中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2011年
2期
110-113
,共4页
黑素细胞%角蛋白细胞%真皮%组织工程%皮肤
黑素細胞%角蛋白細胞%真皮%組織工程%皮膚
흑소세포%각단백세포%진피%조직공정%피부
Melanocytes%Keratinocytes%Dermis%Tissue engineering%Skin
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.
目的 探討體外構建含黑素的組織工程皮膚.方法 採用兩步酶消化法處理健康小兒包皮,穫得錶皮細胞懸液,分彆用人角質形成細胞(KC)無血清培養基(SFM)及改良的M254黑素細胞培養基培養KC、黑素細胞(MC),併傳至第3代.將第3代KC接種于培養瓶中,48 h後加入第3代MC(MC:KC為1:10)混閤培養.製備人去錶皮的真皮(DED),將培養第3代的MC、KC製成細胞懸液,按照1:10的比例接種于DED錶麵,採用液下培養和空氣-液麵培養相結閤的方式進行培養,2週後取培養的組織工程皮膚分彆做HE染色、角蛋白免疫組化染色及Masson-Fontana染色.結果 KC、MC混閤培養于培養皿5 d後,于倒置顯微鏡下觀察到KC呈鋪路石狀生長,其間散佈MC,且樹突延伸到KC細胞間.兩種細胞混閤接種于DED 15 d後,HE染色顯示在DED上有3～6層錶皮細胞,併可見角質層,角蛋白免疫組化染色暘性,銀氨染色顯示基底層見黑素著色.結論 MC、KC混閤接種于培養皿可構建MC和KC接觸生長的單細胞層,將MC、KC接種于DED可構建含有黑素成分的組織工程皮膚.
목적 탐토체외구건함흑소적조직공정피부.방법 채용량보매소화법처리건강소인포피,획득표피세포현액,분별용인각질형성세포(KC)무혈청배양기(SFM)급개량적M254흑소세포배양기배양KC、흑소세포(MC),병전지제3대.장제3대KC접충우배양병중,48 h후가입제3대MC(MC:KC위1:10)혼합배양.제비인거표피적진피(DED),장배양제3대적MC、KC제성세포현액,안조1:10적비례접충우DED표면,채용액하배양화공기-액면배양상결합적방식진행배양,2주후취배양적조직공정피부분별주HE염색、각단백면역조화염색급Masson-Fontana염색.결과 KC、MC혼합배양우배양명5 d후,우도치현미경하관찰도KC정포로석상생장,기간산포MC,차수돌연신도KC세포간.량충세포혼합접충우DED 15 d후,HE염색현시재DED상유3～6층표피세포,병가견각질층,각단백면역조화염색양성,은안염색현시기저층견흑소착색.결론 MC、KC혼합접충우배양명가구건MC화KC접촉생장적단세포층,장MC、KC접충우DED가구건함유흑소성분적조직공정피부.
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.
