【摘要】：目的探討PARP1在β-拉帕醌殺傷人AGS胃癌細胞珠中的作用及分子機制。方法1.選擇人AGS胃癌細胞株,設(shè)計并合成3個針對靶基因PARP1的siRNA寡核苷酸基因片段,以及陰性對照組NC-siRNA,采用常規(guī)瞬時轉(zhuǎn)染的方法干擾PARP-1 表達。2.轉(zhuǎn)染后分別對4組加入4μmol/L的β-拉帕醌,采用Western blot實驗檢測四組PARP1裂解產(chǎn)物蛋白的表達變化,采用方差分析進行比較,篩選出穩(wěn)定的轉(zhuǎn)染細胞株;3.選取已篩選出的siRNA重新轉(zhuǎn)染AGS細胞珠,同時設(shè)置陰性對照組NC-siRNA組;采用MTT實驗及平板克隆實驗分別檢測目的基因轉(zhuǎn)染組(PARP1-siRNA轉(zhuǎn)染組)、NC-siRNA對照組中β-拉帕醌對AGS細胞珠增殖及克隆能力的影響;用劃痕實驗檢測PARP1-siRNA轉(zhuǎn)染組、NC-siRNA對照組中β-拉帕醌對AGS細胞珠遷移能力的影響。結(jié)果1.AGS胃癌細胞珠轉(zhuǎn)染后的轉(zhuǎn)染效率:在顯微鏡下觀察轉(zhuǎn)染后48h的細胞形態(tài),與陰性對照組相比,PARP1基因經(jīng)siRNA抑制表達后在形態(tài)上未見明顯改變,貼壁生長,呈圓形,可見PARP1基因沉默對細胞的形態(tài)生長無顯著的影響。Western blot檢測轉(zhuǎn)染(已加入4μmol/Lβ-拉帕醌)'后72h目的基因PARP1裂解產(chǎn)物蛋白的表達:與NC-siRNA對照組相比,siRNA組PARP1的裂解產(chǎn)蛋白的表達顯著降低,PARP1-siRNA 1 下降了 89%(P0.01),PARP1-siRNA 2 下降了 73%(P0.05),PARP1-siRNA3下降了 83%(P0.05),與另外三組相比,轉(zhuǎn)染后蛋白表達水平較低的是PARP1-siRNA 1組,說明PARP1-siRNA 1是最有效和最特異的序列,可以用于干擾沉默PARP1基因的表達,并用于后續(xù)實驗。2.PARP1-siRNA轉(zhuǎn)染對AGS細胞珠增殖、克隆及遷移能力的影響:MTT、平板克隆實驗、劃痕實驗可以觀察到β-拉帕醌可顯著抑制NC-siRNA對照組組細胞的增殖、克隆及遷移能力,而對PARP1-siRNA轉(zhuǎn)染組的抑制效果明顯減弱;siRNA沉默PARP1基因表達后可顯著降低β-拉帕醌對胃癌細胞的殺傷力。結(jié)論1.使用RNA干擾技術(shù)可以成功抑制AGS胃癌細胞珠中PARP 1基因的表達。2.過度活化的PARP1在β-拉帕醌殺傷胃癌細胞中發(fā)揮著關(guān)鍵作用。
[Abstract]:Objective to investigate the role of PARP1 in killing human AGS gastric cancer cells and its molecular mechanism. Method 1. Three siRNA oligonucleotide fragments targeting the target gene PARP1 and NC-siRNAs of negative control group were designed and synthesized from human AGS gastric cancer cell lines. The expression of PARP-1 was interfered with by conventional transient transfection. After transfection, 4 渭 mol / L 尾 -lappa quinone was added to the four groups. The protein expression of the four groups of PARP1 cleavage products was detected by Western blot assay, and the stable transfected cell line was screened by variance analysis. The selected siRNA was retransfected into AGS cells, and the negative control group was set up in NC-siRNA group. MTT assay and plate cloning assay were used to detect the effects of 尾 -lapraquinone on the proliferation and cloning ability of AGS cells in the target gene transfected group (PARP1-siRNA transfected group). The effect of 尾 -lapraquinone on the migration of AGS cells was detected by scratch test in the control group of PARP1-siRNA transfection group. Results 1. Transfection efficiency after transfection of AGS gastric cancer cell beads: the morphology of the cells was observed under microscope at 48h after transfection. Compared with the negative control group, the expression of PARP1 gene did not change significantly after siRNA inhibition, and the cells grew round and adherent to the wall. 2. It can be seen that paRP1 gene silencing has no significant effect on cell morphological growth. Western blot was used to detect the protein expression of the target gene PARP1 cleavage product 72 h after transfection (4 渭 mol / L 尾 -lapraquinone): compared with NC-siRNA control group, the cleavage of PARP1 produced protein in siRNA group. The expression of PARP1-siRNA1 decreased by 89% (P0.01) and PARP1-siRNA2 decreased by 73% (P0.05), and the expression of PARP1-siRNA3 decreased by 83% (P0.05), compared with the other three groups. The protein expression level of PARP1-siRNA1 group was lower after transfection, indicating that PARP1-siRNA1 was the most effective and specific sequence, which could be used to interfere with the silencing of PARP1 gene expression, and to further experiment .2.PARP1-siRNA transfection on the proliferation of AGS cells. The effects of cloning and migration ability on cell proliferation, cloning and migration of NC-siRNA control group were observed by using 尾 -lapraquinone as control group, and the proliferation, cloning and migration of NC-siRNA control group were significantly inhibited by 尾 -lapraquinone. The inhibitory effect of PARP1-siRNA transfection group on PARP1 gene expression was significantly decreased after silencing PARP1 gene expression by siRNA. The inhibitory effect of 尾 -lapraquinone on gastric cancer cells was significantly decreased. Conclusion 1. The expression of PARP1 gene in AGS gastric cancer cells was successfully inhibited by RNA interference. Overexpression of PARP1 plays a key role in killing gastric cancer cells by 尾-lapanone.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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