利用siRNA沉默SET8基因的表達,探討其對肝癌SMMC-7721細胞在裸鼠體內(nèi)生長的影響。方法:1設(shè)計并合成用于人SET8基因沉默表達的siRNA(SET8-siRNA)及對照的control-si RNA;并采用熒光標記法標記SET8-siRNA及control-siRNA,利用脂質(zhì)體(Lipofectamine TM2000)將兩種siRNA轉(zhuǎn)染入人肝癌SMMC-7721細胞內(nèi)。2嘌呤霉素篩選穩(wěn)定表達SET8-siRNA及control-siRNA的SMMC-7721細胞株。3利用Western blot檢測siRNA對SET8基因沉默表達作用。4裸鼠荷瘤實驗觀察SET8基因沉默對肝細胞肝癌的作用:將穩(wěn)定轉(zhuǎn)染SET8-siRNA及control-si RNA細胞株分別種植到裸鼠皮下,每周觀察裸鼠體內(nèi)腫瘤的大小及生長速度,計算瘤體體積,并利用小動物活體成像系統(tǒng)進行觀察。5采用SPSS 21.0統(tǒng)計學軟件進行統(tǒng)計分析,t檢驗和單因素方差分析(One-way ANOVA)比較組間差異,以P0.05表示差異有統(tǒng)計學意義。結(jié)果:1成功構(gòu)建SET8-siRNA及control-siRNA穩(wěn)定轉(zhuǎn)染的細胞株,熒光顯微鏡下可觀察轉(zhuǎn)染率為100%。2與control-siRNA組及blank-control組相比,SET8-siRNA組SET8蛋白表達量受到顯著抑制(P0.05);與control-siRNA組相比抑制率高達約49%。3與control-siRNA組及blank-control組比較,SET8-siRNA組于裸鼠體內(nèi)異種種植的瘤體在種植后第7、14、21天生長受到顯著抑制(第7天:P0.05;第14天:P0.05;21天:P0.05)。結(jié)論:體內(nèi)實驗發(fā)現(xiàn)SiRNA介導的SET8基因沉默可以抑制肝細胞肝癌的生長。
[Abstract]:Objective to investigate the role of 1% SET8 in the synthesis and repair of gene transcription DNA, the formation of heterochromatin, the stability of genome, the process of cell cycle and so on. Several other studies have shown that SET8 may be closely related to the occurrence and development of various tumors. Our previous studies have found that miR-502 regulates the expression of SET8 gene, and that the expression of SET8 is related to the prognosis of hepatocellular carcinoma. The expression of SET8 gene in SMMC-7721 cells was silenced by small interfering RNAs. It was found that SET8 gene silencing could significantly inhibit the migration, proliferation and invasion of hepatocellular carcinoma cells in vitro. Therefore, siRNA was also used to silence the expression of SET8 gene. To investigate its effect on the growth of hepatocellular carcinoma (SMMC-7721) cells in nude mice. Methods: 1 designed and synthesized siRNA-SET8-siRNAs for silencing expression of human SET8 gene and control-siRNA as control, and labeled SET8-siRNA and control-siRNAs with fluorescent labeling method, and used liposome Lipofectamine TM2000 to label the two kinds of siRNA. Screening of SMMC-7721 Cell Lines with stable expression of SET8-siRNA and control-siRNA by transfection into Human Hepatocellular carcinoma SMMC-7721 Cell Lines. 4 the effect of SET8 Gene silencing on Hepatocellular carcinoma in Nude mice by detecting the silencing expression of SET8 Gene by Western blot. 4. The effect of SET8 Gene silencing on Hepatocellular carcinoma in Nude mice. Stable transfection of SET8-siRNA and control-si RNA cells were implanted into nude mice subcutaneously. The tumor size and growth rate in nude mice were observed weekly, and the tumor volume was calculated. The small animal living imaging system was used to observe the differences between the two groups using SPSS 21.0 statistical software for statistical analysis and One-way ANOVA (One-way ANOVA). Results the stable transfected SET8-siRNA and control-siRNA cell lines were successfully constructed by 1: 1. The expression of SET8 protein in SET8-siRNA group was significantly inhibited compared with control-siRNA group and blank-control group, and the inhibition rate was about 49.3% in control-siRNA group, 49.3% in control-siRNA group and blank-control group in blank-control group. The growth of the body was significantly inhibited at the 21st day after planting (7 days: P0.05; 14 days: P0.05; 21 days: P0.05.Conclusion: SiRNA mediated SET8 gene silencing can inhibit the growth of hepatocellular carcinoma in vivo.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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本文編號：1595124