摘要
目的 探讨体外构建含黑素的组织工程皮肤.方法 采用两步酶消化法处理健康小儿包皮,获得表皮细胞悬液,分别用人角质形成细胞（KC）无血清培养基（SFM）及改良的M254黑素细胞培养基培养KC、黑素细胞（MC）,并传至第3代.将第3代KC接种于培养瓶中,48 h后加入第3代MC（MC：KC为1：10）混合培养.制备人去表皮的真皮（DED）,将培养第3代的MC、KC制成细胞悬液,按照1：10的比例接种于DED表面,采用液下培养和空气-液面培养相结合的方式进行培养,2周后取培养的组织工程皮肤分别做HE染色、角蛋白免疫组化染色及Masson-Fontana染色.结果 KC、MC混合培养于培养皿5 d后,于倒置显微镜下观察到KC呈铺路石状生长,其间散布MC,且树突延伸到KC细胞间.两种细胞混合接种于DED 15 d后,HE染色显示在DED上有3～6层表皮细胞,并可见角质层,角蛋白免疫组化染色阳性,银氨染色显示基底层见黑素着色.结论 MC、KC混合接种于培养皿可构建MC和KC接触生长的单细胞层,将MC、KC接种于DED可构建含有黑素成分的组织工程皮肤.
Objective To construct tissue-engineered skin containing melanin with mixed culture of human keratinocytes （KCs） and melanocytes （MCs） on de-epidermized dermis （DED） in vitro. Methods Single-cell suspension was obtained by digestion of isolated preputial epidermis with pancreatin. Keratinocyte serum-free medium （K-SFM） and modified M254 culture medium were used to culture KCs and MCs respectively. Third-passage KCs were seeded into cell culture flasks and cultured for 48 hours; then, third-passage MCs were seeded into the same cell culture flasks with the MC/KC ratio being 1： 10 followed by a 5-day coculture. The suspension of third-passage KCs and MCs with the MC/KC ratio of 10：1 were seeded onto the surface of a prepared DED and maintained at the air-liquid interface for 11 days following a 4-day submerged culture.Subsequently, the constructed tissue-engineered skin was examined with HE staining, immunohistochemical staining for keratin and Masson-Fontana staining. Results After coculture in flasks for 5 days, KCs exhibited a typical paving-stone appearance, MCs with projected dendrites were scattered in the extracellular space between KCs. HE staining revealed 3 to 6 layers of cells with the formation of stratum corneum after mixed culture on DED for 15 days. Keratin protein was positive throughout the artificial epidermis, and melanin pigments were located in the basal layer of the epidermis as Masson-Fontana staining showed. Conclusions The co-culture of MCs and KCs can form single-cell layers with the contact between MCs and KCs in flasks, and construct tissue-engineered skin with melanin component on DED in vitro.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2011年第2期110-113,共4页
Chinese Journal of Dermatology
基金
国家自然科学基金（30872283）
