Study Of The Differences On Hair Follicle Between AGA And Healthy

[Background]Androgentic Alopecia(AGA)is one of the most common dermatosis in the world,which affects more than 100 million adult men in our country.With the fast pace of our social life and an ageing population,the incidence of AGA showed a trend of rising.As we know,Androgentic Alopecia is a continuous decrement of thinning or loss of hair induced by androgen or dihydrotestosterone.Recently,it has also been proved by researchers that many factors have played an important role in the occu-rrence and development of AGA,including genetic background,nutritional status,stress,aging degree,immune response,external damage,etc.However,the pathogenesis of Androgentic Alopecia(AGA)is not fully understoodas yet.There are two medical treatments approved by the US Food and Drug Administration(FDA)for androgenetic alopecia(AGA),including topical minoxidil(a vasodilator substance)and oral finasteride(a selective type II 5a inhibitor).But the patients who use finasteride 1 mg for AGA may experience difficulty in achieving an erection that continues after stopping the medication.Besides,it may lead to transient increasing hair shedding after drug withdrawal.We need to get the pathogenesis of AGA and develop its targeted intervention,so as to develop more effective treatment of AGA.The development of male hair loss has a relatively fixed mode.In general AGA commences with a thinning of the frontal and vertex scalp areas,afer then a bitemporal recession of the frontal hair line.however the occipital area of the hair growth remained.This is followed by.Hair follicles transplanted from the pillow to the bald forehead area can still grow normally,it is prompt that the root reason of the hair growth stagnation is hair follicle itself rather than its surrounding environment(the 1950 s theory of " donor dominant ").The hair follicle stem cells which possess characteristics of stem cells,including multipotency,high proliferation potential and ability to enter quiescence,appear to be responsible for regenerating the hair follicle in each hair cycle.Hair follicles in the process of the onset of AGA gradually atrophic,growing seasons shortened,resting period extended,even more eventually lose the ability of regeneration of hair.However,there have been no exact studies on the pathogenic and pathogenesis of hair follicle stem cells of AGA yet.Some findings support the notion that not hair follicle stem cells(KRT15+)but progenitor cells(CD200hiCD34hi)play a role in the pathogenesis of AGA.It is important to clarify the molecular mechanism of this abnormal resting state,which will provide clues for the development of its targeted treatment options.It is mainly focused on the morphological differences in the hair folliculars between the alopecic frontal scalp and occipital regions.But there usually be full of structural abnormalities,steatorrhea,infection,inflammation in completely bald scalp hair follicles exist.The aim of this study is to investigate roles of the morphological differences in the hair folliculars between the alopecic frontal scalp(with follicular miniaturization)and occipital regions(AGA and healthy).Then,I will also develop an effective technical for purification of hair follicle stem cells from human hair follicles sample,and try to get the comparison analysis of transcriptome on hair follicle stem cells for the first time in the world.Through these studies,I will further reveal the mechanism of hair follicle stem cells of AGA,and lay a foundation for developing a new type of targeted therapy for AGA.[Object](1)To investigate the morphological differences in the hair folliculars between the alopecic frontal scalp(with follicular miniaturization)and occipital regions(AGA and healthy),including morphological structure,the amount of hair follicle stem cells(HFSc),location of HFSc,proliferation and differentiation.(2)To build a pure hair follicle model of human with no defection,and to develop an effective technical for purification of hair follicle stem cells from human hair follicles sample.(3)To get the comparison analysis of transcriptome on hair follicle stem,and explore the differences of gene expression on HFSc between critical porehead of AGA and occipital regions(AGA and healthy).[Method](1)Measuring the length of the hair follicle in growth period and the number of HF(hair follicle)in hair follicle unit to investigate the morphological differences on the hair folliculars between the alopecic frontal scalp(with follicular miniaturization)and occipital regions(AGA and healthy).Immunohistochemistry was used to explore different tissue about morphological structure,the amount of hair follicle stem cells(HFSc),location of HFSc,proliferation and differentiation in order to evaluate the differency of HF among several areas.(2)Selecting skin with HFs as experimental material and using Enzyme digestion combined with micromanipulation as the preparation method to Build a model of hair follicles.we isolated hair follicle through DispaseII from the skin.By building the hair follicle model,we use immunomagnetic sand method to develop an effective technical for purification of hair follicle stem cells from human hair follicles sample.(3)Using REPLI-g WTA Single Cell Kitwound to amplify gene transcription of trace hair follicle cells.Then using NEBNext DNA Library Prep Master Mix Set for Illumina to get the comparison analysis of transcriptome on hair follicle stem cells.Efficiency and feasibility of transcriptome method were analyzed by glue leaking,PCR method,and TA cloning..[Result](1)The study measuring the length of the hair follicle in growth period and the number of HF(hair follicle)in hair follicle unit to investigate the morphological differences have shown that there were statistically significant differences in the length of the hair follicle in four groups(P<0.001).There were statistically significant differences in the number of HF(hair follicle)in hair follicle unit between the alopecic frontal scalp(with follicular miniaturization)and occipital regions(AGA and healthy)(P<0.01).(2)Analyzing respectively from the structure of hair follicle,stem cell and proliferation etc.I found: firstly,there is no obvious difference among the structure of the four groups of hair follicle about CK14 protein staining;the expression of CD200 in the alopecic frontal scalp(with follicular miniaturization)and occipital regions(AGA and healthy)all exists in bulge area of HFs and presents strong expression,and I also found CD200 in the alopecic frontal scalp(with follicular miniaturization)presents positive expression but weaker than other groups.Second,Ki67 protein of occipital regions,both AGA and healthy,presents positive expression mainly in 1/4~1/5 of HFs,but Ki67 of alopecic frontal scalp(with follicular miniaturization)presents higher positive expression in half of HFs,obviously stronger than others.(3)Choosing human scalp hair follicles as experiment material and the micro separation technology,enzymatic digestion as the preparation methods of the standard hair follicle model makes the hair follicle models have good integrity,high purity,and small damage.(4)We combined three methods micro separation technology,enzymatic digestion and immune magnetic beads method together to get the hair follicle stem cells,and evaluated the system by flow cytometry analysis and cell smear staining method and found that it can further purify hair follicle stem cells and reduce cell loss rate to use this kind of method to obtain hair follicle stem cells.(5)We did cell content analysis of the cell groups which we get on the basis of the establishment of a standardized model of hair follicles,and the results show the four experimental groups of hair follicles within a larger difference of the CD200 percentage of individuals,it is possible because of individual differences.But the CD200 percentage of hair follicles in the posterior part of the occipital was higher than that in the frontal part of each AGA patient.[Conclusion](1)In contrast to four groups,the hair follicle of AGA forehead groups(critical hair follicle)decreased obviously,and become thinner and shorter.And hair follicle of occipital regions on AGA is smaller and shorter than occipital regions on healthy group.(2)the expression of CD200 in the alopecic frontal scalp(with follicular miniaturization)is weaker than occipital regions(AGA and healthy).while,Ki67 of alopecic frontal scalp(with follicular miniaturization)presents higher positive expression than others.(3)Choose enzymatic digestion conbined with micromanipulation as the preparation methods of the standard hair follicle model makes the hair follicle models have good integrity,high purity,and small damage.(4)We combined three methods to get the hair follicle stem cells and found that it can further purify hair follicle stem cells and reduce cell loss rate to use this kind of method to obtain hair follicle stem cells.