【作者】 扈廷茂；

【导师】 陈明杰；

【作者基本信息】 内蒙古大学 ， 微生物学， 2005， 硕士

【摘要】 人内皮抑素（Endostatin）是第一个进入临床研究的内源性血管生成抑制剂,是胶原蛋白ⅩⅧC末端非胶原区NCl结构域的20KDa的片段。它可以抑制内皮细胞的增殖和转移,从而降低肿瘤内血管生成,阻断营养通路,达到杀死肿瘤细胞的目的。其最大的优点为无毒性和无抗药性。本文利用反转录PCR克隆人Endostatin基因,分别构建到分离表达载体pIRES₂-EGFP和融合表达载体pEGFP-C₁中,然后把两个重组表达载体利用脂质体介导法分别转染真核细胞hela,48小时后可在荧光显微镜下观察到被转染细胞发出的绿色荧光,传代10次后,绿色荧光仍持续表达,说明是稳定转染。并且,利用兔抗人Endostatin抗体对已转染的细胞作免疫细胞化学分析,DAB显色后,在显微镜下观察,结果显示,转染的细胞显棕红色,没有转染的细胞不显色。这说明,Endostatin在转染的细胞内稳定遗传与表达。从而为基因药物和基因治疗的研究奠定了基础。更多还原

【Abstract】 Endostatin, a carboxy-terminal 20KDa fragment of the non-coliagenous (NC) 1 domain of collagen ⅩⅧ, is the first endogenous angiogenesis inhibitor entering human clinical trials. It can inhabit endothelial cell migration and multiplication to reduce the vascularization of tumors and finally kill tumors. The great advantage of the agent lies in the lack of toxicity and drug resistance. Human Endostatin gene cloned by RT-PCR was inserted into separate expression vector pIRES2-EGFP and fusion expression vector pEGFP-Ci, which were then transfected into hela cell with lipofectamine? regent. After 48 hours, under the fluorescence microscope, the green fluorescence was spreading in the transfected hela cell. After culturing ten generations, the green fluorescence expressed persistently. This illustrated that the transfection was steady. Further more, after immunocytochemistry in the fixed tranfected hela cell with rabbit anti-Endostatin, under the microscope, the transfected hela cell became brown-red and the negative control was colourless, which meant that Endostatin has steadily expressed in the transfected hela cell. Our results provided a basal evidence for gene therapy and gene medicine.更多还原