Sandberg M, Vuorio E
J Cell Biol. 1987 Apr;104(4):1077-84. doi: 10.1083/jcb.104.4.1077.
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.
为了通过原位杂交鉴定负责产生I型、II型和III型胶原蛋白的细胞，对人类骨骼组织的石蜡切片进行了研究。利用Northern杂交和序列信息选择对应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切片的杂交中得到证实：已知分别仅产生一种类型的纤维状胶原蛋白（分别为I型和II型）的成骨细胞和软骨细胞仅被相应的cDNA探针识别。光滑结缔组织与I型和III型胶原蛋白cDNA探针表现出不同的杂交强度。该技术用于在长骨生长过程中定位软骨不同区域中II型胶原蛋白产生的活性。目视检查和颗粒计数显示，生长板软骨的下增殖区和上肥大区的软骨细胞中前α1（II）胶原蛋白mRNA水平最高。从骨骺（静止）软骨和生长区软骨分离的RNA的Northern印迹证实了这一发现。骨软骨交界处的分析显示，用I型和II型胶原蛋白mRNA特异性探针获得的杂交模式几乎没有重叠。在退变区，只有一小部分软骨细胞被前α1（II）胶原蛋白cDNA探针识别，而I型胶原蛋白cDNA探针未识别任何细胞。在矿化区，几乎所有细胞都被I型胶原蛋白cDNA探针识别，但似乎只有极少数散在细胞含有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，可用于鉴定在发育、分化和生长的各个阶段积极产生不同类型胶原蛋白的结缔组织细胞。
