本文選題：前列腺癌 + 雙氫青蒿素　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討雙氫青蒿素(Dihydroartemisinin,DHA)作用人前列腺癌PC-3細胞株后,泛素樣含PHD和環(huán)指域1(Ubiquitin-like with PHD and ring finger domains 1,UHRF1)的表達改變及可能誘導(dǎo)的甲基化調(diào)控機制。方法:不同濃度(0、25、50和100μmol/L)DHA處理PC-3細胞株48 h,DMSO處理的未加藥組為空白對照組。MTT法檢測細胞增殖活性。FCM法檢測細胞凋亡率、周期分布及活性氧水平(Reactive oxygen species,ROS)。亞硫酸氫鹽基因組測序法(BGS)檢測p16INK4A基因啟動子區(qū)域的甲基化水平。實時熒光定量PCR檢測UHRF1、甲基化轉(zhuǎn)移酶1(DNA methyltransferase 1,DNMT1)及p16INK4A mRNA表達。蛋白質(zhì)印跡法檢測UHRF1、DNMT1及p16INK4A蛋白的表達水平。細胞免疫熒光法檢測p16INK4A蛋白的定位及表達。結(jié)果:1.MTT和FCM結(jié)果表明DHA能通過誘導(dǎo)PC-3細胞凋亡和G1/S期阻滯而抑制細胞增殖,并伴有ROS水平的升高。2.BGS結(jié)果顯示DHA作用PC-3細胞后,p16INK4A基因啟動子區(qū)域的甲基化水平明顯降低。3.RT-PCR和Western blotting結(jié)果顯示DHA誘導(dǎo)UHRF1和DNMT1 mRNA及蛋白表達水平明顯降低,而p16INK4A表達水平升高。4.細胞免疫熒光結(jié)果顯示p16INK4A蛋白在細胞核及細胞質(zhì)中均有表達。DHA作用PC-3細胞后,p16INK4A蛋白定位未發(fā)生改變,但p16INK4A蛋白的熒光強度顯著增加。結(jié)論:DHA可能通過抑制UHRF1和DNMT1的表達,下調(diào)p16INK4A基因啟動子區(qū)域的甲基化水平,從而解除p16INK4A蛋白的表達抑制。最終DHA抑制PC-3細胞增殖,誘導(dǎo)細胞凋亡及G1/S期阻滯,并伴有ROS的生成。DHA作用PC-3細胞的機制可能與UHRF1對p16INK4A的調(diào)控有關(guān)。
[Abstract]:Aim: to investigate the effect of dihydroartemisinine dihydroartemisinin (DHA) on the expression of PHD and 1(Ubiquitin-like with PHD and ring finger domains 1 UHRF1 in human prostate cancer cell line PC-3 and the possible mechanism of methylation regulation induced by dihydroartemisinine dihydroartemisin (DHA). Methods: the PC-3 cell lines treated with different concentrations of 渭 mol/L)DHA and 25 渭 mol/L)DHA for 48 h were treated with DMSO for 48 h. The proliferative activity of PC-3 cells was detected by MTT assay. The apoptosis rate, cell cycle distribution and reactive oxygen speciesrose were detected by FCM method. The methylation level of promoter region of p16INK4A gene was detected by bisulfite genome sequencing. The expression of UHRF1, 1(DNA methyltransferase 1 DNMT1 and p16INK4A mRNA were detected by real-time fluorescence quantitative PCR. The expression of UHRF1 DNMT1 and p16INK4A protein was detected by Western blot. The localization and expression of p16INK4A protein were detected by immunofluorescence assay. Results 1. The results of MTT and FCM showed that DHA could inhibit the proliferation of PC-3 cells by inducing apoptosis and G 1 / S arrest. The results showed that the methylation level of the promoter region of p16INK4A gene in PC-3 cells was significantly decreased after DHA treatment. 3. The results of RT-PCR and Western blotting showed that DHA induced a significant decrease in UHRF1, DNMT1 mRNA and protein expression, while the expression level of p16INK4A was increased by .4. The results of cellular immunofluorescence showed that the localization of p16INK4A protein in PC-3 cells did not change after the expression of p16INK4A protein in nucleus and cytoplasm, but the fluorescence intensity of p16INK4A protein increased significantly. ConclusionDHA may inhibit the expression of p16INK4A protein by inhibiting the expression of UHRF1 and DNMT1 and down-regulating the methylation level in the promoter region of p16INK4A gene. Finally, DHA inhibited the proliferation of PC-3 cells, induced apoptosis and arrest of G 1 / S phase, accompanied by the production of ROS. DHA may play a role in the regulation of p16INK4A by UHRF1.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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