Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model
Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model
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DOI：
10.3171/jns.2001.94.1.0080
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发表时间：
2001-01-01
影响因子：
4.1
通讯作者：
Rutka, JT
中科院分区：
文献类型：
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作者：
Jung, S;Ackerley, C;Rutka, JT
目的。尽管已知恶性星形细胞瘤广泛浸润到正常脑区域，但通常很难在组织病理学制剂或实验性星形细胞瘤模型中明确识别孤立的侵袭性星形细胞瘤细胞。作者描述了一种实验系统，该系统通过使用非肿瘤性脑组织作为侵袭的基质来促进星形细胞瘤细胞的追踪。方法。将脑组织切成 1 毫米厚的切片，并在 Transwell 培养皿的上室中培养，该培养皿位于聚酯膜（孔径为 OA 毫米）的顶部，该膜浸泡在下室提供的培养基中。两种星形细胞瘤细胞系。选择 U-87 MG (U87) 和 U343 MG-A (U343) 是因为它们在单层培养物中的基底细胞运动不同。星形细胞瘤细胞用表达绿色荧光蛋白 (GFP) 的载体稳定转染，无论是单独的还是作为与透明质酸介导的运动受体 (RHAMM) 的融合蛋白，以有义或反义方向。使用荧光显微镜和荧光激活细胞分选仪分析提供的直接可视化选择具有高水平 GFP 表达的稳定转染克隆。将表达GFP的星形细胞瘤细胞克隆植入脑切片中央，利用共聚焦激光显微镜提供的光学切片测量不同时间点星形细胞瘤侵入脑组织的程度。作者观察到，在该模型系统中可以轻松追踪和跟踪表达 GFP 的星形细胞瘤细胞。表现出绿色荧光的单个星形细胞瘤细胞在迁移穿过脑切片后可以很容易地被识别出来。带有 GFP 标记的 U87 星形细胞瘤细胞比 U343 星形细胞瘤细胞迁移到脑切片中更远。 RHAMM转染的GFP标记的星形细胞瘤细胞也比GFP标记的星形细胞瘤细胞本身浸润得更远。反义RHAMM I的表达实际上消除了两种星形细胞瘤细胞系对脑切片的侵袭。结论。作者认为，这种器官典型培养系统在研究星形细胞瘤侵袭过程中可能具有相当大的实用性，不仅因为它更好地代表了侵袭性星形细胞瘤细胞通常遇到的细胞外基质分子，而且还因为 GFP 标签能够在直视下跟踪高度迁移和侵袭性的星形细胞瘤细胞。
Object. Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion.Methods. Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane (OA-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines. U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid-mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-laheled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM I virtually abrogated the invasion of the brain slices by both astrocytoma cell lines.Conclusions. The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion, net only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive ostrocytoma cells under direct vision.