靶向沉默HIF-1α和Survivin基因?qū)Ρ茄拾┞闶笠浦擦龇暖熋舾行缘挠绊?/H1>
發(fā)布時(shí)間：2018-04-04 01:55
本文選題：缺氧誘導(dǎo)因子-1a 切入點(diǎn)：生存素(Survivin) 出處：《貴州醫(yī)科大學(xué)》2017年碩士論文
【摘要】：目的:通過siRNA技術(shù)靶向沉默人鼻咽癌細(xì)胞株CNE-2缺氧誘導(dǎo)因子-1a(Hypoxia inducible factor 1-alpha,HIF-1α)與生存素(Survivin),觀察HIF-1α與Survivin低表達(dá)的CNE-2裸鼠移植瘤對放療敏感性的影響,為探討鼻咽癌基因治療奠定一定的基礎(chǔ);方法:設(shè)計(jì)合成靶向沉默HIF-1α與Survivin的microRNA干擾質(zhì)粒,采用脂質(zhì)體轉(zhuǎn)染法將含HIF-1α、Survivin的microRNA干擾質(zhì)粒轉(zhuǎn)染CNE-2細(xì)胞,熒光顯微鏡觀察其轉(zhuǎn)染率,RT-qPCR檢測其基因干擾效率。取35只4~6周齡的雌性裸鼠,隨機(jī)分為5組,每組7只,分為HIF-1α干擾組,Survivin干擾組,HIF-1αSurvivin干擾組(聯(lián)合干擾組)、空載組、陰性對照組。將轉(zhuǎn)染48h后的各組CNE-2細(xì)胞分別注射至BALA/c裸鼠體內(nèi)成瘤,待各組裸鼠成瘤后,每次每只接受5Gy X射線照射,每隔三天放療一次,共15Gy X,觀察各組裸鼠放射治療后不同時(shí)間腫瘤體積,3天后處死動(dòng)物,迅速剝離瘤體測量體積,觀察放射治療對腫瘤的抑制作用,分析HIF-1α和Survivin基因沉默對腫瘤放射敏感性的影響。RT-qPCR檢測瘤體組織中的HIF-1α和Survivin表達(dá)水平,蛋白免疫印記(Western Blot)檢測HIF-1α和Survivin蛋白表達(dá)情況,TUNEL試劑盒檢測鼻咽癌裸鼠移植瘤中細(xì)胞凋亡。結(jié)果:HIF-1α干擾組、Survivin干擾組與HIF-1αSurvivin干擾組經(jīng)過放射治療后腫瘤體積增加率明顯小于空載體組與陰性對照組(P0.05);HIF-1α和Survivin的mRNA、蛋白質(zhì)在基因干擾組中較空載組與陰性對照組均明顯下降;凋亡率較空載組與陰性對照組升高(P0.05),HIF-1α干擾組、Survivin干擾組與聯(lián)合干擾組三者之間無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:單獨(dú)干擾HIF-1α、Survivin基因與聯(lián)合干擾HIF-1α和Survivin基因均能夠增加鼻咽癌裸鼠移植瘤對放療敏感性,但聯(lián)合干擾HIF-1α和Survivin基因并沒有進(jìn)一步提高鼻咽癌裸鼠移植瘤放療敏感性。
[Abstract]:Objective: to investigate the effects of siRNA targeting CNE-2 hypoxia inducible factor 1-alpha-HIF-1 偽 and survivin survivin on the radiosensitivity of CNE-2 xenografts with low expression of HIF-1 偽 and Survivin in nude mice.Methods: microRNA interference plasmids targeting silencing HIF-1 偽 and Survivin were designed and synthesized, and microRNA interference plasmids containing HIF-1 偽 survivin were transfected into CNE-2 cells by liposome transfection.The transfection rate was observed by fluorescence microscope and RT-qPCR was used to detect its gene interference efficiency.Thirty-five female nude mice aged 4 and 6 weeks were randomly divided into 5 groups, 7 in each group, and divided into HIF-1 偽 interference group, survivin interference group and HIF-1 偽 Survivin interference group (combined interference group, no-load group, negative control group).After 48 hours of transfection, the CNE-2 cells of each group were injected into BALA/c nude mice for tumorigenesis. After tumorigenesis, each nude mouse was irradiated by 5Gy X-ray every time, and every three days was treated with radiotherapy.15Gy X was used to observe the tumor volume of the nude mice at different time after radiotherapy for 3 days. The tumor volume was quickly stripped off and the tumor inhibitory effect was observed.The effect of HIF-1 偽 and Survivin gene silencing on radiosensitivity of nasopharyngeal carcinoma was analyzed. RT-qPCR was used to detect the expression of HIF-1 偽 and Survivin in tumor tissues. Western blot was used to detect the expression of HIF-1 偽 and Survivin protein. Tunel kit was used to detect apoptosis in transplanted nasopharyngeal carcinoma (NPC) nude mice.Results the tumor volume increase rate of survivin interference group and HIF-1 偽 Survivin interference group was significantly lower than that of empty vector group and negative control group after radiotherapy. The protein in gene interference group was significantly lower than that in no-load group and negative control group.The apoptosis rate was higher than that in the no-load group and the negative control group. There was no significant difference between the survivin interference group and the combined interference group.Conclusion: interference of HIF-1 偽 survivin gene and HIF-1 偽 and Survivin genes alone can increase the radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice, but the combined interference of HIF-1 偽 and Survivin genes does not further improve the radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.63
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 趙明;王U，
本文編號：1707859
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/1707859.html
本文選題：缺氧誘導(dǎo)因子-1a 切入點(diǎn)：生存素(Survivin) 出處：《貴州醫(yī)科大學(xué)》2017年碩士論文
【摘要】：目的:通過siRNA技術(shù)靶向沉默人鼻咽癌細(xì)胞株CNE-2缺氧誘導(dǎo)因子-1a(Hypoxia inducible factor 1-alpha,HIF-1α)與生存素(Survivin),觀察HIF-1α與Survivin低表達(dá)的CNE-2裸鼠移植瘤對放療敏感性的影響,為探討鼻咽癌基因治療奠定一定的基礎(chǔ);方法:設(shè)計(jì)合成靶向沉默HIF-1α與Survivin的microRNA干擾質(zhì)粒,采用脂質(zhì)體轉(zhuǎn)染法將含HIF-1α、Survivin的microRNA干擾質(zhì)粒轉(zhuǎn)染CNE-2細(xì)胞,熒光顯微鏡觀察其轉(zhuǎn)染率,RT-qPCR檢測其基因干擾效率。取35只4~6周齡的雌性裸鼠,隨機(jī)分為5組,每組7只,分為HIF-1α干擾組,Survivin干擾組,HIF-1αSurvivin干擾組(聯(lián)合干擾組)、空載組、陰性對照組。將轉(zhuǎn)染48h后的各組CNE-2細(xì)胞分別注射至BALA/c裸鼠體內(nèi)成瘤,待各組裸鼠成瘤后,每次每只接受5Gy X射線照射,每隔三天放療一次,共15Gy X,觀察各組裸鼠放射治療后不同時(shí)間腫瘤體積,3天后處死動(dòng)物,迅速剝離瘤體測量體積,觀察放射治療對腫瘤的抑制作用,分析HIF-1α和Survivin基因沉默對腫瘤放射敏感性的影響。RT-qPCR檢測瘤體組織中的HIF-1α和Survivin表達(dá)水平,蛋白免疫印記(Western Blot)檢測HIF-1α和Survivin蛋白表達(dá)情況,TUNEL試劑盒檢測鼻咽癌裸鼠移植瘤中細(xì)胞凋亡。結(jié)果:HIF-1α干擾組、Survivin干擾組與HIF-1αSurvivin干擾組經(jīng)過放射治療后腫瘤體積增加率明顯小于空載體組與陰性對照組(P0.05);HIF-1α和Survivin的mRNA、蛋白質(zhì)在基因干擾組中較空載組與陰性對照組均明顯下降;凋亡率較空載組與陰性對照組升高(P0.05),HIF-1α干擾組、Survivin干擾組與聯(lián)合干擾組三者之間無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:單獨(dú)干擾HIF-1α、Survivin基因與聯(lián)合干擾HIF-1α和Survivin基因均能夠增加鼻咽癌裸鼠移植瘤對放療敏感性,但聯(lián)合干擾HIF-1α和Survivin基因并沒有進(jìn)一步提高鼻咽癌裸鼠移植瘤放療敏感性。
[Abstract]:Objective: to investigate the effects of siRNA targeting CNE-2 hypoxia inducible factor 1-alpha-HIF-1 偽 and survivin survivin on the radiosensitivity of CNE-2 xenografts with low expression of HIF-1 偽 and Survivin in nude mice.Methods: microRNA interference plasmids targeting silencing HIF-1 偽 and Survivin were designed and synthesized, and microRNA interference plasmids containing HIF-1 偽 survivin were transfected into CNE-2 cells by liposome transfection.The transfection rate was observed by fluorescence microscope and RT-qPCR was used to detect its gene interference efficiency.Thirty-five female nude mice aged 4 and 6 weeks were randomly divided into 5 groups, 7 in each group, and divided into HIF-1 偽 interference group, survivin interference group and HIF-1 偽 Survivin interference group (combined interference group, no-load group, negative control group).After 48 hours of transfection, the CNE-2 cells of each group were injected into BALA/c nude mice for tumorigenesis. After tumorigenesis, each nude mouse was irradiated by 5Gy X-ray every time, and every three days was treated with radiotherapy.15Gy X was used to observe the tumor volume of the nude mice at different time after radiotherapy for 3 days. The tumor volume was quickly stripped off and the tumor inhibitory effect was observed.The effect of HIF-1 偽 and Survivin gene silencing on radiosensitivity of nasopharyngeal carcinoma was analyzed. RT-qPCR was used to detect the expression of HIF-1 偽 and Survivin in tumor tissues. Western blot was used to detect the expression of HIF-1 偽 and Survivin protein. Tunel kit was used to detect apoptosis in transplanted nasopharyngeal carcinoma (NPC) nude mice.Results the tumor volume increase rate of survivin interference group and HIF-1 偽 Survivin interference group was significantly lower than that of empty vector group and negative control group after radiotherapy. The protein in gene interference group was significantly lower than that in no-load group and negative control group.The apoptosis rate was higher than that in the no-load group and the negative control group. There was no significant difference between the survivin interference group and the combined interference group.Conclusion: interference of HIF-1 偽 survivin gene and HIF-1 偽 and Survivin genes alone can increase the radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice, but the combined interference of HIF-1 偽 and Survivin genes does not further improve the radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.63
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 趙明;王U，
本文編號：1707859
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/1707859.html
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