| Vitiligo is a common depigmented skin disease involving the skin and(or)mucous membranes,with a prevalence of approximately 0.5～2%of the global population,and is characterised by progressive autoimmune destruction of mature epidermal melanocytes.Vitiligo is now clearly classified as an autoimmune disease and is often seen as a cosmetic problem,often causing a considerable burden on daily life.The etiology of vitiligo depigmentation is not clear and the origin of repigmentation at the lesion and its stability are not fully understood,so the treatment of vitiligo remains one of the challenges of dermatology.In vitiligo patients,the occurrence of repigmentation has different patterns,and perifollicular repigmentation is its important repigmentation pattern,suggesting that the repigmented melanocytes may originate from the hair follicle,but the exact cellular and molecular mechanisms of repigmentation are still unknown;foreign studies have shown that human epidermal neural crest stem cells(hEPI-NCSCs)are present in the hair follicle bulges,which can be induced to differentiate into melanocytes.This suggests that hEPI-NCSCs may play an important role in the process of perifollicular re-colourisation in vitiligo.Further confirmation of the presence of hEPI-NCSCs in the hair follicle bulges and investigation of their renewal,proliferation and differentiation mechanisms will be of great significance for the treatment of vitiligo.The hair follicle is often compared to a miniature organ that is continuously renewed during periodic growth and is also a ’niche’ for tissue regeneration and is often considered a unique model lineage for the study of physiological tissue regeneration processes.The outer root sheath of the hair follicle contains many different types of stem cells,such as epithelial stem cells,melanocyte stem cells and neural crest-like stem cells,so the hair follicle represents an accessible and abundant source of different types of human stem cells.The advantages of EPI-NCSCs from their own source,being widely available and not involving ethical and immune rejection issues,have attracted much attention in recent years.We have previously confirmed that the emigrated cells from human hair follicle bulge expressed Nestin and Sox10.In order to further confirm the presence of NCSCs in the human hair follicle bulges,we isolated individual hair follicles by microdissection technique,intercepted the hair follicle bulges and cultured them in vitro,microscopically observed the timing,morphology and growth of the migrated cells in the hair follicle bulges,and examined the expression of neural crest stem cell markers(P75,MSX2),self-renewal markers(OCT4,NANOG),and their proliferative activity of the migrated cells in hair follicle bulges,which further provides a basis for further investigation into the mechanism of vitiligo repigmentation,a more effective therapeutic approach and the development of human epidemlal neural crest stem cells(hEPI-NCSCs)as a target for the treatment of vitiligo.Objetives1.To detect the expression of neural crest stem cell markers(P75,MSX2)and self-renewal markers(OCT4,NANOG)of the emigrated cells from the bulges of human hair follicles.2.To detect the proliferation activity of the emigrated cells from the bulges of human hair follicles.Methods1.Scalp tissues containing a certain number of hair follicles were collected from plastic surgery,dermatology,neurosurgery,etc.Selection criteria:(1)age 18～60 years,male or female;(2)vigorous hair and good hair follicle growth;(3)no local pigmentary diseases and no organic disease;(4)no history of local drug use within the last 1～2 months.Exclusion criteria:(1)those with thinning hair,baldness,gray hair and other hair diseases;(2)incomplete hair follicles.2.Under aseptic environment,cleaned specimens were rinsed with DPBS buffered with double antibodies,trimmed(excess fatty tissue around the specimen and surrounding connective tissue sheaths were cut off),and the trimmed scalp tissue was cut into small tissue blocks(about 3 cm×3 cm)along the direction of the hair follicle,and individual hair follicles were separated with microscope to intercept the follicular augmentation area.3.The bulges of human hair follicles.were cultured in vitro with neural crest stem cell medium to observe the time,morphology,and growth of the cells in the hair follicle augmentation area,and the expression and localization of neural crest stem cell markers(P75,MSX2)and self-renewal markers(OCT4,NANOG)in the emigrated cells in human hair follicle bulges were detected by immunofluorescence.4.The primary cultured neural crest stem cells were passaged,and 5-bromo-2-deoxyuridine(Brdu)was added to the corresponding culture dishes at 24h,48h and 72h,respectively,after incubation in the cell incubator for 4 h.Five fields of view were taken at each time point under a fluorescent microscope after fluorescent staining,and the The number of Brdu red-st?ined cells and the number of DAPI blue-stained nuclei were counted,and the positive rate of Brdu of cells migrating out of the hair follicle augmentation zone was calculated to detect their proliferative activity,respectively.Results1.When human hair follicle bulges are cultured in vitro,a small number of cells migrated around the follicle bulge at 5 days,and the cells were star-shaped,shuttle-shaped and round;at 8 days,the migrated cells increased,and the cell morphology was shuttle-shaped;and the cells were active.2.Emigrated cells from human hair follicle bulges positively expressed neural crest stem cell markers P75 and MSX2,P75 was located in the cytoplasm and MSX2 was located in the nucleus.3.Emigrated cells from human hair follicle bulges positively expressed neural crest stem cell markers P75 and MSX2,P75 was located in the cytoplasm and MSX2 was located in the nucleus.4.Emigrated cells from human hair follicle bulges showed strong proliferative activity after passaged culture,with(49±5)%positive for Brdu at 24h;(33 ± 11)%positive for Brdu at 48h;and(18±9)%positive for Brdu at 72h.conclusions1.When human hair follicle bulges are cultured in vitro in neural crest stem cell medium,the migrated cells are neural crest stem cells.2.Human epidemlal neural crest stem cells have the ability of self-renewal.3.Human epidemlal neural crest stem cells have strong proliferative activity. |
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