Biological characteristics of the normal human keratinocytes during the aging process

Xuejun GAO; Xia CAI; Peng ZHANG; Shengjian TANG

ABSTRACT

BACKGROUND:

At present, there is still lack of related reports about the aging process of in vitro cultured epidermal cells, since epidermal cells are seed cells necessary for the construction of tissue engineered skin, this articleis is aimed to investigate the biological property of normal human epidermal cells during aging process so as to provide a foundation for the selection of seed cells for tissue engineered skin

OBJECTIVE:

To observe the in vitro proliferation and aging property of human epidermal cells in order to provide a foundation for the proper selection of seed cells for tissue engineered skin.

DESIGN:

A self-comparative experiment.

SETTING:

Orthopedic Surgery Research Instioute of Weifang Medical College and the General Surgery Department of Weifang Medical College Affiliated Hospital.MATERIALS This experiment was carried out at the Orthopedic Surgery Research Institute of Weifang Medical College, between September 2000and September 2002. Healthy foreskin tissue was obtained from 20 normal boys of 6-8 years old who received peritomy at the General Surgery Department of Weifang Medical College Affiliated Hospital.

METHODS:

Epidermal cells were obtained from normal young people for subculture. Cells were collected from different culture passages and taken as subjects, and their aging characteristics were assessed through morphological observation, population doubling time (PDT), immune cytochemistry and beta-galactosidase staining. MAIN OUTCOME

MEASURES:

① The changes of the epidermal cell growth characteristics. ② The morphological changes of the epidermal cells. ③ The epidermal cell phenotypic changes.

RESULTS:

① The clanges of the epidermal cell growth characteristics Cells were in vitro cultured by monolayer for 9 passages, and PDT of P2 was the shortest. The cells showed strong proliferation in the first 5 passages.From P6, PDT was obviously prolonged, but the cells from P8 did not proliferate any longer. ② The morphological changes of epidermal cells The primary cultured cells began to proliferate 3 days later, which accelerated 4 days later. The cells became approximately fused in about 1 week. The growth of epidermal cells was identified with a microscope and the immuno histological techniques. ③ The epidermal cell phenotypic changes Along with the consecutive subculture, histological expression of beta-galactosidase was found to show an increasing tendency from weak expression (occupying 9% of the young cells) to strong expression (occupying 65% of aging cells), and the positive expression rate of beta-galactosidase was found to be remarkably correlated with cell passage age (r=0.87, P ＜ 0.01).

CONCLUSION:

① Compared with young cells, aging cells displayed more obvious aging morphology and enzyme cyto-chemical characteristics.During the cell aging process, the PDT of cells showed an increasing tendency. ②Compared with young cells, the expression of beta-galactosidase in aging cells was remarkably increased, and this increase paralleled with the appearance of cell aging phenotype and the loss of cell proliferation capability, and reflects the aging degree of cells. ③ The in vitro cultured normal human epidermal cell aging model was established in this experiment. The results of this experiment indicated that epidermal cells from the 1st -5th passage (donators aged 16-18 years old) can be taken as the optimal seed cells for tissue engineered skin construction.