本文選題：Survivin + 真核表達載體��； 參考：《南華大學(xué)》2012年碩士論文

【摘要】：第一部分 目的:通過構(gòu)建靶向Survivin的shRNA真核表達載體的方法來探討RNAi技術(shù)對鼻咽癌細胞生長抑制作用，為鼻咽癌的基因治療提供新的實驗依據(jù)。 方法:以凋亡抑制基因Survivin為靶位，構(gòu)建表達小干擾RNA(siRNA)的真核表達載體，經(jīng)脂質(zhì)體轉(zhuǎn)染鼻咽癌細胞系CNE-2，應(yīng)用RT-PCR檢測鼻咽癌細胞中SurvivinmRNA表達抑制情況，初步鑒定干擾效果。 結(jié)果:重組載體pGPU6/GFP/Neo-shRNA1,2,3,4,N經(jīng)雙酶切和基因測序檢測,證實堿基序列與模板序列相符，無基因突變，載體構(gòu)建成功。重組載體經(jīng)脂質(zhì)體Lipofectamine2000介導(dǎo)轉(zhuǎn)染鼻咽癌細胞，細胞發(fā)生不同程度凋亡，轉(zhuǎn)染效率約為55%。RT-PCR方法初步鑒定其干擾效果，以Survivin/GAPDH的相對表達來表示SurvivinmRNA的變化,各組的mRNA表達相差較大，24h測得的mRNA相對于轉(zhuǎn)染前有一定下降，大約在50%左右；但是隨著時間延長，重組質(zhì)粒的干擾作用逐漸減弱。 結(jié)論：本實驗通過構(gòu)建靶向Survivin的shRNA真核表達載體的方法，經(jīng)脂質(zhì)體介導(dǎo)，，轉(zhuǎn)染鼻咽癌細胞系CNE-2，在一定程度上抑制細胞中Survivin的基因表達水平。證實了應(yīng)用RNAi技術(shù)對鼻咽癌細胞生長有一定程度的抑制作用，并為鼻咽癌的基因治療提供了新的實驗數(shù)據(jù)。 第二部分 目的：通過化學(xué)合成的小干擾RNA(smallinterferingRNA，siRNA)的方法來探討RNAi技術(shù)對鼻咽癌細胞生長抑制作用，為鼻咽癌的基因治療提供新的實驗依據(jù)。 方法：根據(jù)siRNA的設(shè)計原則設(shè)計針對survivin基因序列特異性的siRNA，在脂質(zhì)體介導(dǎo)下轉(zhuǎn)染CNE-2細胞。采用RT-PCR和Westernblotting檢測Survivin在干擾后mRNA和蛋白的表達情況，并篩選出干擾效果最好的siRNA序列進行細胞生長抑制試驗（CCK-8實驗）檢測對細胞生長抑制作用。 結(jié)果：轉(zhuǎn)染siRNA24h、48h和72h后，用RT-PCR和Westernblotting檢測，CNE-2Z細胞survivin表達明顯下降，并篩選出抑制效果最好1號序列進行CCK-8實驗，結(jié)果顯示能明顯抑制細胞生長增殖。 結(jié)論：靶向Survivin的siRNA轉(zhuǎn)染人鼻咽癌CNE-2細胞有效抑制細胞中Survivin基因的表達水平，并對細胞生長增殖等生物學(xué)行為產(chǎn)生抑制作用。證實了應(yīng)用通過化學(xué)合成的siRNA的RNAi技術(shù)對鼻咽癌細胞生長有顯著的抑制作用為鼻咽癌的基因治療提供了新的實驗數(shù)據(jù)。
[Abstract]:Part oneAim: to investigate the inhibitory effect of RNAi on the growth of nasopharyngeal carcinoma cells by constructing shRNA eukaryotic expression vector targeting Survivin, and to provide a new experimental basis for gene therapy of nasopharyngeal carcinoma.Methods: the eukaryotic expression vector expressing small interfering RNAs siRNAs was constructed with Survivin as the target. The nasopharyngeal carcinoma cell line CNE-2 was transfected with liposome. The inhibition of SurvivinmRNA expression in nasopharyngeal carcinoma cells was detected by RT-PCR, and the interference effect was preliminarily evaluated.Results: the recombinant vector pGPU6 / GFPU / Neo-shRNA1, 2, 2, 3, 4N was detected by double enzyme digestion and gene sequencing. It was confirmed that the base sequence was consistent with the template sequence, and there was no gene mutation. The vector was successfully constructed.The recombinant vector was transfected into nasopharyngeal carcinoma cells mediated by liposome Lipofectamine2000. The transfection efficiency was about 55%.RT-PCR method to identify its interfering effect. The relative expression of Survivin/GAPDH was used to express the changes of SurvivinmRNA.The expression of mRNA in each group was significantly lower than that before transfection, but the interference effect of the recombinant plasmid gradually weakened with the prolongation of time, compared with that before transfection, the expression of mRNA decreased to a certain extent (about 50%).Conclusion: in this experiment, shRNA eukaryotic expression vector targeting Survivin was constructed and transfected into nasopharyngeal carcinoma cell line CNE-2 by liposome, and the expression level of Survivin gene was inhibited to some extent.It is confirmed that RNAi can inhibit the growth of nasopharyngeal carcinoma cells to a certain extent and provide new experimental data for gene therapy of nasopharyngeal carcinoma.Part twoAim: to investigate the inhibitory effect of RNAi on the growth of nasopharyngeal carcinoma cells by chemically synthesized small interfering RNAs, and to provide a new experimental basis for gene therapy of nasopharyngeal carcinoma.Methods: according to the design principle of siRNA, survivin gene sequence specific siRNAs were designed and transfected into CNE-2 cells by liposome.RT-PCR and Westernblotting were used to detect the expression of mRNA and protein in Survivin after interference, and the best siRNA sequence was screened for cell growth inhibition test (CCK-8).Results: after transfection of siRNA24 h for 48 h and 72 h, the expression of survivin in CNE-2Z cells was detected by RT-PCR and Westernblotting, and the best inhibitory sequence was selected for CCK-8 assay. The results showed that the expression of survivin was significantly inhibited by RT-PCR and Westernblotting.Conclusion: siRNA targeting Survivin can effectively inhibit the expression of Survivin gene in human nasopharyngeal carcinoma CNE-2 cells and inhibit cell growth and proliferation.It is confirmed that the application of chemically synthesized siRNA RNAi technique can significantly inhibit the growth of nasopharyngeal carcinoma cells and provide new experimental data for gene therapy of nasopharyngeal carcinoma.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.63

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