PROLACTIN AN ANTIANGIOGENIC FACTOR

肿瘤发生的多步骤过程的一个重要组成部分是 一种新的血液供应的发展，血管生成。 的战略 抑制肿瘤形成和生长是抑制 新生血管形成 我们已经证明，16 kD的N末端片段 催乳素（16KPRL）是一种特异性的体外血管溶解因子。 两 大鼠和重组人16K PRL抑制基础和bFGF或VEGF 刺激牛和人血管内皮细胞的生长。16k PRL还抑制毛细血管内皮细胞的组织化， 在胶原凝胶中培养时的毛细血管样结构。 最后16K 催乳素抑制鸡微血管发育 绒毛尿囊膜 我们将确定16K PRL是否抑制蛋白酶的激活， 毛细血管内皮细胞的bFGF。 在鸡胚绒毛尿囊膜 我们将确定16K PRL是否也抑制血管生成。 bFGF和VEGF的作用。 我们将确定16K PRL是否抑制肿瘤生长和相关的 在两个模型系统中的血管生成。 转化NIH 3T3细胞 用信号肽FGF表达载体或HCT转染 将16个人结肠癌细胞皮下移植到 裸鼠。将全身给予16K PRL，或 与肿瘤的生长和血管化 组织学评价。 在另一个模型中， 将开发16K hPRL表达定向于β 胰腺细胞。 这将通过构建一个 由胰岛素启动子/增强子元件（In）组成的嵌合基因 在16K hPRL编码区的上游。 然后这些老鼠 与表达SV40 T抗原的转基因小鼠杂交， 胰腺细胞（In-Tag）。 In-Tag小鼠被很好地表征 并发展成时间依赖性胰腺肿瘤。 频率、比率 评价肿瘤的生长和血管形成。 16K PRL的血管溶解作用是通过一种新的受体介导的， 不是催乳素或FGF受体。 我们将尝试克隆 两种方法的接收器。(1)COS细胞中的表达克隆。一 牛脑毛细血管内皮细胞cDNA文库 载体将在COS细胞中表达并通过放射自显影筛选 I-125 16K PRL结合分析。(2)与GH/PRL/白细胞介素同源 受体家族：用保守的引物获得的PCR片段 受体家族的区域将用于筛选 BBE或人脐静脉内皮细胞。

An essential component of the multistep process of tumorigenesis is the development of a new blood supply, angiogenesis. A strategy in inhibition of tumor formation and growth is to inhibit neovascularization. We have shown that the 16 kd n terminal fragment of prolactin (16K PRL) is a specific angiolytic factor in vitro. Both rat and recombinant human 16K PRL inhibits basal and bFGF or VEGF stimulated growth of bovine and human vascular endothelial cells. 16K PRL also inhibits organization of capillary endothelial cells into capillary like structures when cultured in collagen gels. Finally 16K PRL inhibits development of the microvasculature in the chick chorioallantoic membrane. We will determine if 16K PRL inhibits activation of proteases in capillary endothelial cells by BFGF. In the chick chorioallantoic membrane we will determine if 16K PRL also inhibits the angiogenic actions of bFGF and VEGF. We will determine if 16K PRL inhibits tumor growth and associated angiogenesis in two model systems. NIH 3T3 cells transformed by transfections with a signal peptide FGF expression vector or the HCT 16 human colon carcinoma cells will be transplanted subcutaneously into nude athymic mice. 16K PRL will be administered systemically or intratumorally and the growth and vascularization of tumor histologically evaluated. In another model lines of transgenic mice will be developed in which 16K hPRL expression is directed to beta cells of the pancreas. This will be accomplished by constructing a chimeric gene consisting of the insulin promoter/enhancer elements (In) upstream from the coding region for 16K hPRL. These mice will then be crossed with transgenic mice expressing SV40 T antigen in the beta cells of the pancreas (In-Tag). The In-Tag mice are well characterized and develop time dependent tumors of the pancreas. Frequency, rate of growth and vascularization of tumors will be evaluated. The angiolytic action of 16K PRL is mediated via a novel receptor which is not the prolactin or FGF receptors. We will attempt to clone the receptor by two approaches. (1) Expression cloning in COS cells. A bovine brain capillary endothelial cell (BBE) cDNA library in pcDNA1 vector will be expressed in COS cells and screened by autoradiographic analysis of I-125 16K PRL binding. (2) Homology to GH/PRL/Interleukin receptor family: PCR fragments obtained with primers to conserved region of the receptor family will be used to screen cDNA libraries of BBE or human umbilical vein endothelial cells.