本文選題：MCF-7 + RNAi　； 參考：《南京理工大學》2012年碩士論文

【摘要】：血管內皮生長因子(vascular endothelial growth factor, VEGF)是促血管生長的最關鍵因子之一,而血管的生成在腫瘤的生長過程中起著至關重要的作用。RNA干擾(RNA interference, RNAi)技術是一種高效地、特異性地抑制目的基因表達的技術,因此利用RNAi技術抑制VEGF基因表達,可以抑制血管的生成,抑制腫瘤細胞的增殖。本實驗將構建的三種靶向VEGF的質粒及四種化學合成的siRNA,利用陽離子脂質體轉染的方法將其體外轉染到人的乳腺癌MCF-7細胞中,應用細胞生物學的方法檢測其抑制VEGF基因表達及抑制腫瘤細胞增殖的作用,以期為RNA干擾在腫瘤治療方面的研究提供有效的實驗依據(jù)。 本論文主要通過以下幾個方面對靶向VEGF的質粒及siRNA進行研究： (1)首先通過GFP標記的VEGF高表達質粒(pVEGF—GFP)轉染MCF-7細胞,以轉染時間、轉染試劑濃度比及細胞接種密度為條件進行轉染最佳條件的篩選實驗； (2)將靶向VEGF的三個pSilencer質粒分別與GFP標記的VEGF高表達質粒依照最佳轉染條件進行共轉染,利用倒置熒光顯微鏡觀察pSilencer質粒對VEGF基因表達的抑制情況,利用MTT法檢測pSilencer質粒對MCF-7細胞增殖的抑制情況； (3)將化學合成的四種siRNA轉染MCF-7細胞,分別利用MTT、ELISA、RT-PCR檢測siRNA對細胞增殖的抑制、VEGF mRNA的合成及VEGF蛋白表達抑制情況,從四種不同的siRNA中篩選得到抑制效果最好的一種siRNA; (4)將篩選得到抑制效果較好的化學合成的asiRNA21/23進行進一步研究,利用活體細胞在線培養(yǎng)系統(tǒng)、Annexin V-FITC/PI雙染色及流式細胞術檢測asiRNA21/23對MCF-7細胞的增殖抑制情況及其促凋亡作用。
[Abstract]:Vascular endothelial growth factor (endothelial growth factor, VEGF) is one of the most important factors in promoting vascular growth, and angiogenesis plays an important role in tumor growth. The technique of inhibiting the expression of target gene specifically, so RNAi can inhibit the expression of VEGF gene, which can inhibit the angiogenesis and the proliferation of tumor cells. In this study, three kinds of VEGF targeting plasmids and four chemically synthesized siRNAs were constructed and transfected into human breast cancer MCF-7 cells by cationic liposome transfection in vitro. Cell biology was used to detect the inhibition of VEGF gene expression and tumor cell proliferation in order to provide an effective experimental basis for the study of RNA interference in tumor therapy. In this thesis, the plasmids and siRNA targeting VEGF were studied in the following aspects: Firstly, MCF-7 cells were transfected with GFP labeled VEGF high expression plasmid pVEGF-GFP. The optimal transfection conditions were selected under the conditions of transfection time, transfection reagent concentration ratio and cell inoculation density. (2) three pSilencer plasmids targeting VEGF were cotransfected with VEGF high expression plasmids labeled with GFP in accordance with the optimal transfection conditions. The inhibition of VEGF gene expression by pSilencer plasmids was observed by inverted fluorescence microscope. The inhibitory effect of pSilencer plasmid on the proliferation of MCF-7 cells was detected by MTT assay. (3) four kinds of chemically synthesized siRNA were transfected into MCF-7 cells. The inhibition of siRNA on cell proliferation and the expression of VEGF protein were detected by RT-PCR. The best one was obtained from four kinds of siRNA. (4) further study on the screening of chemically synthesized asiRNA21/23 with better inhibition effect was carried out. In vivo cell line culture system, Annexin V-FITC/PI double staining and flow cytometry were used to detect the inhibitory effect of asiRNA21/23 on the proliferation and apoptosis of MCF-7 cells.
【學位授予單位】：南京理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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