| ObjectivesTo study the effect of full thickness skin wound healing with oophorectomy, and the expression of the estrogen receptors in rat skin during full-thickness skin wound healing; further, to observe the expression of estrogen receptors in human epidermal stem cells, to investigate the effect of estrogen on the three-dimensional culture of epidermal stem cell proliferation and migration, and to elucidate its potentially important role during skin wound healing.Methods1. Animal experiment 40 two-month old Wistar rats were used in this research, divided into 3 groups at randomly. Group A consisted of 10 rats which received a placebo procedure, Group B consisted of 15 rats that underwent the oophorectomy procedure in addition to the surgery, and Group C consisted of 15 rats which only had the surgery performed on them. After 8 weeks, the full-thickness skin wounds were developed in the back of each rat and were excised and longitudinally cut in half through the least-healed portion. The wound healing rate was observed by measuring the wound area from day 1 to 21, and wound cavity volume of 3 groups were observed under histology. The expression of estrogen receptors was determined by immunofluorescence staining technique. 2. Cell experiment Human embryonic stem cells (hESCs) were isolated from normal human foreskin and cultured in medium, and second generation hESCs were identified through flow cytometry after being marked with integrin ?1,CK14,CK19 and CK10 antigens, then hESCs were randomly divided into 3 groups. hESCs were cultured with Epilif and mixed with 0.1nmol/l Diethylstilbestrol in the estrogen group(n=9)[Group A]. hESCs were cultured with Epilif and mixed with 1nmol/l Raloxifene hydrochloride in the estrogen receptor blocking agent group(n=8)[Group B]. hESCs were cultured with only Epilif in blank group(n=8)[Group C]. ER-? of hESCs were identified with flow cytometry for the 3 groups. A 100μm"wound"of epidermis was subsequently reproduced by scraping confluent hESCs cells under an inverted microscope. The number of cells migrating across the edge of the"wound"was counted, and the rate of"wound healing"was calculated by SigmaScan Pro5 software. The proliferating effect hESCs had on estrogen was determined with the MTT method.Results1. Animal experiment To compare the injury cavity volume and rate of wound healing at each interval, Group A and Group C had an injury cavity volume significantly smaller than that of Group B; Group A and Group C had a healing rate that was significantly higher than Group B (P <0.01), while the difference between Group A and Group C was not significant. During the early injury stage (days 1-3), ER-βgradually increased, mainly in the dermis layer. Furthermore, there was no significant difference in the expression of ER-βbetween the three groups. After 7 days, the expression of ER-βgradually increased, mainly in the dermis, but some in the epidermis. After 14 days, the expression of ER-βremained high, particularly in the epidermis. Furthermore, expression in Group A and Group C was significantly higher. After 21 days, the expression of ER-βhad been weakened, but the expression in the epidermis and dermis vascular was still strong.2. Cell experiment Flow cytometry revealed that hESCs were positive for integrin ?1,CK19 and CK14 (96.63%,95.47%,94.27%), and negative for CK10 (1.32%). Flow cytometry showed hESC were ER-? in A, B or C (66.43%, 43.47%,54.27%). In Groups A, B, and C, the number cells crossing the edge of wound was (33.6±0.34), (8.5±0.75) and (11.4±0.45) cells/HP at 24 hours; (67.5±0.24), (27.5±0.35) and (33.5±0.45) cells/HP at 48 hours; (117.5±0.35), (63.5±0.35) and (78.5±0.45) cells/HP at 72 hours after culturing(P<0.01), respectively. The rate of wound healing in the respective groups was (69.00±0.05)%, (35.00±0.05)% , and (48.00±0.06)% (P<0.01), and the proliferation speed of hESCs in the three groups had a significant difference (P<0.01).ConclusionsWound healing was slowed down after oophorectomy; therefore, the female hormone system was involved in all stages of wound healing, and potentially affected the rate of wound healing in rats. Estrogen is capable of promoting the proliferation of epidermal stem cells and migration capabilities. As an important role in skin damage repair activities, estrogen also accelerates the clinical rate of wound healing and improves the quality of tissue repair; this provides a new way for the treatment of refractory wounds.
|
PDF Full Text Request