Booth B A, Polak K L, Uitto J
Biochim Biophys Acta. 1980 Mar 28;607(1):145-60. doi: 10.1016/0005-2787(80)90228-2.
Skin fibroblasts in culture can provide a convenient means to study aberrations of collagen metabolism in a variety of clinical conditions. In the present study, the culture conditions for the synthesis of procollagen by cultured human skin fibroblasts were optimized by independently varying parameters in the cell culture environment. To study the synthesis of procollagen the cell cultures were labeled with [3H]proline and the collagenous polypeptides were determined either by measuring the synthesis of hydroxy[3H]proline or by assaying the 3H-labeled proteins digested into dialyzable 3H-labeled peptides by bacterial collagenase. On the basis of the experimental results, the following culture conditions are suggested for optimal synthesis of procollagen: (a) cell culture medium should be supplemented with ascorbic acid (25--50 micrograms/ml) and fetal calf serum (20%); (b) the pH of the culture medium should be kept above 7.2 and preferably in the pH range 7.5--7.8; (c) the cell cultures should be used one to two days after reaching visual confluency. Under these conditions the synthesis and secretion of [3H]procollagen was found to be linear through a 24 h incubation period, and procollagen was demonstrated to be a major gene product of the fibroblasts. The relative synthesis of type I and type III procollagens was also monitored by isolating these genetically distinct procollagens by DEAE-cellulose chromatography or by measuring type I and III collagens by sodium dodecyl sulfate polyacrylamide gel electrophoresis after limited pepsin proteolysis. No marked changes were observed in type I/III procollagen ratios in situations where the total formation of hydroxy[3H]proline was significantly affected. The average coefficient of variance for procollagen synthesis between replicate cultures was found to be relatively small (14%), and the optimization of the culture conditions for the control cells has, therefore, created a reliable and reproducible basis for employing human skin fibroblasts to study collagen metabolism in acquired and inherited diseases.
培养的皮肤成纤维细胞可为研究多种临床病症中胶原代谢异常提供便利手段。在本研究中，通过独立改变细胞培养环境中的参数，优化了培养的人皮肤成纤维细胞合成前胶原的培养条件。为研究前胶原的合成，用[3H]脯氨酸标记细胞培养物，通过测量羟[3H]脯氨酸的合成或通过检测经细菌胶原酶消化成可透析的3H标记肽的3H标记蛋白来测定胶原多肽。根据实验结果，建议采用以下培养条件以实现前胶原的最佳合成：(a)细胞培养基应补充抗坏血酸(25 - 50微克/毫升)和胎牛血清(20%)；(b)培养基的pH值应保持在7.2以上，最佳为7.5 - 7.8；(c)细胞培养物应在达到肉眼可见的汇合状态后1至2天使用。在这些条件下，发现[3H]前胶原的合成和分泌在24小时孵育期内呈线性，并且前胶原被证明是成纤维细胞的主要基因产物。还通过DEAE - 纤维素色谱法分离这些基因不同的前胶原，或在有限的胃蛋白酶水解后通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳测量I型和III型胶原，来监测I型和III型前胶原的相对合成。在羟[3H]脯氨酸的总形成受到显著影响的情况下，未观察到I/III型前胶原比率有明显变化。发现重复培养之间前胶原合成的平均变异系数相对较小(14%)，因此，优化对照细胞的培养条件为用人皮肤成纤维细胞研究获得性和遗传性疾病中的胶原代谢创造了可靠且可重复的基础。
