PRODUCTION OF COMPONENTS OF EXTRACELLULAR-MATRIX BY CULTURED RAT SERTOLI CELLS
PRODUCTION OF COMPONENTS OF EXTRACELLULAR-MATRIX BY CULTURED RAT SERTOLI CELLS
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DOI：
10.1095/biolreprod35.4.997
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发表时间：
1986-11-01
影响因子：
3.6
通讯作者：
HALL, PF
中科院分区：
文献类型：
--
作者：
BORLAND, K;MUFFLY, KE;HALL, PF
在塑料培养皿中培养15、20和25日龄大鼠的支持细胞，并用Triton X-100（0.1% w/v）或脱氧胆酸钠（2% w/v）提取。发现提取后留下的残留物含有细胞外基质的三种蛋白质特征（纤连蛋白、胶原IV和层粘连蛋白）。通过四种方法鉴定这些蛋白质：间接免疫荧光、与标准蛋白质在聚丙烯酰胺凝胶中电泳的共迁移、免疫印迹（Western印迹）和用[35 S]甲硫氨酸孵育支持细胞后的免疫沉淀。此外，纤连蛋白通过免疫电子显微镜与第二抗体结合到胶体金。在相同的细胞残留物中，通过这些方法中的第一种初步鉴定了硫酸乙酰肝素。这些研究中使用的细胞通过电子显微镜显示为基本上纯的支持细胞培养物（> 95%纯）。由于100%的细胞检查显示阳性和特异性免疫荧光染色的四种成分的细胞外基质的良好特性的抗体，由于胶体金的研究揭示了纤维连接蛋白的存在密切相关的细胞内鉴定的电子显微镜支持细胞，它必须得出结论，支持细胞合成这四种蛋白质，并推测硫酸乙酰肝素。显然，培养的支持细胞可以合成和分泌细胞外基质的一些组分。
Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (> 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.