【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(jiàn)的惡性腫瘤之一。中國(guó)等亞洲國(guó)家的前列腺癌發(fā)病率雖遠(yuǎn)低于歐美，但隨著生活以及飲食習(xí)慣的改變，近年來(lái)我國(guó)男性前列腺癌的發(fā)病率呈逐年增長(zhǎng)趨勢(shì)。 組蛋白去乙�；敢种苿℉DACIs）作為一類新興的抗腫瘤藥物，具有低毒、高效的特點(diǎn)，通過(guò)抑制腫瘤細(xì)胞的增殖、阻抑細(xì)胞周期、促進(jìn)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(xì)胞中檢測(cè)到HDAC1蛋白過(guò)表達(dá)。HDAC1與RB1和E2F1聯(lián)系密切，RB家族蛋白通過(guò)募集HDAC1來(lái)抑制E2F1的活性，進(jìn)而調(diào)控下游基因轉(zhuǎn)錄。但HDAC1在此復(fù)合物中的具體調(diào)控關(guān)系仍不確定。 本研究通過(guò)構(gòu)建針對(duì)于HDAC1基因表達(dá)的shRNA（short hairpinRNA，短發(fā)夾RNA）重組干擾質(zhì)粒，脂質(zhì)體轉(zhuǎn)染shRNA HDAC1重組干擾質(zhì)粒，成功導(dǎo)入PC-3M細(xì)胞，抑制HDAC1基因轉(zhuǎn)錄水平，運(yùn)用Real-time PCR檢測(cè)細(xì)胞轉(zhuǎn)染后RB1和E2F1基因mRNA表達(dá)水平，運(yùn)用流式細(xì)胞儀檢測(cè)沉默HDAC1基因表達(dá)對(duì)PC-3M細(xì)胞周期的影響。 研究結(jié)果顯示，shRNAHDAC1重組干擾質(zhì)粒轉(zhuǎn)染進(jìn)入PC-3M細(xì)胞后，HDAC1基因mRNA表達(dá)水平降低，，RB1和E2F1基因mRNA表達(dá)水平雖均有不同程度的下降，但均未達(dá)到顯著水平。提示HDAC1在復(fù)合物中不處于中心調(diào)控位置，HDAC1、RB1和E2F1基因分別受其各自的上游基因調(diào)控。RB1、E2F1與HDAC1在細(xì)胞內(nèi)以形成復(fù)合物的形式來(lái)發(fā)揮調(diào)控細(xì)胞周期的作用，且RB1蛋白通過(guò)磷酸化與去磷酸化兩種形式的改變來(lái)參與細(xì)胞周期調(diào)控。更進(jìn)一步的蛋白水平研究還將繼續(xù)。PC-3M細(xì)胞中HDAC1基因表達(dá)降低后，細(xì)胞周期阻抑于G1期，從而促進(jìn)腫瘤細(xì)胞發(fā)生凋亡，提示HDAC1可作為有效的腫瘤抑制劑分子靶點(diǎn)。 本研究得到以下結(jié)論： 1.實(shí)驗(yàn)采用RNA（iRNA interference，RNA干擾）技術(shù)，以HDAC1為靶基因，成功設(shè)計(jì)、構(gòu)建了兩條能夠在真核細(xì)胞內(nèi)表達(dá)針對(duì)HDAC1基因的shRNA表達(dá)載體，為前列腺癌的基因治療提供了物質(zhì)基礎(chǔ)。 2. shRNA HDAC1重組干擾質(zhì)粒干擾降低PC-3M細(xì)胞中HDAC1mRNA的表達(dá)水平，對(duì)RB1和E2F1基因mRNA的表達(dá)無(wú)顯著影響。 3. shRNA HDAC1重組干擾質(zhì)粒抑制PC-3M細(xì)胞中HDAC1mRNA的表達(dá)，能夠阻抑PC-3M細(xì)胞周期，進(jìn)而誘導(dǎo)細(xì)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 朱剛,劉明,萬(wàn)奔;早期前列腺癌的診斷與治療[J];中華男科學(xué)雜志;2005年09期

2 劉春艷,孫海晶,陸軍,黃百渠;組蛋白乙�；c癌癥[J];生物化學(xué)與生物物理進(jìn)展;2003年01期

3 張孟賢;韓娜;于世英;;RNA干擾沉默HDAC1基因?qū)Υ竽c癌細(xì)胞增殖和凋亡的影響[J];世界華人消化雜志;2008年11期

4 葉定偉;李長(zhǎng)嶺;;前列腺癌發(fā)病趨勢(shì)的回顧和展望[J];中國(guó)癌癥雜志;2007年03期

5 李峰;楊森;周鵬飛;陳映鶴;;siRNA沉默PRL-3基因?qū)η傲邢侔㏄C-3細(xì)胞生物學(xué)行為的作用[J];醫(yī)學(xué)研究雜志;2012年11期

6 范校周;郭燕麗;;前列腺癌的分子影像學(xué)進(jìn)展[J];中國(guó)醫(yī)學(xué)影像技術(shù);2013年01期

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