Linsenmayer T F, Igoe F, Gibney E, Gordon M K, Birk D E
Department of Anatomy and Cellular Biology, Tufts University Medical School, Boston, Massachusetts 02111, USA.
Exp Cell Res. 1996 Oct 10;228(1):36-43. doi: 10.1006/excr.1996.0296.
Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for alpha1(V) chain mRNA, and this effect is at least somewhat cell specific.
我们之前的研究表明，V型胶原蛋白至少是导致角膜基质中条纹状胶原纤维直径特征性小且均匀的一个因素。这些纤维是I型和V型胶原蛋白的异型组合，与肌腱和巩膜的纤维相比，其V型胶原蛋白含量多四到五倍。肌腱和巩膜的纤维要大得多且更加异质分散。角膜中V型胶原蛋白的这种高含量反映在角膜成纤维细胞中α1(V)链mRNA的含量同样升高。因此，角膜中该分子产量的增加似乎在转录水平和/或mRNA稳定性水平受到调控。对此的一种可能解释是，角膜成纤维细胞含有特异性上调V型胶原蛋白基因转录和/或增加其mRNA稳定性的正调控因子。为了验证这种可能性，我们通过将鸡角膜成纤维细胞与两种选定的几乎不含α1(V)mRNA的人非角膜细胞系融合，产生了瞬时异核体。如果鸡角膜细胞含有能够跨物种起作用的正调控因子，那么这些调控因子应该会导致人α1(V)转录本水平升高。通过使用一对因其能够特异性扩增人α1(V)mRNA的一部分而选择的引物进行逆转录-聚合酶链反应来评估结果。在鸡角膜成纤维细胞与人细胞系的融合中，经过10 - 14小时的延迟后，与亲代细胞系相比，含有异核体的培养物显示出人α1(V)链mRNA的从头出现或上调。未融合的混合细胞类型培养物未显示出这种上调，因此这种效应不是由细胞间作用的可扩散物质介导的。在相同检测中测试时，V型胶原蛋白低产的鸡肌腱成纤维细胞未引起人转录本的可检测增加。因此，角膜细胞确实含有α1(V)链mRNA的正调控因子，并且这种效应至少在某种程度上具有细胞特异性。
