本文選題：Cadherin-5　切入點：基因　出處：《江西醫(yī)學(xué)院》2005年碩士論文

【摘要】：目的:研究粘附素5 N 端CED-4(cadherin extracellular domain-4)基因經(jīng)重組逆轉(zhuǎn)錄病毒介導(dǎo)轉(zhuǎn)染人乳腺癌細(xì)胞MDA-MB-435 及其誘導(dǎo)MDA-MB-435 細(xì)胞凋亡的分子調(diào)控機制。方法:前期工作已完成CED-4 基因克隆和鑒定,并與克隆載體連接轉(zhuǎn)化大腸桿菌,獲取復(fù)制質(zhì)粒。本實驗承接前期工作,主要實驗方法:⑴酶切鑒定質(zhì)粒:限制性內(nèi)切酶BspHⅠ、BglⅡ、AflⅡ分別酶切質(zhì)粒pVSV-G、pGAG-POL、pMSCV、pMSCV-CED4,電泳鑒定質(zhì)粒。⑵293包裝細(xì)胞包裝產(chǎn)生重組逆轉(zhuǎn)錄病毒:脂質(zhì)體介導(dǎo)質(zhì)粒pVSV-G、pGAG-POL、pMSCV 、pMSCV-CED4,轉(zhuǎn)入293 包裝細(xì)胞,經(jīng)G418 篩選,293 細(xì)胞包裝產(chǎn)生含目的基因和不含目的基因的重組逆轉(zhuǎn)錄病毒。⑶病毒感染靶細(xì)胞MDA-MB-435 細(xì)胞:兩組重組逆轉(zhuǎn)錄病毒(實驗組和實驗對照組)分別感染MDA-MB-435 細(xì)胞,增強型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)作為基因轉(zhuǎn)染陽性細(xì)胞標(biāo)記,第三天觀察綠色熒光,2 周后密切觀察細(xì)胞形態(tài)變化。⑷細(xì)胞凋亡的檢測:光學(xué)顯微鏡下顯微觀察凋亡細(xì)胞形態(tài),Annexin V-BIOTIN apoptosis detection kit 細(xì)胞染色,流式細(xì)胞儀檢測細(xì)胞凋亡; ⑸RT-PCR:提取實驗組、實驗對照組和空白對照組三組細(xì)胞總體RNA,設(shè)計引物,經(jīng)反轉(zhuǎn)錄PCR 擴增目的基因CED-4 mRNA、Integrinβ1 亞基mRNA、原癌基因c-fos mRNA,瓊脂糖凝膠電泳;⑹Westhen blot 檢測c-fos 蛋白的表達(dá)。結(jié)果:⑴質(zhì)粒DNA 大小正常:酶切質(zhì)粒,電泳結(jié)果表明,質(zhì)粒pVSV-G、pGAG-POL、pMSCV、pMSCV-CED4 大小正常,未發(fā)生降解。⑵293 細(xì)胞包裝產(chǎn)生重組逆轉(zhuǎn)錄病毒:質(zhì)粒轉(zhuǎn)化293 細(xì)胞,48小時后,熒光顯微鏡下可見綠色熒光。⑶重組逆轉(zhuǎn)錄病毒感染MDA-MB-435細(xì)胞:逆轉(zhuǎn)錄病毒感染MDA-MB-435 細(xì)胞,48 小時后熒光顯微鏡下可見綠色熒光,提示病毒介導(dǎo)基因轉(zhuǎn)染成功。⑷RT-PCR 檢測到目的基因CED-4 基
[Abstract]:Aim: to investigate the molecular regulatory mechanism of CED-4(cadherin extracellular domain-4) gene transfection into human breast cancer cell line MDA-MB-435 mediated by recombinant retrovirus and its mechanism of inducing apoptosis of MDA-MB-435 cells.Methods: the CED-4 gene was cloned and identified in the previous work and transformed into Escherichia coli by ligation with the clone vector to obtain the replication plasmid.This experiment undertakes the preliminary work,涓昏瀹為獙鏂規(guī)硶:鈶撮叾鍒囬壌瀹氳川綺，

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