| Object:This study was designed to investigate the effect of ACE inhibitors on the migration,proliferation of epidermal stem cells during wound healing. We also observed thelocation of ACE and epidermal stem cells in normal rat skin and wounded site, and tryto get a new information to improve the wound healing.Methods:1. To label the ESCs of wistar rat by Label-retaining technique: Rats were injectedintraperitoneally with50mg/kg body weight5-bromo-2’-deoxyuridine (BrdU;Sigma Chemical Co) twice a day (at8:00AM and6:00PM) for4consecutivedays. Two months later, they were prepared for experiments.2. To develop animal model:1) The experimental rats were injected intraperitoneallywith10mg/kg body weight with Captopril (Sigma Chemical Co), begining on oneday before wounding, once a daily, for a period of the day before killed.2) Thecontrol rats were injected intraperitoneally with physiological saline. All ratswere anesthetized with an intraperitoneal injection of sodium pentobarbital(30mg/kg body weight). The dorsal regions (1cm below scapula and1cm besidespine) were shaved with a razor blade after subs lubrication and the surgical areawas disinfected with70%alcohol. A round section of full-thickness skin (diameter:1.5cm) was resected with scissors and hemostasis was obtained bydirect pressure using sterile gauze. Wound closure was measured at0,3,7,11days (five rats per group per time point) after wounding. Animals wereeuthanized at each time point and the wound samples including adjacent normalskin were harvested and fixed in10%formalin for histological andimmunochemical assay, or snap-frozen in liquid nitrogen for further analysis.3. Estimation of wound healing: Curative effect on the wound (wound closure) wasevaluated by tracing the outer margins of the wound on each rat. Wound areawas measured at0,3,7,11days (5rats per treatment group per time point) afterwounding and the wound closure rate was expressed as the percentage of woundarea compared with that on postoperative day. The following formula was usedto calculate the percentage of wound contraction.Wound contraction%=wound area day0-wound area day(n)wound area day0×100%4. Using the H&E (haematoxylin and eosin) sections, the extent of re-epithelizationof wounds was evaluated by measuring the epidermal migration from the normalwound margin to the point where the migrating epithelium stopped processing.The re-epithelization index was determined by the length of the new epitheliumpresent in the one side wound.5. Five-micrometer sections were cut for immunohistochemical study using thelabeled streptavidin-biotin-peroxidase method as described previously. Theprimary antibody used in this study was Mouse monoclonal anti-ACE antibody(ab-77990,1:100)(abcam Co) and Mouse monoclonal anti BrdU (sc-32323,1:100)(Santa Cruz, CA, U.S.A).Result:1、By viewing the five random visual field of each normal skin sample, wecalculated the rate of the LRCs which just lied in basal layer. The rate of the LRCs was between1%and5%, and the average rate was (2.25±1.34)%.2、The macroscopic evaluation of the wound revealed that both groups required atotal period of about11days for near complete epithelialization and healing.Both of them showed an almost similar pattern of wound contraction at3,7and11days after wound. The area of unhealed wound and the wound closure ratewere measured based on the image using Image-J software. The area of unhealedwound of Captopril group was inhibited as to the control group at3,7and11daysafter wound, also the wound closure rate of Captopril group had a significantdecrease at7days compared to control group (P<0.05).3、The effect of captopril on re-epithelization in wound edge was evaluatedkinetically. On day11, the control group and the group with captopril show100%re-epithelization of the wound. However, the group with captopril, on day3and7, show delayed re-epithelization of the wound. On day7, administration ofcaptopril significantly inhibited (P<0.05) the keratinocyte re-epithelization.4、During the wound healing process, the wound edge of both group was began toobserve LRCs on day3after wound, and it increased to peak on7th day.However, the administration of Captopril inhibited the rate of LRCs at eachselected time point. The LRCs rate of wound edge significantly suppressed(P<0.05) on7th day compared to the control.5、In normal skin of rat, the location of the ACE was almost seen in basal layer. Onthe day3after wound, ACE positive cells were seen in the spinous and granularlayer of regenerated-epithele of wound edge, and seem to be increase， with apeak on day7. Additionally, there was the colocalization of expression of ACEand BrdU during wound healing.Conclusion:1、ACE share the common localization with BrdU positive cells，in normal skin andwound healing process.2、Epidermal stem cell participated in the healing of wound and promoted the healing of granulating wounds through migrating and differentiating in the wound.And the epidermal stem cells maybe the main source of re-epithelialization ofgranulating wounds.3. Administration of Captopril could impair wound healing by inhibiting themigration and proliferation of epidermal stem cells. This phenomenon suggestedthat ACE could affect the wound healing through regulating the biologicalbehavior of epidermal stem cells during wound healing. |
PDF Full Text Request