Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes.
Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes.
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培养的人类细胞表达细胞外基质基因：信使 RNA 和抗原表位的定位。
DOI：
10.1016/0003-2697(89)90377-1
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发表时间：
1989
影响因子：
2.9
通讯作者：
Uitto,J
中科院分区：
文献类型：
--
作者：
Peltonen,J;Jaakkola,S;Gay,K;Olsen,DR;Chu,ML;Uitto,J
开发了利用原位杂交和过氧化物酶-抗过氧化物酶免疫检测在mRNA和蛋白质水平上检查细胞基因表达的简化和方便的方法。这些技术首先被优化用于检测由培养的人皮肤成纤维细胞和角质形成细胞（人皮肤的两种主要细胞类型）表达的细胞外基质基因。原位杂交和北方转移分析与人类序列特异性cDNA编码胶原和非胶原蛋白序列表现出不同的基质基因的选择性表达，这两种细胞类型，表明不同的生物合成能力，这些细胞和证明杂交的特异性。原位杂交的效用也被证明在混合的原代细胞培养建立从皮肤神经纤维瘤组成的雪旺细胞，神经束膜细胞，成纤维细胞。本文所开发的方法进一步用于同时检测成纤维细胞培养物中的纤连蛋白mRNA和免疫反应蛋白。该程序允许检测代表放射性标记的cDNA-mRNA杂合体和蛋白质表位的颗粒，如通过相同细胞上的过氧化物酶-抗过氧化物酶免疫检测所观察到的。这种方法，适当的修改，可适用于其他细胞类型以及组织标本。
Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.