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

【摘要】：人內(nèi)皮抑素(Endostatin)是第一個進入臨床研究的內(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)皮細胞的增殖和轉(zhuǎn)移,從而降低腫瘤內(nèi)血管生成,阻斷營養(yǎng)通路,達到殺死腫瘤細胞的目的。其最大的優(yōu)點為無毒性和無抗藥性。本文利用反轉(zhuǎn)錄PCR克隆人Endostatin基因,分別構(gòu)建到分離表達載體pIRES_2-EGFP和融合表達載體pEGFP-C_1中,然后把兩個重組表達載體利用脂質(zhì)體介導(dǎo)法分別轉(zhuǎn)染真核細胞hela,48小時后可在熒光顯微鏡下觀察到被轉(zhuǎn)染細胞發(fā)出的綠色熒光,傳代10次后,綠色熒光仍持續(xù)表達,說明是穩(wěn)定轉(zhuǎn)染。并且,利用兔抗人Endostatin抗體對已轉(zhuǎn)染的細胞作免疫細胞化學分析,DAB顯色后,在顯微鏡下觀察,結(jié)果顯示,轉(zhuǎn)染的細胞顯棕紅色,沒有轉(zhuǎn)染的細胞不顯色。這說明,Endostatin在轉(zhuǎn)染的細胞內(nèi)穩(wěn)定遺傳與表達。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.

【學位授予單位】：內(nèi)蒙古大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

【引證文獻】

中國碩士學位論文全文數(shù)據(jù)庫 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細胞中的融合表達[D];華中農(nóng)業(yè)大學;2006年

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本文編號：1480344