RNAi介导抑制MCF-7细胞中VEGF基因表达

发布时间：2018-05-01 21:28

本文选题：MCF-7 + RNAi　；参考：《南京理工大学》2012年硕士论文

【摘要】：血管内皮生长因子(vascular endothelial growth factor, VEGF)是促血管生长的最关键因子之一,而血管的生成在肿瘤的生长过程中起着至关重要的作用。RNA干扰(RNA interference, RNAi)技术是一种高效地、特异性地抑制目的基因表达的技术,因此利用RNAi技术抑制VEGF基因表达,可以抑制血管的生成,抑制肿瘤细胞的增殖。本实验将构建的三种靶向VEGF的质粒及四种化学合成的siRNA,利用阳离子脂质体转染的方法将其体外转染到人的乳腺癌MCF-7细胞中,应用细胞生物学的方法检测其抑制VEGF基因表达及抑制肿瘤细胞增殖的作用,以期为RNA干扰在肿瘤治疗方面的研究提供有效的实验依据。本论文主要通过以下几个方面对靶向VEGF的质粒及siRNA进行研究： (1)首先通过GFP标记的VEGF高表达质粒(pVEGF—GFP)转染MCF-7细胞,以转染时间、转染试剂浓度比及细胞接种密度为条件进行转染最佳条件的筛选实验； (2)将靶向VEGF的三个pSilencer质粒分别与GFP标记的VEGF高表达质粒依照最佳转染条件进行共转染,利用倒置荧光显微镜观察pSilencer质粒对VEGF基因表达的抑制情况,利用MTT法检测pSilencer质粒对MCF-7细胞增殖的抑制情况； (3)将化学合成的四种siRNA转染MCF-7细胞,分别利用MTT、ELISA、RT-PCR检测siRNA对细胞增殖的抑制、VEGF mRNA的合成及VEGF蛋白表达抑制情况,从四种不同的siRNA中筛选得到抑制效果最好的一种siRNA; (4)将筛选得到抑制效果较好的化学合成的asiRNA21/23进行进一步研究,利用活体细胞在线培养系统、Annexin V-FITC/PI双染色及流式细胞术检测asiRNA21/23对MCF-7细胞的增殖抑制情况及其促凋亡作用。
[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.
【学位授予单位】：南京理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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