Cell biological and biochemical study on the mechanism and oxidative regulation of photoaging

由于与光老化过程相关的生化变化尚不完全清楚，积累的光氧化细胞损伤在皮肤光老化过程中起重要作用的理论仍然存在争议。在本研究中，我们证明了重复UVB照射可提高SOD活性，并逐渐恢复到对照水平，但不受UVA照射的影响。据报道，在急性光化损伤后，SOD活性会下降，这可能是由于反复暴露于UVB导致的，可能是对慢性光氧化应激的反应，然而，持续的累积应激可能会使这一系统的能力不堪重负，从而导致光老化症状。我们还用新开发的高效液相色谱方法研究了无毛小鼠皮肤经长期重复紫外线照射后GAG主要二糖单元的变化。我们发现，主要双糖单位…的总量36周时，UVA照射增加幅度更大。重复暴露36周后，DELTADI-HA(HA)含量依次降低，DELTADI-4S(DS)含量依次升高，UVA和UVB照射组DELTADI-HA(DS)含量依次升高。这些结果首次证明了GAG在总量和组成上的精确变化，证实了先前的组织化学发现。我们还建立了添加L-抗坏血酸-2-磷酸(AsC2-P)的真皮成纤维细胞三维培养体系。将ASC2-P修饰的成纤维细胞加入到体外真皮样三维结构的组织中，而不需要对塑料培养皿进行任何预处理。与单层培养体系相比，细胞外基质包裹的多层成纤维细胞易于观察到，其主要二糖单位和羟脯氨酸的含量均显著增加，且其组成更接近真皮。利用这种培养体系，可以研究从光老化皮肤获得的真皮成纤维细胞所产生的胶原的生化变化。较少

Since the biochemical changes associated with the photoaging process are not fully understood, the theory that accumulated photo-oxidative cellular injury contributes significantly to the photoaging process in skin still remains open to debate.In the present study, we have demonstrated that SOD activity was increased by repeated UVB irradiation and gradually returned to the control levels but unaffected by UVA irradiation. The SOD activity, which has been reported to decrease after acute actinic injury, is induced by repeated exposures to UVB presumably in response to the chronic photo-oxidative stress, however, continual cumulative stress may overwhelm the capacity of this system with resultant photoaging symptoms. We have also investigated the alteration of main disaccharide units of GAGs from hairless mice skin after chronic and repeated UV radiation using high performance liquid chromatography by our newly developed method. We found that the total amount of main disaccharide units … More increased by UVA irradiation at 36th week. Concerning the changes in the disaccharide composition after 36 weeks'repeated exposure, DELTADi-HA (HA) decreased and DELTADi-4S (DS) increased in the order of control, UVA- and UVB- irradiated group. These results, for the first time, demonstrate the precise alterations of GAGs both in total amount and composition confirming the previous histochemical findings. We have also developed the three-dimensional culture system supplemented with L-ascorbic acid 2-phosphate (Asc 2-P) using dermal fibroblasts. The addition of Asc 2-P rendered fibroblasts to the organization of the dermis-like three-dimensional structure in vitro without any pretreatments of plastic dish. Multilayred fibroblasts surrounded by extracellular matrices were readily observed and the amounts of both main disaccharide units and hydroxyproline are significantly increased and, furthermore, the composition revealed more similar to that of dermis when compared with monolayr culture system. Using this culture system, it should be possible to investigate the biochemical changes of collagen produced by dermal fibroblasts obtained from photoaged skin. Less