Mechanistic Studies On Delayed Hair Regeneration And Accelerated Hair Follicle Aging Caused By Lgr4 Deficiency

The significance of hair to human beings has surpassed that of the general mammals in terms of warmth and protection,and more in maintaining a good social image.Hair growth is a lifelong but cyclical process.A complete hair cycle consists of telogen,anagen and telogen.Hair follicle is an important structure supporting hair growth,and its periodical structural change is the basis of hair cycle.Hair follicle disease and aging will lead to hair cycle disorders,resulting in hair loss and other hair diseases.Thus the health of hair follicle and the normal cycle of hair growth are prerequisites to guarantee its physiological function.It is of great significance for developing hair disorder therapeutic medicine and hair follicles aging preventive drugs to specify genes regulating hair follicle growth and to reveal their specific mechanism.In this thesis,we show that a member gene of the G protein coupled receptor family,Lgr4（Gpr48）,plays a positive role in hair follicle growth and vitality maintainance.On the one hand,Lgr4 could ensure normal cyclic regeneration of hair follicle;on the other hand,it could also prevent hair follicle from premature aging.First of all,through the transcriptional level detection and the staining analysis of the reporter gene LacZ,we identified the dynamic expression profile of Lgr4 with the hair cycle in the skin of mice.Then,three kinds of animal models(including Lgr4whole body knockout mice Lgr4^-/-,Lgr4 epidermis conditional knockout mice K14Cre/Lgr4^fl/fl and Lgr4 whole body knockout mice with LGR5⁺hair follicle stem cells autofluorescence Lgr5^EGFP/Lgr4^-/-)were used to explore the function of Lgr4 in the hair follicle.According to our results,we found a delayed hair cycle and an accelerated hair follicle aging in Lgr4 knockout mice.The two distinct hair follicle dysfunctions caused by Lgr4 deficiency are discussed separately in this thesis.By immunofluorescence,immunohistochemical,westernblot and other detection methods,the skin samples of juvenile mice during first telogen to anagen（P23-P27）were taken and analyzed,and we found that the proliferation of LGR5⁺hair follicle stem cells in the secondary germ of Lgr4 knockout mice is restrained,while the forcible activation of hair follicle stem cells by hair plucking reverses the inhibition.At histological and cellular levels（including the HaCaT gene silence cell model and the primary epidermal cells）,we further confirmed that Lgr4 plays its role in cell activation and proliferation control by regulating AKT/mTOR and BMP signaling pathways.In addition,through the flow cytometry analysis on the number of stem cells in the adult mouse skin sample,we found that Lgr4 deficiency may result in a reduction in the replenishment of LGR5⁺active hair follicle stem cell pool by CD34⁺quiescent hair follicle stem cells,thus attenuating LGR5⁺stem cell pool but accumulating CD34⁺cells,further leading to a weak response of the hair follicle to activation signals.By immunofluorescence,immunohistochemical,real-time fluorescence quantitative detection and other detection methods,with the addition of hair follicle reconstruction experiment,the skin samples of aged mice（1-1.5 years of age）were taken and analyzed,and we found that Lgr4 knockout mice showed premature hair follicle aging and hair loss.Although hair follicle regeneration deficiency may be one of the important factor,the differentation deviation and property weakening of bulge stem cells resulted from Lgr4 deficiency may also contribute to hair follicle premature aging.Based on the above results,we confirmed that the absence of Lgr4 will lead to uncontrolled hair follicular regeneration and accelerated hair follicle aging,and therefore,activating Lgr4 may be beneficial to the growth of hair and the protection from hair follicle aging.According to previous reports,Lgr4 in hair follicle has a similar expression pattern in mice and human,suggesting that the conclusion in mice may be consistent with human.Therefore,this article provides some theoretical basis for confirming Lgr4 as a potential medicine targets in the treatment of hair loss diseases.