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大多数应用动物模型或成纤维细胞培养的研究中显示糖皮质激素降低胶原的合成,但也有报告无影响或相反的结果。在临床上糖皮质激素与维甲酸(RA)有时合并应用。有实验证明RA 能拮抗糖皮质激素对胶原合成的影响。基于上述情况,作者用人类成纤维细胞(HSFs)培养研究地塞米松(Dex)与RA 对结缔组织生物合成的影响。将HSFs 分别在含有Dex、13-顺维甲酸、依曲替酯及乙醇的培养基中孵育4～48小时。再用~(14)C脯氨酸标记细胞4～16小时,然后测定总的及~(14)C羟脯氨酸的放射活性,一部分细胞在声波处理后用作DNA 分析。纤维结合素及前胶原的相对量用光密度计扫描分析。应用Chirgwin 等的方法从孵育48小时的HSFs 中提取总的RNA,并分别以PHCAL1、PHFS3、PFH-1及PRGAPDH-13等cDNA 探针探测前α_1(Ⅰ)胶原mRNA、前α_1(Ⅲ)胶原mRNA、纤维结合素mRNA 及3-磷酸甘油醛脱氢酶(3-GAPDH)mRNA。特异性mRNA 的量借X 线底片多次曝光后行光密度计扫描。
Most studies using animal models or fibroblast cultures have shown that glucocorticoids reduce the synthesis of collagen, but there are also reports of no or adverse consequences. Clinically glucocorticoid and retinoic acid (RA) are sometimes used in combination. Experiments show that RA can antagonize glucocorticoid on collagen synthesis. Based on the above, the authors used human fibroblasts (HSFs) to study the effects of dexamethasone (Dex) and RA on connective tissue biosynthesis. HSFs were incubated for 4 to 48 hours in media containing Dex, 13-cis-retinoic acid, ettringit and ethanol, respectively. The cells were then labeled with ~ (14) C-proline for 4 to 16 hours and then the total and ~ (14) C hydroxyproline radioactivity was measured, and some of the cells were used for DNA analysis after sonication. The relative amounts of fibronectin and procollagen were scanned by densitometry. Total RNA was extracted from HSFs incubated for 48 hours by Chirgwin et al. The mRNA of pre-α1 (Ⅰ) collagen and pre-α1 (Ⅲ) collagen were detected with cDNA probes such as PHCAL1, PHFS3, PFH-1 and PRGAPDH- mRNA, fibronectin mRNA and glyceraldehyde-3-phosphate dehydrogenase (3-GAPDH) mRNA. The amount of specific mRNA by X-ray film after multiple exposure scanning densitometer.