Isolation,Culture And Preliminary Identification Of Neural Crest Stem Cells From Human Hair Follicle

BackgroundVitiligo is the most common depigmentation disease,the pathogenesis is unknown.Due to the destruction of the basal layer of functional melanocytes,the skin,mucous membranes and even hair pigmentation.At present,about 0.5%~1% of the global population suffering from the disease.Although vitiligo lesions without symptoms,did not give patients with physical pain,but its mental health and quality of life are affected.Therefore,to explore the pathogenesis of vitiligo and to seek the treatment of vitiligo have scientific and social significance.Vitiligo pathogenesis,including decolorization mechanism and complex color mechanism,is now focused on the decolorization mechanism,but decolorization mechanism has the same important complex mechanism is rarely studied.Therefore,to further explore the pathogenesis of vitiligo(including decolorization mechanism and complex mechanism),to find a new method of treatment of vitiligo will be far-reaching.We through clinical observation,found that vitiligo in the complex color around the hair follicle is the most complex pattern.Thus,we will be foucsed by follicular vitiligo complex mechanism for the research direction.It is well known that hair follicle(HF)is an important skin attachment structure of mammals,its structure is small,but its role is huge.It is not only able to regulate the growth of hair,shedding,or to maintain the body’s biological function of an important dynamic micro-organ.The results showed that there were many kinds of stem cells in HF,including hair follicle stem cells(HF-SC),mesenchymal stem cells(MSCs),pigmented stem cells,neurogenic stem cells,And studies have confirmed that hair follicle stem cells in vitro can be highly proliferated,and have the potential for differentiation.In addition to the above stem cells,recent studies have found that there are neural crest stem cells in the hair follicle,and neural crest stem cells are a kind of progenitor cells with pluripotency differentiation potential,which can differentiate into different cell lines.This topic will be from the perspective of stem cell biology,human hair follicles as the object of study,confirmed that human hair follicles not only the existence of neural crest stem cells,and through primary culture,it will be separated from the hair follicle,the next step to differentiate into melanocytes.Objective1.The aim of experiment is to prove the presence of neural crest stem cells in human hair follicles.2.Isolation,culture and preliminary identification of neural crest stem cells from human hair follice.Methods1.Specimens came from the First Affiliated Hospital of Zhengzhou University,orthopedic surgery and dermatology surgery obtained scalp tissue,the skin lesions of local was non-organic disease,and no pigmented disease.2.Specimens washed with PBS and Hank’s solution,cut the skin tissue into small pieces of tissue,the use of 0.48 U / ml of neutral protease(Dispase II)for digestion overnight,the next day separation of a single free Hair follicle.3.The expression of SOX10 and Nestin mRNA in single hair follicle tissue was detected by RT-PCR.4.The free hair follicles were made into frozen sections and sections were sectionally analyzed.Immunofluorescence was used to detect the expression of SOX10 and Nestin in hair follicles5.In the sterile environment,digestion and separation of the hair folliclecontaining skin tissue,cut off the hair follicle tissue excess hair,placed in the neural crest stem cell culture medium for primary culture,observe the cell from the hair follicle rupture zone free time,remove the hair follicle tissue,after digestion of cells though immunofluorescence identified expression of sox10,Nestin.Results1.RT-PCR results show that SOX10,Nestin express positive in isolated single hair follicle tissue.2.Immunofluorescence showed that SOX10 and Nestin were positive in the middle of hair follicle tissue,SOX10 was expressed in the nucleus,was green fluorescence.Nestin was expressed in the cytoplasm,was red fluorescence.3.Though primary culture,we observed in the neural crest stem cell culture medium,cell move out from hair follicle bulge in 2 to 3 days,in the first 4 days which appear accelerated proliferation.Cells were spindle-shaped,oval,nucleus full,similar to fibroblast,cell immunofluorescence showed that SOX10 and Nestin positive.Conclusions1.At the mRNA and protein levels,there are SOX10 and Nestin positive expression of neural crest stem cells in human hair follicles.2.In vitro culture,human hair follicles can move out of cells,the initial identification of migrating cells was neural crest stem cells.