Youn Sang Woong, Kim Dong Seok, Cho Hyun Joo, Jeon Sang Eun, Bae In Hwa, Yoon Ho Joon, Park Kyoung Chan
Department of Dermatology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-city, Kyounggi-do 463-707, South Korea.
J Dermatol Sci. 2004 Aug;35(2):113-23. doi: 10.1016/j.jdermsci.2004.04.002.
It is known that cellular senescence could affect culture results. A previous study on K19 found that the loss of stem cell proportion is the reason for difficulties experienced when culturing aged keratinocytes. But the situation is unclear, because K19 is not generally accepted as an epidermal stem cell marker.
The aim of this study was to investigate the effects of cellular senescence caused by chronological aging or by repeated subcultures.
The effects of cellular senescence were investigated using monolayer cultures of keratinocytes and reconstructed epidermis. We prepared keratinocytes from donors of different ages and by repeated subcultures. Flow cytometric analysis was performed using alpha6 integrin and CD71 antibodies, and candidate keratinocyte stem cell proportions were separated according to reactivities to these antibodies. Living skin equivalents (LSEs) were reconstructed using keratinocytes from child, adult and elderly donors.
Flow cytometric analysis showed a decrease in the candidate stem cell proportion in an age- or culture passage-dependent manner. LSE experiments showed that a reconstructed epidermis using child's keratinocytes was well formed compared to epidermis reconstructed using an elderly donor's keratinocytes. Different expression of proliferation markers was also observed according to donor age.
Our results showed that cellular senescence by chronological aging or repeated sub-culture induced the loss of candidate stem cell proportion in keratinocyte cultures. This seems to be the reason why it is difficult to culture keratinocytes from the elderly or by repeatedly culturing keratinocytes in vitro.
已知细胞衰老会影响培养结果。先前一项关于K19的研究发现，干细胞比例的降低是培养老年角质形成细胞时遇到困难的原因。但情况尚不清楚，因为K19并未被普遍接受为表皮干细胞标志物。
本研究旨在调查自然衰老或反复传代培养所导致的细胞衰老的影响。
使用角质形成细胞单层培养和重建表皮来研究细胞衰老的影响。我们从不同年龄的供体以及通过反复传代培养制备角质形成细胞。使用α6整合素和CD71抗体进行流式细胞术分析，并根据对这些抗体的反应性分离候选角质形成干细胞比例。使用来自儿童、成人和老年供体的角质形成细胞重建活皮肤替代物（LSE）。
流式细胞术分析显示，候选干细胞比例以年龄或传代培养依赖性方式降低。LSE实验表明，与使用老年供体角质形成细胞重建的表皮相比，使用儿童角质形成细胞重建的表皮形成良好。根据供体年龄还观察到增殖标志物的不同表达。
我们的结果表明，自然衰老或反复传代培养导致的细胞衰老会导致角质形成细胞培养中候选干细胞比例的丧失。这似乎是难以培养老年角质形成细胞或通过体外反复培养角质形成细胞的原因。
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