Vasostatin相互作用蛋白质的筛选与鉴定

发布时间：2018-06-06 04:31

本文选题：vasostatin + 细菌双杂交系统　；参考：《第二军医大学》2005年硕士论文

【摘要】：1998年一种内皮细胞抑制因子从经EB病毒转化的B细胞系培养液上清中提纯出来,经鉴定证实为Calreticulin [Calreticulin主要作为ER(内质网)中Ca~(2+)的贮存器参与Ca~(2+)的信号转导]N-端的一个片段。该片段经纯化重组可以抑制内皮细胞增殖,但对其它细胞系没有作用,并且在体内抑制新生血管生成。因而将Calreticulin N-端的这个片段命名为vasostatin,它包含1～180aa,是Calreticulin中最保守的区域,与其它蛋白序列没有任何同源性。将vasostatin接种到无胸腺小鼠体内可明显抑制人Burkitt淋巴瘤、结肠癌的生长。与其它血管生成抑制因子相比,vasostatin与其所在的大分子Calreticulin都具有血管生成抑制作用。vasostatin是一个分子量小,可溶于水且稳定的分子,便于生产和运输。作为一个专门作用于增生内皮细胞的血管生成抑制因子,vasostatin在癌症的治疗上具有潜在的应用价值。 Vasostatin不与Ca~(2+)结合,但它可与整合素α亚单位的胞内区相结合调节细胞的粘附,还可以与糖皮质激素、雄激素、视黄酸的核受体相互作用影响其与DNA结合。有报道称calreticulin在体外专一与内皮细胞可逆性结合,在体内选择性停留在血管内皮表面,提示这可能与Vasostatin与内皮细胞的特异作用有关。Vasostatin还被发现可促进内皮细胞释放NO(一氧化氮)。尽管NO在血管生成中的作用还有争议,但研究表明它具有抑制血管生成和内皮细胞迁移的作用。体外实验尚未证明Vasostatin对内皮细胞生长抑制是由NO介导的。然而我们相信vasostatin对血管增生和肿瘤生长的抑制作用主要还是通过对内皮细胞增生的抑制实现的。体外培养中,加入vasostatin不会抑制肿瘤细胞的生长。而经vasostatin治疗的小鼠肿瘤组织形态上与对照组无差异,肿瘤远端的血管未见异常,肿瘤内部的血管损伤也无证据表明是由vasostatin引起的。这些都表明vasostatin对肿瘤细胞无直接作用,它的抗肿瘤作用与它的抑制新血管生成有关,而不是由它对肿瘤血管的细胞毒作用引起的。目前关于Vasostatin抗血管生成的几种可能机制包括产生自由基如阴离子和NO,上调Fas/FasL系统等。但Vasostatin抑制内皮细胞生长的确切机制还不清楚。 Laminin(层粘连蛋白)是ECM(细胞外基质)中的多功能糖蛋白,构成BM(基底膜)的主要结构,在细胞的粘附、生长、迁移、分化中发挥重要作用。我们知道内皮细胞表达细胞外基质蛋白laminin,而Vasostatin特定的作用于laminin的α5γ1链。
[Abstract]:In 1998, an endothelial cell inhibitory factor was isolated from the supernatant of B cell line transformed by EB virus. It was identified as a fragment of the N-terminal of Calreticulin [Calreticulin mainly acts as a storage unit of Ca~(2 in ER (endoplasmic reticulum) to participate in the signal transduction of Ca~(2]. This fragment can inhibit endothelial cell proliferation, but has no effect on other cell lines, and inhibits angiogenesis in vivo. Therefore, the N- terminal fragment of Calreticulin is named vasostatin, which contains 1T 180aa and is the most conserved region in Calreticulin and has no homology with other protein sequences. Inoculating vasostatin into thymus-free mice significantly inhibited the growth of human Burkitt lymphoma and colon cancer. Compared with other angiogenesis inhibitors, vasostatin and its macromolecule Calreticulin have angiogenesis inhibition. Vasostatin is a small molecular weight, soluble in water and stable molecule, which is easy to be produced and transported. Vasostatin, an inhibitor of angiogenesis that acts specifically on proliferative endothelial cells, has potential application in the treatment of cancer. Vasostatin does not bind to Ca~(2, but it can bind to the intracellular domain of integrin 伪 subunit to regulate cell adhesion, and it can interact with the nuclear receptors of glucocorticoid, androgen and retinoic acid to affect its binding to DNA. It has been reported that calreticulin specifically reversibly binds to endothelial cells in vitro and selectively stays on the surface of vascular endothelial cells in vivo, suggesting that this may be related to the specific effect of Vasostatin on endothelial cells. Vasostatin has also been found to promote the release of no (nitric oxide) from endothelial cells. Although the role of no in angiogenesis is still controversial, it has been shown to inhibit angiogenesis and endothelial cell migration. In vitro experiments have not yet proved that the inhibition of endothelial cell growth by Vasostatin is mediated by no. However, we believe that the inhibitory effect of vasostatin on vascular proliferation and tumor growth is mainly through the inhibition of endothelial cell proliferation. In vitro culture, the addition of vasostatin does not inhibit the growth of tumor cells. However, there was no difference in the morphology of tumor tissues between the mice treated with vasostatin and that in the control group. There was no abnormal blood vessel in the distal part of the tumor, and no evidence of vascular injury within the tumor was found to be caused by vasostatin. These results suggest that vasostatin has no direct effect on tumor cells and its antitumor effect is related to its inhibition of neovascularization rather than its cytotoxicity to tumor vessels. Several possible mechanisms of Vasostatin against angiogenesis include the production of free radicals such as anions and no, and the up-regulation of Fas/FasL systems. However, the exact mechanism of Vasostatin inhibiting endothelial cell growth is unclear. Laminin (Laminin) is a multifunctional glycoprotein in ECM (extracellular matrix), which constitutes the main structure of BMN and plays an important role in cell adhesion, growth, migration and differentiation. We know that endothelial cells express extracellular matrix protein laminin, and Vasostatin specifically acts on the 伪 5 纬 1 chain of laminin.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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