A "NEGATIVE" FACTOR FOR GLIOMA CELLS FROM LEPTOMENINGEAL AND MESENCHYMAL CELLS

本研究的目的是从软脑膜和/或其他细胞的组织培养液中寻找一种抑制人脑胶质细胞生长的“抑制剂”。除人软脑膜外，所有外植体培养均成功传代，并能检测其条件培养液。生长实验的基本靶细胞是永久性的人脑胶质瘤细胞系KNS81，生长实验采用MTT比色法。尽管皮肤成纤维细胞的细胞生长潜力较小，但即使在外植体培养物与KNS81的混合培养中，条件培养液中也没有检测到任何抑制活性。在制备条件培养液之前，用无血清培养液冲洗细胞两次，但这不足以完全去除用作其生长介质的血清因素。从人胶质瘤细胞系KNS 81中成功分离出一株在无血清培养条件下生长的人脑胶质瘤细胞系KNS 81-FD，并将其抗性为KNS 81-FD。对KNS 81-FD的自分泌生长因子进行了细胞内研究，并在细胞外胰酶作用下大量释放。细胞生长和形态依赖于细胞外基质。在人细胞外基质蛋白中，血浆纤维连接蛋白和层粘连蛋白促进细胞生长，而Tenascin抑制细胞生长和铺展，胶原1居中。结果表明，细胞外基质蛋白对细胞的生长和形态起着关键作用。

The research project was aimed to find out a "suppressor agent" for human gliom cell growth from tissue culture medium of leptomeningeal and/or other cells.Explant cultures of human skin, leptomeninges, meningiomas and gliomas, and of newborn rat leptomeninges were newly performed for the present project. All explant cultures except human leptomeninges were successfully subcultured and able to examine their conditioned media. The basic target cells of the conditioned media for growth assay was a permanent human glioma cell line KNS81 and the growth assay was performed by a colorimetric assay using MTT.Any inhibiting activy was not detected in the conditioned media even in the mixed cultures of the explant cultures and KNS81, although the medium from skin fibroblast was less potential for cell growth than the others. The cells were rinsed two times with a serum-free medium prior to preparing conditioned medium, yet it was insufficient for complete removal of serum factors used as their growth media. It was mandatory to establish serum-independent cell lines for studying growth inhibitors.A human glioma cell line growing in a serum-free medium was successfully isolated from a permanent cell line, KNS 81 and resistered as KNS 81-FD.The autocrine growth factors of KNS 81-FD were intracellularly researved and were massively released extracellularly trypsinisation.The cell growth and morphology were depended on the extracellular matrix. Among of human extracellular matrix proteins, plasma fibronectin and laminin enhanced cell growth, while tenascin suppresses cell growth and spreading, and collagen 1 was intermediate. The results indicated that extracellular matrix proteins act as a key role for cell growth and morphology.