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The analysis of the structure and the funtion of AIP and gene therapy using AIP

AIP的结构和功能分析及AIP基因治疗

基本信息

批准号：
11557087
负责人：
ISHIDA Hisao
金额：
$ 8.64万
依托单位：
Tazuke Kofukai Medical Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557087/
关键词：
AIP angiogenesis HUVEC glycosylation 糖鎖 metastasis 血管新生血管新生因子

项目摘要

The molecular basis of cell motility is obviously highly complex and is believed to be controlled by a number of molecular systems, while angiogenesis is an important biological component of fumor progression. The aims of this study were to systematically investigate the possible involvement of proteins at the, cell surface in controlling cell motility and angiogenesis, and further identify the cell surface molecules involved in malignant tumors. The present study utilized an approach based on the selection of HUVEC, which showed motility. We have addressed this study using functional monoclonal antibodies (MAbs), which inhibit HUVEC motility is probes The monoclonal antibody HM7 3 was established after immunization of mice with HUVEC, and was selected on the basis of its ability to inhibit HUVEC migration in a malrigel transwell penetration assay and tube formation. HM7-3 inhibited augiogenesis using eggs and cornea of rats. cDNA cloning revealed that HM7-3 recognizes a new specific protein structure and we named this protein Angiogenesis Inhibiting Protein (AlP). However, the extent of suppression of angiogenesis of malignant tumors is not directly related to the level of ALP expression. A repIication-deficient adenovirus vector was used for the in vivo transfer of All cDNA. Intratumor injection of an adenovirus vector (rAd-AIP) expressing AlP resulted in no inhibition of tumor angiogenesis. As ALP has several N-glycosyIation sites, the function of ALP may be, in general, highly controlled by N-glycosylation. De-N-glycosyltion of ALP results in the loss of functions. The terminal structure, sialic acid might have an important role on angiogenesis. On the other hand, ALP inhibits angiogenesis of the cornea of the rat, but cannot inhibit angiogenesis of malignant cells. The hypothesis may be considered that ALP may have an effect on angiogenesis of normal cells. The further examination will be necessary for the analysis of the mechanism of ALP.

细胞运动的分子基础显然是高度复杂的，并且被认为是由许多分子系统控制的，而血管生成是肿瘤进展的重要生物学组成部分。本研究旨在系统探讨细胞表面蛋白可能参与细胞运动和血管生成的调控，并进一步确定与恶性肿瘤有关的细胞表面分子。本研究采用基于选择HUVEC的方法，HUVEC具有运动性。我们利用抑制HUVEC运动的功能性单克隆抗体（mab）来进行这项研究。单克隆抗体HM7 3是在用HUVEC免疫小鼠后建立的，并根据其在malrigel transwell渗透试验和管形成中抑制HUVEC迁移的能力而被选择。HM7-3抑制大鼠卵和角膜的自生。cDNA克隆显示HM7-3识别了一个新的特异性蛋白结构，我们将该蛋白命名为血管生成抑制蛋白（Angiogenesis inhibitory protein, AlP）。然而，恶性肿瘤对血管生成的抑制程度与ALP表达水平无直接关系。利用复制缺陷腺病毒载体进行所有cDNA的体内转移。肿瘤内注射表达AlP的腺病毒载体（rAd-AIP）对肿瘤血管生成无抑制作用。由于ALP具有多个n -糖基化位点，因此ALP的功能通常受n -糖基化的高度控制。ALP的去n -糖基化导致功能丧失。末端结构，唾液酸可能在血管生成中起重要作用。另一方面，ALP抑制大鼠角膜的血管生成，但不能抑制恶性细胞的血管生成。可以认为ALP可能对正常细胞的血管生成有影响。进一步的研究将是分析ALP机制的必要条件。