【作者】 陈洪栋；

【导师】 李红民；

【作者基本信息】 西北大学 ， 细胞生物学， 2011， 硕士

【摘要】 恶性肿瘤和糖尿病都是严重危害人类健康的常见疾病,但目前仍然缺少有效的治疗和预防手段。因此开发新的高效治疗药物与手段,建立有效的预防措施一直是各国学者共同努力的方向。Arresten是人Ⅳ型胶原α1链羧基末端非胶原蛋白结构域断裂产生的多肽片段,能特异性诱导血管内皮细胞凋亡,抑制其增殖、迁移和管腔形成,可有效抑制肿瘤的生长和转移,是一个具有潜在临床应用价值的强效血管生成抑制因子,有望成为具有良好应用前景的抗肿瘤血管生成药物。目前有多个研究机构正在寻求适合重组Arresten生产的外源蛋白表达方法以满足未来治疗用药需求。本研究通过引物设计、PCR扩增和限制性核酸内切酶作用,将实验室保存的克隆化Arresten编码基因分别亚克隆至原核表达载体和哺乳动物表达载体,为建立合适的Arresten外源生产表达系统、进一步开展Arresten结构与功能关系研究奠定研究基础。另外,实验过程中还通过动物细胞培养、MTT法、ELISA、放射性免疫测定等方法考察了多甲氧基黄酮对大鼠胰岛β细胞系INS-1细胞分泌胰岛素的影响作用,旨在为阐明多甲氧基黄酮抗糖尿病的作用机理、寻求天然抗糖尿病膳食补充剂、拓宽国内资源丰富的柑橘属植物的利用效率提供理论和技术依据。已经取得以下研究结果：1、成功构建了重组Arresten原核表达载体。通过引物设计、PCR扩增和限制性核酸内切酶作用,将实验室保存的克隆化Arresten编码基因定位克隆到原核表达载体pET22b (+) HindⅢ/BamH I位点之间,经核酸限制性内切酶消化和序列测定,获得重组Arresten原核表达载体。2、采用CaCl2转化法实现重组Arresten原核表达载体对大肠杆菌细胞BL21(DE3)的转化,经SDS-PAGE电泳检测和Western bloting杂交检测,获得高效表达重组Arresten的E.coli B121 (DE3)稳定转化细胞,并通过镍柱亲和层析获得纯化的重组Arresten。3、成功构建了重组Arresten哺乳动物细胞表达载体。通过引物设计、PCR扩增和限制性核酸内切酶作用,将实验室保存的克隆化Arresten编码基因定位克隆到哺乳动物表达载体pEGFP-N1质粒绿色荧光蛋白编码框上游的HindⅢ/BamH I位点之间,经核酸限制性内切酶消化和序列测定,构建出与绿色荧光蛋白融合表达的重组Arresten哺乳动物细胞表达载体。4、采用脂质体转染法,实现重组Arresten在哺乳动物细胞HEK293A细胞中的瞬时表达,建立了重组Arresten哺乳动物细胞HEK293A瞬时表达体系。5、通过多甲氧基黄酮刺激大鼠胰岛β细胞系INS-1细胞,经MTT法检测,多甲氧基黄酮在10gg/mL-80μg/mL作用浓度范围内,与对照组相比,对大鼠胰岛β细胞系INS-1细胞增殖的影响无显著统计学差异。6、采用ELISA、放射性免疫测定和RT-PCR分析,初步揭示出多甲氧基黄酮作用不能直接刺激大鼠胰岛p细胞系INS-1细胞胰岛素分泌,药物作用24小时,对胰岛素表达无明显影响作用。更多还原

【Abstract】 Malignancies and diabetes are two critical diseases which do serious harm to human healthy, and to each of them there were no efficient therapy and preventing method. So, exploiting new high effective drugs and methods to establish effective prevention measures is a longterm goal for every researcher.Arresten is a 26-kDa anti-angiogenic fragment proteolysis from the a 1 chain of type IV collagen. This molecule inhibits endothelial cell proliferation, migration, tube formation and matrigel neovascularization. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models. As a new potential clinical anti-angiogenic factor, Arresten is expected to be a prospective anti-angiogenic drug. By now, many research institutions are looking for a suitable protein expression method in order to satisfy future therapy. In this study, by using primer designing, PCR and restrict digestion, we cloned Arresten gene into prokaryotic and eukaryotic expression plasmid respectively. This lays foundation for constructing suitable Arresten expression system and further study of the relationship between structure and function. We also analyzed the effect of Polymethoxylated Flavones on insulin secretion in mouse pancreatic islandβcell line INS-1.Animal cell culture、MTT、ELISA and radioimmunoassay et al. were adopted in this process. Collectively, these data sheds more light into the research of the antidiabetic mechanism of Polymethoxylated Flavones, also it provides theoretical and technical basis for looking for natural antidiabetic supplements and making full use of resourceful citrus.Result:1. The recombinant Arresten-pET22b (+) prokaryotic expression was successfully constructed. Preservative Arresten was amplified by PCR. PCR product was digested with HindⅢ/BamH I, and ligated into predigested pET22b (+). Recombiant plasmid was identified by restriction PCR, enzyme digestion and sequenceing.2. The recombinant Arresten-pET22b(+)was successfully transformed into E. coli B121 (DE3) using CaCl2 transformation method for expressing, The expression level of soluble Arresten was detected by quantitative SDS-PAGE and Western blotting analysis.This recombinant protein(with His-tag) was isolated using affinity chromatography.3. Recombinant Arresten eukaryotic expression was successfully constructed. Preservative Arresten was was amplified by PCR. After digestion, it was ligated into predigested pEGFP-N1, leaving Arresten on the upstream of GFP. Restriction enzyme digestion and sequenceing were adopted to identify recombinant plasmid.4. The pEGFP-N1-Arresten recombinant vector was used to transfect HEK293 A using liposome. The recombinant protein was successfully expressed in it as Arresten-GFP fusion protein. We successfully obtained a transient expression system of HEK293A.5. Polymethoxylated Flavones was used to stimulate INS-1, and cell viability was analyzed by MTT. Compared with control group, there are no significant difference when the concentration was at the range of 10μg/mL～80μg/mL.6. The result of ELISA, radioimmunoassay and RT-PCR primarily revealed that Polymethoxylated Flavones can not stimulate insulin secretion of INS-1 derectly. Result showed that after 24h affected by Polymethoxylated Flavones, insulin secretion in INS-1 had no obvious change.更多还原