Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization.
Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization.
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DOI：
10.1083/jcb.104.4.1077
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发表时间：
1987-04
影响因子：
7.8
通讯作者：
Vuorio, E
中科院分区：
文献类型：
--
作者：
Sandberg, M;Vuorio, E
研究了人骨骼组织的石蜡切片，以通过原位杂交鉴定负责生产I型、II型和III型胶原的细胞。使用北方杂交和序列信息来选择相应mRNA的cDNA克隆的限制性片段，以获得具有最小交叉杂交的探针。探针的特异性在与发育中的手指的部分杂交中得到证明：已知仅产生一种类型的纤维状胶原蛋白的成骨细胞和软骨细胞（分别为I和II）仅被相应的cDNA探针识别。光滑结缔组织表现出可变的杂交强度与I型和III型胶原蛋白cDNA探针。该技术被用来本地化的活动II型胶原蛋白的生产在不同区域的软骨在长骨的生长。目视检查和颗粒计数显示生长板软骨的下部增殖区和上部肥大区的软骨细胞中的前α 1（II）胶原mRNA水平最高。这一发现通过从骨骺（静止）软骨和生长区软骨分离的RNA的北方印迹证实。骨软骨交界处的分析显示，几乎没有重叠的杂交模式与探针特异性I型和II型胶原mRNA。只有一小部分的软骨细胞在退变区被认可的前α 1（II）胶原蛋白的cDNA探针，和没有由I型胶原蛋白的cDNA探针。在矿化区，几乎所有的细胞都被I型胶原蛋白cDNA探针识别，但只有极少数分散的细胞似乎含有II型胶原蛋白mRNA。这些数据表明，原位杂交是一种有价值的工具，用于鉴定结缔组织细胞，这些细胞在发育、分化和生长的各个阶段积极产生不同类型的胶原。
Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross- hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.