A monoclonal antibody recognizes a form of intermediate filament protein in rat Sertoli cells that is not present in seminiferous peritubular cells.
A monoclonal antibody recognizes a form of intermediate filament protein in rat Sertoli cells that is not present in seminiferous peritubular cells.
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单克隆抗体可识别大鼠支持细胞中的一种中间丝蛋白，该蛋白不存在于生精管周细胞中。
DOI：
10.1095/biolreprod35.1.227
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发表时间：
1986
影响因子：
3.6
通讯作者：
Tres,LL
中科院分区：
文献类型：
--
作者：
Kierszenbaum,AL;Crowell,JA;Shabanowitz,RB;Smith,EP;Spruill,WA;Tres,LL
我们提取中间丝蛋白（IFP），不溶于洗涤剂从培养的大鼠睾丸支持细胞和管周细胞标记后，[35 S]蛋氨酸和解决他们的双向聚丙烯酰胺凝胶电泳，放射自显影和Western印迹。我们发现，波形蛋白是主要的IFP在支持细胞和管周细胞。然而，虽然两个等位变异的波形蛋白观察支持细胞，只有一个被检测到在管周细胞。此外，波形蛋白在Sertoli细胞产生了大量的分解产物，许多不同的分子量和等电点相比，管周细胞。这些研究结果表明，钙激活蛋白酶，通常发现在细胞中含有波形蛋白，能够产生特定的细胞类型的蛋白水解片段模式。从培养的支持细胞中提取IFP产生单克隆抗体，并使用支持细胞、小管周围细胞和完整睾丸通过间接免疫荧光监测其反应性。一种单克隆抗体，命名为IFP-SC，是一种免疫球蛋白（IgM），在培养的支持细胞中产生波形蛋白样免疫荧光模式。这种模式包括一个广泛的丝状网络，延伸到整个细胞，并包围细胞核。在培养的管周细胞，IFP-SC产生了弥漫性，非丝状免疫反应。IFP-SC与生精小管的成分反应，并显示与支持细胞相关的免疫反应性，但不与生精小管周细胞壁的元素。由于免疫荧光模式对培养细胞和完整组织中的细胞类型具有明显特异性，因此单克隆抗体IFP-SC可用于定量评价支持细胞和管周细胞的细胞交叉污染，因此，允许（1）精确表征细胞类型特异性功能事件（2）并监测培养中细胞随时间的分化状态。
We extracted intermediate filament proteins (IFP) that were insoluble in in detergent from cultured rat Sertoli and peritubular cells after labeling with [35S] methionine and resolved them by two-dimensional polyacrylamide gel electrophoresis, autoradiography and Western blotting. We found that vimentin was the predominant IFP in both Sertoli and peritubular cells. However, while two isoelectric variants of vimentin were observed in Sertoli cells, only one was detected in peritubular cells. In addition, vimentin in Sertoli cells generated a large number of breakdown products, many differing in molecular weight and isoelectric point when compared to peritubular cells. These findings suggested that a Ca2+-activated protease, usually found in cells containing vimentin, was capable of generating proteolytic fragment patterns that were specific to cell type. Monoclonal antibodies were generated against IFP extracted from cultured Sertoli cells and their reactivity monitored by indirect immunofluorescence using Sertoli cells, peritubular cells and intact testis. One monoclonal antibody, designated IFP-SC, was an immunoglobulin (IgM) that produced a vimentin-like immunofluorescent pattern in cultured Sertoli cells. This pattern consisted of an extensive filamentous network that extended throughout the cell and surrounded the nucleus. In cultured peritubular cells, IFP-SC generated a diffuse, nonfilamentous immunoreactivity. IFP-SC reacted with components of seminiferous tubules and displayed immunoreactivity associated with Sertoli cells but not with elements of the seminiferous peritubular cell wall. Because immunofluorescent patterns were distinctly specific to cell type in cultured cells and in the intact tissue, monoclonal antibody IFP-SC is useful for the quantitative evaluation of cell cross-contamination of Sertoli and peritubular cells and, therefore, allows (1) a precise characterization of functional events specific to cell type (2) and monitoring of the differentiation state of cells with time in culture.