Expressions Of SOX9 And ETS-1 Proteins And Migration Capabilities In The Emigrating Cells From Human Hair Follicles Bulge

BackgroundVitiligo is a acquired skin and mucosal disease of depigmentation.The clinical manifestation is leukoplakia,and the hair follicles are affected as gray hair and white vellus hair.Vitiligo can be involved in any parts of the body,and it usually occurs in exposed,rubbed,and wrinkled areas,such as the face,neck,hands,wrists,forearms,abdomen,and vulva.Because of its impairing appearance,it brings mental pressure and social burden to patients.Even if the skin lesions occur in the clothing-covered area,patients often worry about progressing to the exposed area or expanding the area,accompanied by psychological disorders,such as anxiety and depression.But at present,vitiligo is incurable.Common clinical treatments,such as topical corticosteroids,calcineurin inhibitors,narrow band ultraviolet(NB-UVB)and 308 nm excimer laser have different effects from person to person.Therefore,further study in exploring the mechanism of vitiligo repigmentation can be effective for vitiligo treatment methods and achieve the goal that vitiligo is curable.Hair follicle(HF)is a unique structure.During morphogenesis,epithelial tissue and mesenchymal tissue recombine to form a fully differentiated fur fat unit.Recent studies have shown that there are neural crest stem cells(NCSCs)in the hair follicles bulge area formed by the outer root sheath(ORS),which express ETS-1,MSX2,NESTIN,SOX9,SOX10 and other stem cells markers.NCSCs has a variety of differentiation potentials and can differentiate into melanocytes,bone cells,chondrocytes,neurons and Schwann cells after induction.A number of studies have confirmed that neural crest stem cells(NCSCs)in the human hair follicles bulge area can differentiate into melanocytes in vitro.Based on this,we assume that the melanin from melanocyts which are derived from EPI-NCSCs can promote the repigmentation of vitiligo skin lesions.The neural crest stem cells markers SOX9 and ETS-1 proteins play the role of maintaining the characteristics of stem cells and promoting stem cells migration.This study intends to detect the expressions of SOX9 and ETS-1 proteins in the emigrating cells form cultured normal human hair follicle bulge and identified them as neural crest stem cells.Likes embryonic stem cells,human hair follicle neural crest stem cells may also have migratory capabilities and can emigrate and purify from the hair follicles bulge region cultured in vitro.This research is aim at exploring the significances of the expressions of SOX9 and ETS-1 proteins and migration capabilities in human hair follicles neural crest stem cells,then provide a scientific basis for the use of human hair follicle neural crest stem cells in the treatment of vitiligo.Objectives1.This experiment was designed to observe the expressions and localizations of the neural crest stem cells markers SOX9 and ETS-1 proteins in the emigrating cells from human hair follicles bluge.2.An investigation into the migration capabilities in the emigrating cells from human hair follicles bluge.Methods1.The specimens were normal human scalp tissues containing about 20-30 hair follicles,and were taken from trauma surgery patients in the Department of Neurosurgery and Plastic Surgery of the First Affiliated Hospital of Zhengzhou University.Selection criteria:(1)No gender limitation,age 18-60 years old;(2)Those with non-pigmented,infectious and tumorous diseases on the scalp;(3)Those who have no use of medicated or bathing history on the scalp in the past 1 month.In a sterile environment,rinse the scalp tissue block with 5%volume fraction penicillin-streptomycin DPBS buffer,and cut into small tissue blocks containing several hair follicles along the direction of the hair follicles,then dissect and separate a single hair follicle under a stereomicroscope within magnify 10-20 times,and cut off the bulges area of the hair follicles.Select neural crest stem cells culturing medium to culture hair follicles bluge in vitro,observing the time,morphology,number and distribution of cells emigrating from the hair follicles bulge under a microscope.2.Use indirect immunofluorescence staining technology to detect the expressions and localizations of the SOX9 and ETS-1 proteins in the well-growing emigrating cells from human hair follicles bulge which are neural crest stem cells markers.3.Inoculate the emigrating cells from human hair follicles bulge into a 6-well plate,then use the scratch test method to detect their 48-hour migration capabilities,and take pictures under a microscope to collect 0 hours,24 hours,and 48 hours pictures centered on the cell scratches.Use Image J software measures the scratch area and calculates the percentage of scratch healing area at 24 hours and 48 hours.The percentage of scratch healing area=[(h1 scratch area-h2 scratch area)/h1 scratch area]×100%.4.Inoculate the emigrating cells from human hair follicles bluge into the upper chamber of the CIM-Plate 16 assay plate and use the unlabeled real time cell analysis system(RTCA)to automatically record the their cell index(CI)of the experimental groups and the self-control groups for detecting further study of 24 hours migration capabilities.Results1.When the hair follicle bluge is cultured in vitro,a small amount of cells can be seen emigrating around the hair follicles bulge area under the microscope on the 3rd day;on the 4th day of culture,more cells have emigrated out of the hair follicles bulge area than on the 3rd day,and cells nucleis are visible;On the 6th day,the cells morphology was a long spindle with full cytoplasm;on the 9th day of culture,the cells densities around the hair follicles bulge reached 80%.After the cells resuspended and continued to be cultured,the overgrowth time and overgrowth density of the emigrating cells were[(3.17± 1.17)/d]and[(2.79±0.48)106 cells/mL]respectively.2.Immunofluorescence stain results showed that the neural crest stem cells markers SOX9 and ETS-1 proteins were expressed in the emigrating cells from the hair follicles bulge and their were both localized in the nucleus.3.The scratch method results showed that the healing percentages of the scratch area of emigrating cells from human hair follicles bulge were 62.8%and 95.6%at 24 hours and 48 hours respectively,and the scratches of emigrating cells from human hair follicles bulge were basically healed at 48 hours.4.The unlabeled real time cell function analysis technology showed that the Cell index curve of the emigrating cells from human hair follicles bulge gradually increased and remained stable within 24 hours,and the cell index of different cells densities was[(4×104)个/mL]>[(2×104)个/mL]>[(1×104)个/mL].Conclusions1.There are neural crest stem cells expressing SOX9 protein and ETS-1 protein in the human hair follicles bulge.2.The neural crest stem cells from the human hair follicles bulge possess excellent migration capabilities in vitro.