本文關(guān)鍵詞： Endostatin RT-PCR 基因克隆 脂質(zhì)體轉(zhuǎn)染 免疫細(xì)胞化學(xué)　出處：《內(nèi)蒙古大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：人內(nèi)皮抑素(Endostatin)是第一個(gè)進(jìn)入臨床研究的內(nèi)源性血管生成抑制劑,是膠原蛋白ⅩⅧC末端非膠原區(qū)NCl結(jié)構(gòu)域的20KDa的片段。它可以抑制內(nèi)皮細(xì)胞的增殖和轉(zhuǎn)移,從而降低腫瘤內(nèi)血管生成,阻斷營(yíng)養(yǎng)通路,達(dá)到殺死腫瘤細(xì)胞的目的。其最大的優(yōu)點(diǎn)為無(wú)毒性和無(wú)抗藥性。本文利用反轉(zhuǎn)錄PCR克隆人Endostatin基因,分別構(gòu)建到分離表達(dá)載體pIRES_2-EGFP和融合表達(dá)載體pEGFP-C_1中,然后把兩個(gè)重組表達(dá)載體利用脂質(zhì)體介導(dǎo)法分別轉(zhuǎn)染真核細(xì)胞hela,48小時(shí)后可在熒光顯微鏡下觀察到被轉(zhuǎn)染細(xì)胞發(fā)出的綠色熒光,傳代10次后,綠色熒光仍持續(xù)表達(dá),說(shuō)明是穩(wěn)定轉(zhuǎn)染。并且,利用兔抗人Endostatin抗體對(duì)已轉(zhuǎn)染的細(xì)胞作免疫細(xì)胞化學(xué)分析,DAB顯色后,在顯微鏡下觀察,結(jié)果顯示,轉(zhuǎn)染的細(xì)胞顯棕紅色,沒(méi)有轉(zhuǎn)染的細(xì)胞不顯色。這說(shuō)明,Endostatin在轉(zhuǎn)染的細(xì)胞內(nèi)穩(wěn)定遺傳與表達(dá)。從而為基因藥物和基因治療的研究奠定了基礎(chǔ)。
[Abstract]:Human endostatin (Endostatin) is the first to enter the clinical study of the endogenous angiogenesis inhibitor is terminal collagen XVIII C noncollagenous domain NCl domain fragment of 20KDa. The proliferation and it can inhibit endothelial cell migration, thereby reducing tumor angiogenesis, blocking nutritional pathway to kill the tumor cells. Its biggest advantage is non-toxic and without resistance. In this paper, using reverse transcription PCR cloning of human Endostatin gene, were cloned into the expression vector pIRES_2-EGFP separation and fusion expression vector pEGFP-C_1, then the two recombinant expression vector by liposome mediated method were transfected into eukaryotic cells HeLa, after 48 hours can be observed in fluorescence under the microscope, green fluorescence was transfected cells from the 10 passage, the green fluorescence continued to express, that is stable transfection. And anti human Endostatin Using Rabbit anti body to turn Immunocytochemical analysis of stained cells, DAB staining, observed under the microscope showed that the transfected cells were transfected cells without brownish red, no color. This shows that the expression of Endostatin and genetic stability in transfected cells. It laid the foundation for the study of gene therapy for drug and gene.

【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

【引證文獻(xiàn)】

中國(guó)碩士學(xué)位論文全文數(shù)據(jù)庫(kù) 前1條

1 張明富;禽傳染性支氣管炎病毒S1基因與豬IgGFc基因的克隆及在Hela細(xì)胞中的融合表達(dá)[D];華中農(nóng)業(yè)大學(xué);2006年

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本文編號(hào)：1480344