Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons.
Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons.
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DOI：
10.1083/jcb.112.6.1205
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发表时间：
1991-03
期刊：
影响因子：
--
通讯作者：
Liem RK
中科院分区：
文献类型：
--
作者：
Weinstein DE;Shelanski ML;Liem RK
胶质细胞酸性蛋白（GFAP）是一种胶质细胞特异性中间丝蛋白，在中枢神经系统的星形胶质细胞以及星形细胞瘤细胞系中表达。为了研究GFAP的功能，我们研究了人星形细胞瘤细胞系U251，它组成型表达GFAP和波形蛋白在相同的10纳米细丝。这些细胞在体外以与原代星形胶质细胞相同的方式对神经元作出反应：它们退出细胞周期，支持神经元细胞存活和轴突生长，并且它们延伸复杂的GFAP阳性过程。为了确定GFAP在这些反应中的作用，我们通过用反义GFAP构建体稳定转染U251细胞来特异性抑制其表达。从单独转染中分离出两种稳定的反义细胞系，并通过北方和Western印迹分析以及免疫荧光研究显示为GFAP阴性。反义细胞系在其响应神经元而延伸显著胶质过程的能力方面受到抑制。在与原代神经元的培养中，U251细胞的突起长度的平均增加接近400%，而反义转染子仅增加14%。另一个神经元诱导星形胶质细胞的反应，即，增殖停滞和神经元支持在这些细胞系中不受影响。这些数据支持的结论是，胶质细胞特异性的中间丝蛋白，GFAP，所需的形成稳定的星形胶质细胞的过程中响应神经元。
The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.