【摘要】：前列腺癌（prostate cancer）是人類特有的腫瘤疾病，是歐美男性最常見的惡性腫瘤之一。中國等亞洲國家的前列腺癌發(fā)病率雖遠低于歐美，但隨著生活以及飲食習慣的改變，近年來我國男性前列腺癌的發(fā)病率呈逐年增長趨勢。 組蛋白去乙酰化酶抑制劑（HDACIs）作為一類新興的抗腫瘤藥物，具有低毒、高效的特點，通過抑制腫瘤細胞的增殖、阻抑細胞周期、促進凋亡等方式抑制腫瘤的發(fā)生與發(fā)展。有研究顯示，在前列腺癌組織和細胞中檢測到HDAC1蛋白過表達。HDAC1與RB1和E2F1聯(lián)系密切，RB家族蛋白通過募集HDAC1來抑制E2F1的活性，進而調控下游基因轉錄。但HDAC1在此復合物中的具體調控關系仍不確定。 本研究通過構建針對于HDAC1基因表達的shRNA（short hairpinRNA，短發(fā)夾RNA）重組干擾質粒，脂質體轉染shRNA HDAC1重組干擾質粒，成功導入PC-3M細胞，抑制HDAC1基因轉錄水平，運用Real-time PCR檢測細胞轉染后RB1和E2F1基因mRNA表達水平，運用流式細胞儀檢測沉默HDAC1基因表達對PC-3M細胞周期的影響。 研究結果顯示，shRNAHDAC1重組干擾質粒轉染進入PC-3M細胞后，HDAC1基因mRNA表達水平降低，，RB1和E2F1基因mRNA表達水平雖均有不同程度的下降，但均未達到顯著水平。提示HDAC1在復合物中不處于中心調控位置，HDAC1、RB1和E2F1基因分別受其各自的上游基因調控。RB1、E2F1與HDAC1在細胞內以形成復合物的形式來發(fā)揮調控細胞周期的作用，且RB1蛋白通過磷酸化與去磷酸化兩種形式的改變來參與細胞周期調控。更進一步的蛋白水平研究還將繼續(xù)。PC-3M細胞中HDAC1基因表達降低后，細胞周期阻抑于G1期，從而促進腫瘤細胞發(fā)生凋亡，提示HDAC1可作為有效的腫瘤抑制劑分子靶點。 本研究得到以下結論： 1.實驗采用RNA（iRNA interference，RNA干擾）技術，以HDAC1為靶基因，成功設計、構建了兩條能夠在真核細胞內表達針對HDAC1基因的shRNA表達載體，為前列腺癌的基因治療提供了物質基礎。 2. shRNA HDAC1重組干擾質粒干擾降低PC-3M細胞中HDAC1mRNA的表達水平，對RB1和E2F1基因mRNA的表達無顯著影響。 3. shRNA HDAC1重組干擾質粒抑制PC-3M細胞中HDAC1mRNA的表達，能夠阻抑PC-3M細胞周期，進而誘導細胞凋亡。
[Abstract]:Prostate cancer (prostate cancer) is one of the most common malignant tumors in Europe and America. Although the incidence of prostate cancer in China and other Asian countries is far lower than that in Europe and America, with the change of life and diet habits, the incidence of male prostate cancer in China has been increasing year by year. Histone deacetylase inhibitor (HDACIs), as a new antitumor drug, has the characteristics of low toxicity and high efficiency. It inhibits the occurrence and development of tumor by inhibiting the proliferation of tumor cells, inhibiting cell cycle and promoting apoptosis. Studies have shown that overexpression of HDAC1 protein has been detected in prostate cancer tissues and cells. HDAC1 is closely related to RB1 and E2F1. RB family proteins inhibit E2F1 activity by recruiting HDAC1 and then regulate downstream gene transcription. However, the specific regulatory relationship of HDAC1 in this complex remains uncertain. In this study, we constructed the shRNA (short hairpinRNA, short hairpin RNA (shRNA (short hairpinRNA, short hairpin RNA) interference plasmid for the expression of HDAC1 gene, transfected the shRNA HDAC1 recombinant interference plasmid with liposome, successfully introduced into PC-3M cells, and inhibited the transcription level of HDAC1 gene. The expression of RB1 and E2F1 gene mRNA was detected by Real-time PCR and the effect of silent HDAC1 gene expression on PC-3M cell cycle was detected by flow cytometry. The results showed that after transfection of shRNAHDAC1 recombinant interference plasmid into PC-3M cells, the mRNA expression level of HDAC1 gene decreased, but the mRNA expression level of RB1 and E2F1 genes decreased in varying degrees, but none of them reached significant level. HDAC1,RB1 and E2F1 genes are regulated by their respective upstream genes respectively. RB1,E2F1 and HDAC1 play a role in regulating cell cycle in the form of complex. RB1 protein participates in cell cycle regulation through phosphorylation and dephosphorylation. The further study of protein level will continue. After the expression of HDAC1 gene in PC-3M cells decreases, the cell cycle is inhibited in G1 phase, thus promoting the apoptosis of tumor cells, suggesting that HDAC1 can be used as an effective molecular target of tumor inhibitor. The conclusions of this study are as follows: 1. Using RNA (iRNA interference,RNA interference technique and HDAC1 as target gene, two shRNA expression vectors which can express HDAC1 gene in eukaryotic cells were successfully designed and constructed, which provided a material basis for gene therapy of prostate cancer. 2. ShRNA HDAC1 recombinant interference plasmid interference reduced the expression of HDAC1mRNA in PC-3M cells, but had no significant effect on the expression of RB1 and E2F1 gene mRNA. 3. The recombinant shRNA HDAC1 interference plasmid inhibited the expression of HDAC1mRNA in PC-3M cells, inhibited the cell cycle and induced apoptosis of PC-3M cells.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25

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