Preliminary Study On The Anti-androgen Mechanism Of Caffeine In Human Dermal Papilla Cells In Vitro And Gene Expression Profiling Of Dermal Papilla Cells In Androgenetic Alopecia In 3D Culture And The Investigation Of Minoxidil In Improving The Quality Of

Chapter one Preliminary study on Mechanisms of Caffeine Antiandrogenic on Human Dermal Papilla cells in vitroBackground and Objective: Hair loss is universal problem.Androgenetic Alopeciai(AGA),also called seborrheic alopecia,is one of the most common disorders in hair loss.Pathogenesis of AGA is associated with genetic factor and androgen dependent which attack from puberty with character of backward of frontal hairline and hair progressive decrease in vertex.Data showed that severity of AGA increase with aging.It involves both gender and significantly impair quality of life in patients.AGA is a common problem in the middle age males with a prevalence of approximately 50% in western.An epidemiological survey in north China suggested that the male incidence was 21.3%.Nowaday,finasteride and topical minoxidil are only approved by FDA in AGA treatment since inhibition of the 5?-reductase and androgen receptor is the two main therapeutic targets in the management of AGA.Finasteride inhibit type II 5?-reductase,thus decrease DHT produced in hair follicles.However,it was found out to be invalid in nearly 30% patients.Furthermore,systemic application will bring the side effect of systemic 5?-reductase inhibition such as sexual dysfunction,rash,breast tenderness and lump.A 7-year period of the trial showed that finasteride increased the risk of high-grade postrate cancer comparing to the placebo which raised concerns over the safety of this medicine,which definitely brought us to the attention of security in this treatment again.Furthermore,topical minoxidil can also cause scalp stimulation for side affect.Caffeine is the world's most widely consumed substance for its psychoactive effect.Data from a recent research showed that caffeine ameliorates hair loss in AGA patients.As a phosphodiesterase inhibitor,caffeine increases c AMP levels in dermal papilla cells and therefore promotes proliferation by stimulating metabolism a mechanism which would counteract testosterone/DHT-induced miniaturization of the hair follicle.Fischer confirmed that caffeine with concentration of 0.001% and 0.005% demonstrate the ability of reversing the inhibition of testosterone on hair follicle which revealed that caffeine canbe the one of the prospective treatment for AGA.Our previous study has also verified that certain concentrations of caffeine led to significant stimulation on dermal papilla cells growth which mechanism is still unclear.Human hair follicles,distributed in specific sites of the body,appear to have an inherited susceptibility for androgen-dependent growth.Depending on the body sites,androgens have paradoxically different effects on human hair follicles.Androgens stimulate hair growth in some sites such as the beard,axillary and pubic areas and suppress the growth of frontal scalp hair of genetically disposed individuals which is still remain unclear in mechanism.The development of AGA is predominantly associated with the testosterone metabolite,Wnt signaling pathway,TGF-?/smad signaling pathway and p53 signalling pathway mediating aging and apotosis.At present the common method widely used for isolating and cultivating human dermal papilla cells is enzyme digestion which show the advantages of short time-consuming,high output and purity and better adherence.Microdissection method first invented by Messenger was replaced by enzyme digestion for its long time consuming and technical requirements.However,proper method should be adoptted base on special request in certain site such as axillary hair and pubic hair.The purpose of study in this part is to disscuss the proper method for isolating special site of dermal papilla cells and investigate the possible antiandrogenic mechanisms of caffeine with concentration of 0.0005% on different site of human dermal papilla cells in vitro,which can provide experimental basis for the clinical application of caffeine.Methods: 1.Isolating the dermal papilla cells in scalp and pubic by using enzyme digestion method,microdissection method and modified microdissection method respectively.Identified the pubic dermal papilla cells by alkaline phosphatase staining and ?-SMA immunofluorescent staining.Compare three methods by microscope observation and adhesive rate 3.Human dermal papilla cells,taken from vertex,occiput and pubis,were cultivated for 48 h in vitro with 0.0005% caffeine and 10 n M testostero respectively or combination.Measured the relative growth and apoptotic rate by MTT assay and flowcytometry,and Realtime-PCR was applied to analysis m RNA of 10 candidate genes connecting to possible signaling pathway of AGA.Westernblot and immunofluorescence were used for further confirmation.Results: 1.Enzyme digestion method is superior to other methods with high quality and efficiency in isolating scalp cells while modified method has an advantage over microdissection method with high adherent rate(P<0.005).2.0.0005% caffeine stimulated the proliferation and inhibited the apoptosis of human DPCs in vitro.AR?SRD5A2?p53?Fas R?GSK-3??TGF-?2 show significant regulations in vertex DPC culture treatment concentrations of 10 n M testostero.In pubic cell culture p53 and Fas R were down-regulated with testostero and further apoptosis suppression can be achieved by caffeine treatment.Conclusion: Modified microdissection methods is a proper way for isolating dermal papilla cells in special site.Caffeine probable play a role in antiandrogenic effect on human dermal papilla cellss in vitro by acting on different signalling pathway.Genome-wide Expression profile anlysis of 3D cultured dermal papilla cells from Androgenic alopecia patientsBackground and Objective: Androgenic alopecia is the most common hair disorder which significantly impaire mental health of patients and proved to be associated with several systemic diseases.Androgen,especially DHT has been clear to influence the hair follicle and specific dermal papilla cells with genetic susceptibility pathologically into apoptosis for chronic and dramatic change leading to atrophy and miniaturization of hair follicle in vertex which mechanism is not fully understood.Hair follicle as the smallest unit control hair shaft production and hair cycle.Dermal papilla located in the bottom of the hair follicle rich in androgen receptor and 5?-reductase?,is identified as organs to be the responder of androgen and can release a variety of cell growth factor regulating the hair shaft and follicle cycle.Hair follicle growth and development involved in multiple transcription factors and signalling pathways such as Runx1?Tcf3/4?NFATc1?Lhx2?Sox9?Tbx1?p63?Dlx3?Lef1?Msx2/Foxn1?Gli?Gata3 and Wnt?Bmp?Shh?Notch.BY far pathogenesis of AGA was verified to contain one or more signal transduction pathway with abnormality.For example,Androgen metabolic pathway,Wnt signalling pathway,TGF-?/Smad signalling pathway and p53 pathway.However,whether there are crosslinking between these various signal pathways,through what kind of signal molecule they interacted with whether and are there other signal molecule and signal transduction pathways involved,still need to further study.DPC with testosterone or dihydrotestosterone stimulation represent significantly different genes profile,which definitely provide the valuable clue of AGA attack.Moreover,rare investigation was found by Using genome-wide expression profile analysis in screening target genes related to AGA morbidity.Previous studies on dermal papilla were based on 2 D cultured models.However the biological characteristics of cells in mitosis,aggregation and inducibility of hair follicle formation presents huge difference with intact dermal papilla.Recent study found that 3 D cultured model show advantages on maintain the biological characteristics of dermal papilla cells or restore the inducibility decreased in 2D culture which successfully establish proper cell culture model for further research.The purpose of the study is to screen the target genes involved in mechanisms of AGA by using Gene expression profile anlysis and preliminary explore their molecular functions in corresponding signalling pathways.Methods: 1.Cultured occipital and vertex dermal papilla cells from AGA patients in vitro for 3 passages and continuous cultured for another 72 h by 3D cultured model with hanging drop methods.2.Two groups are divided: with DHT treatment in vertex DPCs and without treatment in occipital DPCs.Total RNA of two groups were extracted and amplified by RT-PCR.The product c DNA was labeled with Cy3 and hybridized on Nimble Gen Homo sapiens12×135K Array whole genechip.Fluorescence scanning,Gene Ontology(GO)and pathway analysis were used to screen the different expressed genes.The analytic result was validated by Realtime PCR.Results : 1.Dermal papilla cells aggregated to spheres presenting the similar formation with intact dermal papilla.And cell emigration with AKP staining positive could be found in sphere after 2D culture again.2.A total of 622 genes showed differentially expressed between two groups including 359 up-regulated genes and 263 down-regulated genes.GO analysis showed that the up-regulated expressed genes mainly involved in antiproliferation,apoptosis process,and signalling inhibition in cell growth,such as CHEK1 and Tob1 while down-regulated genes function as signalling molecular in cell proliferation and epidermal development like BAMBI,EFNA3,Dlx3 and UCGC,which is comfired by Realtime PCR for consistency with microarray respectively.Pathway analysis showed that signalling pathway controlling cell circle are on the top.Conclusion: The change of expression profile between two groups indicated that a small part of genes and signalling pathway were involved in this complex process of androgenic alopecia,and how these gene worked and co-worked to regulate the procedure need further investigations.Topical Minoxidil Improved the Quality of Life of Female pattern hair loss PatientsBackground and objective: Female pattern hair loss(FPHL)characterized by diffused reduction in hair thickness,is the most common cause of hair loss in women,which affects over 50% women at the age of 80.Furthermore,an epidemiological survey in north China suggested that the incidence was 6.0% in females,which was lower than that in Western countries.However,due to the large population in China,the exact number of women suffered from FPHL is in fact greater.Although it is a mild dermatological disorder,psychologists and dermatologists found that even clinically imperceptible hair loss could damage Quality of Life(Qo L)in patients by loss of self-image and low self-esteem.This could be notably obvious in women with hair loss who have being reported to experience adverse psychosocial aspects such as irritability,anger,anxiety and depression due to the significant sexual and social functions of hair on a woman's appearance.Visual Analog Scale(VAS)and Dermatology Life Quality Index(DLQI)were widely applied for assessing quality of life in patients with skin disease but rare in hair disorder.Treatment selections are currently limited and a substantial length of time is required to reach a satisfactory outcome.Minoxidil topical solution,approved by FDA,has been confirmed to be effective and safe to treat female androgenetic alopecia by promoting hair cycling and prolonging aging.However few studies have demonstrated a desired impact of topical minoxidil treatment on Qo L in female patients.This study is designed to assess Qo L in female patients with FPHL(n = 125)and determine the effectiveness of topical minoxidil treatment in improving the Qo L of these patients(n=31).Methods:One hundred and twenty-five female FPHL patients aged 16-72 years answered the Visual Analog Scale and Dermatology Life Quality Index before the adminstration of minoxidil(2ml/day)and 31 patients finishing the treatment of 12 months filled in again.Each patient was assessed by four grades namely,‘significant improvement',‘improvement',‘no change' and ‘worsening'.‘Significant improvement' was defined by two Ludwig score increase while ‘improvement' described one Ludwig score increment.Decreasing one or two grades was considered ‘worsening' by global photograph.Patients assessed as ‘significant improvement' or ‘improvement' were defined as ‘good reactors' while those assessed as ‘no change' or ‘worsening' were defined as ‘poor reactors'.The improved value of each Qo L index was compared to the baseline.Describe the improved value of each QOL index and the changes in QOL before and after the treatment were statistically analyzed.Results: 125 patients showed the correlation between severity and values of the two index(P<0.05).The DLQI and VAS scale were highly correlated with a statistical significant negative correlation in all first visit patients(P<0.000,r =-0.441).There was a statistical difference in the VAS(P<0.01)and DLQI(P<0.01)indices before and after the administration of treatment.With regard to specific life events,there were significant differences in the scores for ‘symptom and feeling'(P<0.01),‘daily activities'(P<0.01),‘leisure'(P<0.05)Comparison of the good responders(23 cases)and poor responders(8 cases)revealed no statistical difference in the improvement of VAS and DLQI scores.Conclusion: Female patients Qo L were impaired seriouly by suffering AGA;DLQI and VAS adopted here are suitable for the evalation of Qo L in AGA patients;Topical minoxidl improved the Qo L of these patients.