Analysis of metastasis mechanism in experimental lung metastasis with heparanase transgenic tumor cells

乙酰肝素酶转基因肿瘤细胞实验性肺转移机制分析

• 批准号: 14571278
• 负责人: HORINOUCHI Hirohisa
• 金额: $ 1.92万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571278/
• 关键词: heparanase; lung metastasis; Heparan sulphate proteoglycan; Interstitial matrix; angiogenesis; ヘパラン硫酸プロテオグリカン; ヘパラナーゼ; 腫瘍内血管密度; 腫瘍循環; ヘパリナーゼ

To establish metastasis to the lung, several steps are considered necessary. Among them, destruction of the existing architecture is thought necessary for tumor cell growth. MMPs (Matrix metalloproteinase) are the most studied molecule which is a key factor of invasion. In existing matrix, heparin sulfate proteoglycan is forming network of skeleton and it attract various heparin binding proteins such as bFGF,PDGF,VEGF which are tightly bound to cell growth and angiogenesis. When bounded they don't influence their activity to the cells around but when released by enzymatic cleavage, their functions are activated. Key enzyme of this mechanism is heparanase.The aim of this study is visualizing the newly generated tumor circulation and for the aid of visualization tumor cells are transected with GFP protein coding plasmid. Transfection of GFP gene was only successful within several days after transfection. Expression of the protein observed as fluorescence faded after tumor cell began to grow.To establish lung metastasis model, 6-10 days' subcutaneous inoculation of LY80 cells and mince the subcutaneous tumor followed by intravenous injection was necessary. Following this method, after 14-16 days, lung metastasis was found.After 14-16 days, rats were euthanized and lung metastasis were dissected, weighed, and stored in -80 degree. Heparanase activity was studied using heparin sulphate degradation kit.Results : In small lung metastasis (less than 3mm in diameter) showed relatively high value of heparanase activity, while large metastasis (more than 3mm in diameter) showed low heparanase activity compare to the normal lung tissue. AdditionalThese result indicate that we may suspect in early stage of metastasis, heparanase may play an important role in degradation of existing heparin sulphate proteoglycan network in the matrix.

要确定肺转移，有几个步骤被认为是必要的。其中，对现有结构的破坏被认为是肿瘤细胞生长所必需的。基质金属蛋白酶(MMPs)是目前研究最多的分子，是肿瘤侵袭的关键因素。在现有的基质中，硫酸肝素蛋白多糖正在形成骨架网络，并能吸引多种与细胞生长和血管生成密切相关的肝素结合蛋白，如bFGF、PDGF、VEGF等。当它们结合时，它们不会影响它们对周围细胞的活性，但当它们被酶切割释放时，它们的功能就会被激活。这一机制的关键酶是肝素酶。本研究的目的是可视化新生成的肿瘤循环，并将GFP蛋白编码质粒横切肿瘤细胞。GFP基因只有在感染后几天内才能转染成功。LY80细胞皮下接种LY80细胞6~10天，切块后静脉注射，建立肺转移模型。14~16天后，发现肺转移灶。14~16天后，处死大鼠，解剖、称量肺转移灶，-80度保存。结果：与正常肺组织相比，小转移灶(直径<3 mm)的乙酰肝素酶活性相对较高，而大转移灶(直径>3 mm)的乙酰肝素酶活性较低。此外，这些结果表明，在转移的早期阶段，肝素酶可能在基质中现有的硫酸肝素蛋白多糖网络的降解中发挥重要作用。