Effect Of Epidermal Growth Factor On Proliferation And Migration Of Amelanotic Melanocytes Of Human Hair Follicle

Objective: To investigate the effect of epidermal growth factor(EGF) in different concentration on proliferation and migration ofamelanotic melanocytes (AMMC) of human hair follicle. Methods: Aftersigning the informed consent document with the scalp providers, thenormal human scalp with hair follicles was harvested. Then the fat andthe tissue about1mm above the dermis were removed. The normal humanscalp with hair follicles was incubated with1%dispase Ⅱ. And thendigested with0.5%pancreatin. Hair follicle cells were cultured inmedium MCDB153with fetal bovin serum (FBS) chelated by chelex100.Hair follicle cells suspensions were isolated by variant enzyme treatment.Then the cells of third passage was confirmed by staining withmonoclonal antibodies of NKI/beteb and HMB-45. The AMMCs wererandomly divided into five groups according to the differentconcentration of EGF, which include0ng/ml(the blank group),1ng/ml,10ng/ml,20ng/ml and50ng/ml respectively. After incubated with fivedifferent concentration of EGF for48h, the proliferation of AMMC wasconfirmed by means of CCK8chromatometry.The third passagedAMMCs were cultured in medium MCDB153without FBS for24h. Thenmigration of AMMC which incubated with five different concentration ofEGF was confirmed by means of transwell chamber migration assay after cultured48h.Using spss11.5statistic software by one-way ANOVA.method to analyse the data.Results: AMMC cultured in mediumMCDB153with FBS chelated by chelex100and other factors couldgrow conveniently,and it can be isolated by variant enzyme treatment.Theantibodies of the cells of third passage were positive to NKI/beteb,whichwere negative to HMB-45.The OD values of CCK8test of AMMCs were0.1968±0.1763in blank group,0.2986±0.1773in1ng/ml EGF group,0.6302±0.2292in10ng/ml EGF group,0.7414±0.2177in20ng/ml EGFgroup and0.8198±0.2604in50ng/ml EGF group. The blank group’sOD values was less than it in10ng/ml EGF group,20ng/ml EGF groupand50ng/ml EGF group respectively(P <0.05). The OD values of1ng/mlEGF group was significantly lower than it in10ng/ml EGF group,20ng/ml EGF group and50ng/ml EGF group respectively(P <0.05). Butthere were no deference of OD values between blank group and1ng/mlEGF group(P>0.05). There were also no deference of OD valuest of10ng/ml EGF group,20ng/ml EGF group and50ng/ml EGF(P>0.05).The numbers of migrating AMMCs by transwell chamber migrationassay were5.88±2.03in blank group,6.75±2.71in1ng/ml EGF group,22.50±5.71in10ng/ml EGF group,54.75±6.54in20ng/ml EGF groupand55.75±6.30in50ng/ml EGF group respectively.The numbers ofmigrating AMMCs of blank group were significantly less than it in10ng/ml EGF group,20ng/ml EGF group and50ng/ml EGF group respectively(P <0.05). The numbers of migrating AMMCs of1ng/mlEGF group were significantly less than it in10ng/ml EGF group,20ng/mlEGF group and50ng/ml EGF group respectively(P <0.05). The numbersof migrating AMMCs of10ng/ml EGF group were significantly less thanit in20ng/ml EGF group and50ng/ml EGF group (P <0.05).But therewere no deference of numbers of migrating AMMCs between blankgroup and1ng/ml EGF group(P>0.05). There were also no deference ofnumbers of migrating AMMCs between20ng/ml EGF group and50ng/mlEGF group(P>0.05). Conclusion:The proliferation and migration ofAMMC could be induced by EGF in certain concentration,rather than inconcentration-dependent manner.