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

为了确定肺转移，需要几个步骤。其中，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，它是肿瘤侵袭的关键因子.在现有的基质中，硫酸肝素蛋白聚糖形成骨架网络，并吸引各种肝素结合蛋白，如bFGF、PDGF、VEGF，这些蛋白与细胞生长和血管生成紧密结合。当它们结合在一起时，它们不会影响周围细胞的活性，但当它们被酶裂解释放时，它们的功能被激活。该机制的关键酶是乙酰肝素酶。本研究的目的是可视化新产生的肿瘤循环，并为了帮助可视化，用GFP蛋白编码质粒横切肿瘤细胞。GFP基因的转染仅在转染后数天内成功。肺转移模型的建立需要皮下接种LY 80细胞6-10天，将皮下肿瘤切碎后静脉注射。14 - 16天后，处死大鼠，解剖肺转移灶，称重，-80 ℃保存。结果：与正常肺组织相比，小转移灶（直径小于3 mm）中乙酰肝素酶活性较高，大转移灶（直径大于3 mm）中乙酰肝素酶活性较低。这些结果提示，我们可能怀疑在转移的早期阶段，乙酰肝素酶可能在基质中存在的硫酸肝素蛋白聚糖网络的降解中起重要作用。

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.