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

【摘要】：血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)是促血管生長的最關(guān)鍵因子之一,而血管的生成在腫瘤的生長過程中起著至關(guān)重要的作用。RNA干擾(RNA interference, RNAi)技術(shù)是一種高效地、特異性地抑制目的基因表達(dá)的技術(shù),因此利用RNAi技術(shù)抑制VEGF基因表達(dá),可以抑制血管的生成,抑制腫瘤細(xì)胞的增殖。本實(shí)驗(yàn)將構(gòu)建的三種靶向VEGF的質(zhì)粒及四種化學(xué)合成的siRNA,利用陽離子脂質(zhì)體轉(zhuǎn)染的方法將其體外轉(zhuǎn)染到人的乳腺癌MCF-7細(xì)胞中,應(yīng)用細(xì)胞生物學(xué)的方法檢測其抑制VEGF基因表達(dá)及抑制腫瘤細(xì)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(shí)驗(yàn)依據(jù)。 本論文主要通過以下幾個(gè)方面對靶向VEGF的質(zhì)粒及siRNA進(jìn)行研究： (1)首先通過GFP標(biāo)記的VEGF高表達(dá)質(zhì)粒(pVEGF—GFP)轉(zhuǎn)染MCF-7細(xì)胞,以轉(zhuǎn)染時(shí)間、轉(zhuǎn)染試劑濃度比及細(xì)胞接種密度為條件進(jìn)行轉(zhuǎn)染最佳條件的篩選實(shí)驗(yàn)； (2)將靶向VEGF的三個(gè)pSilencer質(zhì)粒分別與GFP標(biāo)記的VEGF高表達(dá)質(zhì)粒依照最佳轉(zhuǎn)染條件進(jìn)行共轉(zhuǎn)染,利用倒置熒光顯微鏡觀察pSilencer質(zhì)粒對VEGF基因表達(dá)的抑制情況,利用MTT法檢測pSilencer質(zhì)粒對MCF-7細(xì)胞增殖的抑制情況； (3)將化學(xué)合成的四種siRNA轉(zhuǎn)染MCF-7細(xì)胞,分別利用MTT、ELISA、RT-PCR檢測siRNA對細(xì)胞增殖的抑制、VEGF mRNA的合成及VEGF蛋白表達(dá)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(xué)合成的asiRNA21/23進(jìn)行進(jìn)一步研究,利用活體細(xì)胞在線培養(yǎng)系統(tǒng)、Annexin V-FITC/PI雙染色及流式細(xì)胞術(shù)檢測asiRNA21/23對MCF-7細(xì)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(xué)位授予單位】：南京理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 孫建清;黃桂香;;VEGF-C及受體在子宮內(nèi)膜癌中的表達(dá)及意義[J];包頭醫(yī)學(xué)院學(xué)報(bào);2010年06期

2 馮春瓊,馬文麗,宋艷斌,郭秋野,吳清華,鄭文嶺;細(xì)胞凋亡的MTT染色法檢測[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2002年03期

3 尹玉;曹立宇;李昊;江燕;張洪福;;siRNA沉默VEGF基因?qū)Υ竽c癌細(xì)胞生物學(xué)特性的影響[J];中國組織化學(xué)與細(xì)胞化學(xué)雜志;2010年03期

4 鄧清華;劉國文;付世新;劉磊;王曉旭;朱曉巖;王建國;白鴿;王振全;李小兵;王哲;;RNAi及沉默通路調(diào)控研究進(jìn)展[J];中國畜牧獸醫(yī);2011年01期

5 張會(huì)勇;劉景晶;張笑巖;曹榮月;李泰明;;VEGF165過表達(dá)對小鼠乳腺癌及腫瘤周邊皮膚血管生成模式的影響研究[J];河南師范大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年03期

6 魏星;邢麗娜;;shRNA干擾VEGF的表達(dá)對宮頸癌SiHa細(xì)胞凋亡的影響[J];臨床腫瘤學(xué)雜志;2011年05期

7 高彥;仲偉霞;周庚寅;;淋巴管生成與胃腸道腫瘤轉(zhuǎn)移關(guān)系的研究進(jìn)展[J];中華腫瘤防治雜志;2008年03期

8 張?jiān)?趙暉;;RNA干擾抑制食管鱗癌VEGF表達(dá)的實(shí)驗(yàn)研究[J];中華腫瘤防治雜志;2009年17期

9 尹延梅;于紅;周巖冰;張文卿;呂銳;;慢病毒介導(dǎo)的RNAi對人胃癌細(xì)胞VEGF表達(dá)的影響[J];山東醫(yī)藥;2009年21期

10 郭文忠 ,冉宇靚 ,劉軍 ,遇瓏 ,孫立新 ,楊治華;低氧提高腫瘤細(xì)胞反義VEGF_(165)基因表達(dá)[J];生物化學(xué)與生物物理學(xué)報(bào);2002年05期

相關(guān)博士學(xué)位論文 前1條

1 賀楚峰;siRNA抑制hTERT及VEGF表達(dá)治療鼻咽癌的實(shí)驗(yàn)研究[D];中南大學(xué);2006年

，

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