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

细胞运动的分子基础显然是高度复杂的，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成分。本研究的目的是系统地研究细胞表面蛋白在调控细胞运动和血管生成中的可能作用，并进一步确定参与恶性肿瘤的细胞表面分子。本研究采用了一种基于人脐静脉内皮细胞选择的方法，该方法表现出运动性。我们用抑制HUVEC运动的功能性单抗(MAb)进行了这项研究。HM7-3是用HUVEC免疫小鼠后建立的，根据其在Malrigel跨孔穿透实验和管状形成中抑制HUVEC迁移的能力而被选择。HM7-3可抑制大鼠卵和角膜的新生血管生成。克隆发现HM7-3识别一种新的特异性蛋白质结构，我们将其命名为血管生成抑制蛋白(ALP)。然而，恶性肿瘤血管生成的抑制程度与ALP的表达水平并不直接相关。用复制缺陷型腺病毒载体体内转移所有的c DNA。瘤内注射表达碱性磷酸酶的重组腺病毒载体(rad-AIP)不能抑制肿瘤血管生成。由于碱性磷酸酶有多个N-糖基化位点，因此，碱性磷酸酶的功能一般受N-糖基化的高度控制。碱性磷酸酶的去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，碱性磷酸酶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。这一假设可能被认为是碱性磷酸酶对正常细胞的血管生成有影响。进一步的研究对于分析碱性磷酸酶的作用机制是必要的。

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.