无血清无滋养层培养的人角质形成细胞生物学特征

目的建立一种人角质形成细胞(HKC)无血清、无滋养层培养方法,观察用该方法培养的HKC的生物学特征。方法取5~10岁儿童及20~30岁成人(各5例)环切术后包皮,分为儿童组和成人组。采用二步消化法分离人包皮标本,检测其原代HKC获得数;用KC无血清培养液培养,光镜下观察HKC形态;荧光显微镜下鉴定HKC,并观察其生长速度;用噻唑蓝(MTT)法检测HKC生长曲线,流式细胞仪检测细胞周期。结果儿童组原代HKC获得数为(1.780±0.010)×106/cm2,较成人组(1.490±0.120)×106/cm2高(P<0.01).HKC形态学观察见刚分离的HKC为透亮的小圆形细胞,台盼蓝染色约94%细胞拒染,多次传代的HKC贴壁速度、贴壁率及透亮度明显增加;荧光显微镜下见细胞胞浆呈强黄绿色荧光,细胞核未着色,证明其为KC.儿童组HKC传代次数为(11.0±1.2)次,较成人组(9.2±0.8)次高(P<0.05).HKC生长曲线无明显潜伏期,细胞增殖速度快,扩增倍数高。G1期细胞为36.15%,G2期细胞为25.17%,S期细胞为38.68%,细胞增殖指数为63.85%。结论无血清、无滋养层培养,是一种较理想的培养HKC的方法。

Biological characteristics of human keratinocytes cultured in serum-free medium without feeder layer

OBJECTIVE: To establish an optimal method for serum-free and feeder layer-free culture of human keratinocytes and to investigate their biological characteristics. METHODS: The keratinocytes were harvested from human foreskin of 5 children (aged 5-10 yr) and 5 adults (aged 20-30 yr). The samples were isolated by two-step digestion and the quantities of primary harvested HKCs were determined. The HKCs were then cultured in KCS serum-free culture medium. The morphology of HKCs were observed under light microscope. The HKCs and their growing speed were observed and identified under fluorescent microscope. The growth curve of HKCs was detected with MTT method, and the cell cycle was determined with flow cytometry. RESULTS: The number of harvested HKCs from children [(1.780 +/- 0.010) x 10(6)/cm(2)] was obviously higher than that from adults [(1.490 +/- 0.120) x 10(6)/cm(2)], (P < 0.01). Freshly isolated primary HKCs were round and transparent, and 94% of them were trypan blue resistant. The adherent speed and rate and lucent degree of multiply passaged HKCs increased followed by each passage. Under the fluorescent microscope, the cells exhibited strong Kelly fluorescence in the cytoplasm and with no staining in the nucleolus, thus the cells were identified as HKCs. The HKCs from children for skin could be passaged for more times [(11.0 +/- 1.2) times] than that from adults [(9.2 +/- 0.8) times], (P < 0.05). There was no clear sign of incubation period in the growth curve of HKCs, and both cellular proliferating speed and rate of proliferation were high. The percentage of cells in G1, G2 and S phase and the proliferation index was 36.15%, 25.17%, 38.68% and 63.85%, respectively. CONCLUSION: Serum-free and feeder layer-free culture seems to be an ideal method for the cultivation of HKCs.