Distribution Of Epidermal Stem Cells In Human Skin And Its Separation, Culture And Identification

1. Introduction The homeostasis of human skin mainly depends on the surviving epidermal stemcells (ESC) staying inside the basal layer of epidermis. Though rare (fewer than 10%)in total basal layer population, they have strong ability to proliferate and differentiate.ESC play a crucial role in skin renewal, wound healing, tumor formation andmaintenance of skin internal environment. It was found in clinic that to patients withserious burn and trauma, their healing ability of different part of skin is incompletelysame. With the increase of age, healing ability of skin wound decrease gradually, suchas in deep second degree burn patients, healing ability of the child evidentlyovermatch that of the older. The reason of this phenomenon involves several aspects.Quantity, location, and potential of proliferation and differentiation of ESC are likelyone of the most important factors. Researches have indicated that fetal skin epidermiscontain a large number of stem cells and transit amplifying cells, and the proportionof stem cells and transit amplifying cells decreases with age after birth, which maybea reason of the age associated difference in ability of wound healing. Is there anydifference in the number of ESC in different part of human skin? It is not clear yet. Toravel this question is very important for the study of development, differentiation,mechanism of healing of human skin. For patients suffering mass area burn injury,one of the most critical treatments is to cover the wound timely. Because donor site isscarce, how to create a useful skin substitute is one focus of medical researches. Thereis no special effective skin substitute being used in clinic yet. Skin substitutes beingtried now still have some problems which can not be solved such as metabolicinstability, quick apoptosis etc. If we can isolate large quantities of ESC and use themas seed cells for the constitution of tissue engineering skin, it maybe an expedient forthe study of man-made skin substitute. The study of ESC has extensive perspective. 5第二军医大学 英文接要 硕士学位论文The study of its development, differentiation and mechanisms of regulation isimperative to study the mechanism of wound healing, tumor formation and genetherapy etc.2. Objectives(1)To determine the distribution of ESC in different part of human skin andoptimize the choice of donor site in skin transplantation and isolation of ESC forculture.(2)To study the separation, culture and identification of ESC and observe thegrowth characteristic of ESC in vitro, to make it a basis for the future use of ESC asseed cells of tissue engineering skin.3. Materials and methodsPartⅠ Biopsys were performed on twelve different parts of five adult volunteers(male,18-45 years old). For each volunteer, normal skin of scalp, anterior trunk,posterior trunk, hip, inner side of thigh, outer side of thigh, inter side of the upper arm,outer side of the upper arm, palm, vola, foreskin and scrotum were taken. Thensamples were made into paraffin sections. K19 and integrinβ1 were used as thebiochemical markers for ESC identification. Immunohistochemistry was performed.All sections were processed in EnVision procedure. SPSS was used to analyze thedifference of K19 and integrinβ1 positive cells in different part of skin.PartⅡ Normal skin samples were collected from operation discards duringcircumcision. Epidermis was separated from dermis with 0.5% DispaseⅡ in 37℃for 2 h , then was digested for 10 min in 0.2% trypsin at 37℃ to dissociate into singlecells. Epidermal cells were divided into two group:adhering to collagen Ⅳ in 10min and adhering in 24 h. Two groups were both cultured in KSFM. Adherence rateand colony formation efficiency were observed. K19 and p63 were used as thebiochemical markers for ESC identification. Immunocytochemistry and flowcytometry were performed. SPSS was...