Embryonic neuronal markers in tuberous sclerosis: Single-cell molecular pathology
Embryonic neuronal markers in tuberous sclerosis: Single-cell molecular pathology
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DOI：
10.1073/pnas.93.24.14152
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发表时间：
1996-11-26
影响因子：
11.1
通讯作者：
Eberwine, J
中科院分区：
文献类型：
--
作者：
Crino, PB;Trojanowski, JQ;Eberwine, J
结节性硬化症（TSC）的一个标志是存在由异常形状的神经元和巨细胞组成的高度致癫痫性发育不良的大脑皮层（块茎）。TSC基因（TSC 2）的突变可能破坏神经元前体的分化和成熟，因为TSC 2基因产物块茎素被认为调节细胞增殖，为了验证块茎中的细胞保留胚胎或未成熟神经元的分子表型的假设，用神经元前体中表达的蛋白质的抗体探测来自五名TSC患者的块茎（巢蛋白、Ki-67和增殖细胞核抗原），这些抗体对块茎中的许多畸形神经元和巨细胞进行了染色，而邻近正常和对照皮质中的神经元没有标记，为了进一步表征块茎中细胞的分子表型，我们开发了一种方法，其中从石蜡包埋的脑标本中的化学标记的单细胞扩增poly（A）（+）mRNA。这种方法使我们能够检测编码巢蛋白和其他细胞骨架元件，细胞周期标志物，通过反向北方印迹分析单个巢蛋白染色细胞中存在的合成酶，我们得出结论，块茎内的多个mRNA（mRNA和蛋白质）表明在皮质发育期间TSC中细胞周期调节和神经元成熟的破坏，电化学标记细胞为研究基因表达和许多神经系统疾病的分子病理生理学提供了强有力的工具。
One hallmark of tuberous sclerosis (TSC) is the presence of highly epileptogenic dysplastic cerebral cortex (tubers) composed of abnormally shaped neurons and giant cells, Mutation of the TSC gene (TSC2) may disrupt differentiation and maturation of neuronal precursors, since the TSC2 gene product tuberin is believed to regulate cellular proliferation, To test the hypothesis that cells in tubers mag. retain the molecular phenotype of embryonic or immature neurons, tubers from five TSC patients were probed with antibodies to proteins expressed in neuronal precursors (nestin, Ki-67, and proliferating cell nuclear antigen), Many dysmorphic neurons and giant cells in tubers were stained by these antibodies, while neurons in adjacent normal and control cortex were not labeled, To further characterize the molecular phenotype of cells in tubers, we developed a methodology in which poly(A)(+) mRNA was amplified from immunohistochemically labeled single cells in paraffin-embedded brain specimens, This approach enabled us to detect mRNAs encoding nestin, and other cytoskeletal elements, cell cycle markers, and synthetic enzymes present in individual nestin-stained cells by means of reverse Northern blotting, We conclude that the presence of immature phenotypic markers (mRNAs and proteins) within tubers suggests disruption of cell cycle regulation and neuronal maturation in TSC during cortical development, Characterization of multiple mRNAs within fixed, immunohistochemically labeled cells provides a powerful tool far studying gene expression and the molecular pathophysiology of many neurologic diseases.