本文選題：小激活RNA + Numb　； 參考：《福建醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的基于小激活RNA(small activating RNA,sa RNA)的腫瘤治療策略,為Numb基因篩選出具有激活功能并相對高效的sa RNA分子。繼而探討雄激素及抗雄激素藥物對轉(zhuǎn)染Numb-sa RNA的人前列腺癌PC-3細(xì)胞生長的影響。方法1.設(shè)計(jì)與合成針對Numb基因的3對候選小分子雙鏈RNA(double-stranded RNA,ds RNA)分子,對照組ds RNA(ds Control)設(shè)計(jì)成與人類基因組序列非同源。將ds RNA分子轉(zhuǎn)染人前列腺癌PC-3細(xì)胞。2.采用Real-time quantitative PCR(RT-q PCR)法檢測轉(zhuǎn)染后人前列腺癌PC-3細(xì)胞中靶基因Numb的m RNA表達(dá)水平。3.Western Blot驗(yàn)證轉(zhuǎn)染后人前列腺癌PC-3細(xì)胞靶基因Numb的蛋白表達(dá)水平。4.將篩選出的Numb-sa RNA轉(zhuǎn)染入人前列腺癌PC-3細(xì)胞,實(shí)驗(yàn)組分為轉(zhuǎn)染組和非轉(zhuǎn)染組,以未轉(zhuǎn)染以及未行藥物處理的細(xì)胞作為陰性對照組。分別采用不同濃度的二氫睪酮(dihydrotestosterone,DHT),氟他胺(flutamide)、二氫睪酮-氟他胺混合液(dihydrotestosterone-flutamide mixture,DHT-F)處理實(shí)驗(yàn)組細(xì)胞,采用MTT法在藥物處理48h后分別測定細(xì)胞活性;5.根據(jù)MTT法檢測結(jié)果,篩選出有效的藥物濃度,分別處理轉(zhuǎn)染組和非轉(zhuǎn)染細(xì)胞,培養(yǎng)48h后,采用Annexin V-FITC/PI檢測各組細(xì)胞的凋亡情況。結(jié)果1.ds Numb-870、ds Numb-948均未能上調(diào)人前列腺癌PC-3細(xì)胞中靶基因Numb m RNA水平;而ds Numb-298能上調(diào)細(xì)胞內(nèi)Numb m RNA和蛋白水平。2.采用MTT法檢測細(xì)胞增殖情況,DHT處理細(xì)胞后,發(fā)現(xiàn)轉(zhuǎn)染組在10-3mol/L、10-4mol/L、10-5mol/L三個濃度組的相對存活率均明顯低于陰性對照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);轉(zhuǎn)染組在10-3mol/L和10-4mol/L濃度組的相對存活率明顯低于非轉(zhuǎn)染組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。氟他胺處理細(xì)胞后,轉(zhuǎn)染組在10-3mol/L、10-4mol/L、10-5mol/L三個濃度組的相對存活率均低于陰性對照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);轉(zhuǎn)染組在10-4mol/L濃度組的相對存活率明顯低于非轉(zhuǎn)染組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。二氫睪酮-氟他胺混合液處理后,轉(zhuǎn)染組各個濃度組的相對存活率較陰性對照組均明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);且轉(zhuǎn)染組在各個濃度的相對存活率均明顯低于非轉(zhuǎn)染組,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。3.采用Annexin V-FITC/PI雙染法,利用流式細(xì)胞技術(shù)檢測細(xì)胞凋亡情況,DHT處理后,轉(zhuǎn)染組的凋亡率較陰性對照組下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05);轉(zhuǎn)染組的凋亡率較非轉(zhuǎn)染組明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。氟他胺處理后,轉(zhuǎn)染組的凋亡率較陰性對照組明顯上升,差異有統(tǒng)計(jì)學(xué)意義(P0.05);轉(zhuǎn)染組較非轉(zhuǎn)染組的凋亡率明顯上升,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。二氫睪酮-氟他胺混合液處理后,轉(zhuǎn)染組的凋亡率較陰性對照組明顯上升,差異有統(tǒng)計(jì)學(xué)意義(P0.05);轉(zhuǎn)染組的凋亡率多于非轉(zhuǎn)染組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1.經(jīng)篩選ds Numb-298具有特異性激活人前列腺癌PC-3細(xì)胞中Numb基因表達(dá)的功能。2.將Numb-sa RNA轉(zhuǎn)染入人前列腺癌PC-3細(xì)胞中,能在一定程度上使人前列腺癌PC-3恢復(fù)對激素的敏感性,誘導(dǎo)其凋亡。
[Abstract]:Objective to screen active and relatively efficient sa RNA molecules for numb gene based on small activated (small activating RNA-sa RNA. Then we investigated the effects of androgen and androgen drugs on the growth of PC-3 cells transfected with Numb-sa RNA. Method 1. Three pairs of candidate small molecule double-stranded RNAs (double-stranded RNAs RNAs) were designed and synthesized for numb gene, while the control group DS RNA (DS control) was designed to be not homologous to the human genome sequence. The DS RNA molecule was transfected into human prostate cancer PC-3 cell line. 2. Real-time quantitative polymerase chain reaction (RT-q PCR) was used to detect the mRNA expression level of target gene numb in human prostate cancer PC-3 cells. 3. Western blot was used to verify the protein expression level of the target gene numb of human prostate cancer PC-3 cells. The selected Numb-sa RNA was transfected into human prostate cancer PC-3 cells. The experimental group was divided into transfection group and non-transfection group. Untransfected and untreated cells were used as negative control group. The experimental cells were treated with different concentrations of dihydrotestosterone (DHT) and flutamide (flutamide), mixture (dihydrotestosterone-flutamide mixture- DHT-F), respectively. The cell activity was measured by MTT assay after 48 hours of treatment. According to the results of MTT assay, the effective drug concentration was selected. The transfected cells and non-transfected cells were treated respectively. After 48 hours of culture, Annexin V-FITC / Pi was used to detect the apoptosis of the cells in each group. Results 1.ds Numb-870 DS Numb-948 could not upregulate the level of target gene numm RNA in human prostate cancer PC-3 cells, while DS Numb-298 could up-regulate the level of numb mRNA and protein in PC-3 cells. MTT assay was used to detect the proliferation of cells treated with DHT. It was found that the relative survival rate of the transfected group in 10-3 mol / L 10-4 mol / L 10-5 mol / L group was significantly lower than that in the negative control group. The relative survival rate of transfection group in 10-3 mol / L and 10-4 mol / L groups was significantly lower than that in non-transfection group (P0.05). After treated with flutamide, the relative survival rate of transfected cells in 10-3 mol / L 10-4 mol / L 10-5 mol / L group was significantly lower than that in negative control group (P0.05), and the relative survival rate in 10-4 mol / L group was significantly lower than that in non-transfection group (P0.05). The relative survival rate of each concentration group in transfection group was significantly lower than that in negative control group (P0.05), and the relative survival rate in each concentration of transfection group was significantly lower than that in non-transfection group. The difference was statistically significant (P0.05). Using Annexin V-FITC / Pi double staining method and flow cytometry, the apoptosis rate of transfected group was lower than that of negative control group (P0.05), and the apoptosis rate of transfected group was significantly lower than that of non-transfected group. The difference was statistically significant (P0.05). After flutamide treatment, the apoptosis rate of transfection group was significantly higher than that of negative control group (P0.05); the apoptosis rate of transfection group was significantly higher than that of non-transfection group (P0.05). The apoptosis rate of transfection group was significantly higher than that of negative control group (P0.05), and the apoptosis rate of transfection group was more than that of non-transfection group (P0.05). Conclusion 1. DS Numb-298 has the function of specifically activating the expression of numb gene in human prostate cancer PC-3 cells. Transfection of Numb-sa RNA into human prostate cancer PC-3 cells can restore the hormone sensitivity and induce apoptosis in human prostate cancer PC-3 cells to a certain extent.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 孫穎浩;;前列腺癌診治進(jìn)展[J];上海醫(yī)學(xué);2011年07期

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本文編號：2075343