Experimentation Of Pathogenesis And Therapeutic Intervation Of Gynecomastia

PART 1 THE STUDY OF ERα,ERβAND PTEN EXPRESSION IN GYNECOMASTIAObjective Gynecomastia is a frequent benign male breast disease which presents a breast enlargement resulting from a proliferation of the mammary glandular component.Among its pathophysiological development mechanism and mammary proliferation mechanism,the altered responsiveness of the breast tissues against hormone is an important factor.There have not been reported about estrogen receptor subtypes and phosphatase and tensin homology deleted on chromosome ten (PTEN) protein expression in the breast tissues of gynecomastia at present.To investigate the status of hormone responsiveness to local mammary tissues and then study its pathogenesis further,the present study detects the expressions of ERα, phospho-ERα,ERβand PTEN protein in mammary tissues of gynecomastia.Methods The experiement group includes sixty-eight consecutive mammary tissue specimens of gynecomastia patients.The control group includes twenty-four normal male mammary specimens.All cases were divided into three histological groups:a,florid type(13cases);b,intermediate type(18 cases) and c,fibrous type(37 cases).The cases were also divided into three different age groups:young, middle-aged,and aged group.Immunohistochemical staining SP method was used to detect the expressions of ERα,phospho-ERα(p-ERα),ERβand PTEN protein.The expression level was assessed by the staining intensity and the positive cell rate.Results The expression of ERβprotein was observed in both the mammary ductal epithelial cells and mammary stroma cells(fibroblasts) and its expression level in gynecomastia breasts was up-regulated contrasted to normal male breast.The expression of ERα,p-ERαand PTEN protein were only detected in mammary ductal epithelial cells but not in stromal cells.The expression level of ERαand p-ERαprotein in gynecomastia breast was higher than normal male breast.Nevertheless,the PTEN protein expression level decreased in gynecomastia breast.Moreover,the increase degree of ERβand ERαand the decrease degree of PTEN degraded in fibrous type,intermediate type,and florid type by turns and the difference between them was statistically significant(p＜0.05).The increase degree of ERβand ERαa and decrease degree of PTEN degraded in young group,middle-aged group and aged group by turns,and the difference between them was statistically significant(p＜0.05).Conclusions The expression ERβ,ERα,p-ERαprotein were different in the mammary epithelia cells and stroma fibroblasts of gynecomastia and normal male breast tissues.This confirmed that the improvement of breast local tissue reactivity against estrogen in gynecomastia was one of pathogenesises of gynecomastia.The up-regulated expression levels of ERα,and ERβin gynecomastia reveals the responsiveness of mammary tissues to estrogen may play an important pole in the mammary epithelia cells proliferation and stroma fibroblasts in the breasts of gynecomastia.The weakened expression of PTEN protein in gynecomastia was relatived with the proliferation of ductule of gynecomastia breast,and its expression level was significantly different in three histological groups and three age groups of gynecomastia.PART 2 THE STUDY OF CULTURE OF MAMMARY EPITHELIAL CELLS AND FIBROBLASTS DERIVED FROM GYNECOMASTIA AND NORMAL MANObjective No studies about cell culture in vitro from gynecomastia and normal man breasts have been reported until now.The present study aims to investigate the isolation and culture the male human mammary epithelial cell and fibroblasts from gynecomastia and normal man breast tissues and establish cell model cultured in vitro.Methods Basing on female human mammary epithelial cell(HMEC) culture in vitro,the fresh mammary tissues from gynecomastia(10 cases) and normal man(4 cases) breasts were minced and digested with 50 U/ml collagenase(typeâ… ) on shaker at 37℃over night.The mammary epithelial cells and fibroblasts were isolated collected by Percoll gradient centrifugation isolation technique.And the two isolated cells were planted in their different culture solution.The cell growth morphous was observed by phase contrast microscope,and the ultrastructure was observed by perspective and electron microscope.Immunocytochemistry of cytokeratin 19 and vimentin were used to determine the identification the primary cultured human mammary epithelial cells and fibroblasts respectively.The tow kinds of cells were passaged and frozed reservation,and the cell growth energy was observed.Results The HMEC- and fibroblasts-derived from normal man and gynecomastia mammary tissues were successfully isolated and grew better in vitro in their respective mediums.The immunology marker of cytokeratin 19 and vimentin were expressed specially in HMEC and fibroblasts differently,and the positive cell rate reached 95%.The second passage of HMEC grew well,and the HMEC and fibroblasts after cryopreservation resuscitation maintained good cell activity.The growth energy of fibroblasts was higher than HMEC.Conclusions The isolation and culture HMEC- and fibroblasts derived from normal male and gynecomastia mammary can be successfully carried out in vitro.The culture model in vitro of HMEC- and fibroblasts-derived from normal man and gynecomastia mammary tissues can be successfully established. PART 3 THE STUDY OF EFFECTS OF 17β-ESTRADIOL ON HMEC-DERIVED FROM NORMAL MAN AND GYNECOMASTIA BREAST AND POLE OF PTEN IN GYNECOMASTIA DEVELOPMENTObjective Some researches disclosed that the effects of E₂ on female HMEC depended on ERαthrough PI3K/Akt pathway.PI3K/PTEN/AKt pathway lives in the normal gland alveolus and ductal epithelial cells and ERαcan bind the p85 regulatory subunit of PI3K in a ligand-dependent manner.But,there were no reports on the PI3K/PTEN/AKt pathway in the mammary of gynecomastia,neither the relation between the former pathway and E₂.The purpose of this study is to determine the effects of E₂ on normal male human mammary epithelial cells(nmHMEC) and gynecomastia mammary epithelial cells(gyHMEC) cultured in vitro and on the PI3K/PTEN/AKt pathway.The study also explores the pole of PTEN in the development of gynecomastia by observing PTEN protein expression in nmHMEC and gyHMEC and its relationship with ERα.Methods NmHMEC in logarithmic phase were treated with 10^-12,10^-11,10^-10, 10^-9,10^-8 mol/l E₂ in indicated times.Cell proliferation with DNA synthesis on 0 hour, 12 hour,24 hour,48 hour,and 72 hour as a marker was assessed by BrdU incorporation. So,the suitable concentration and optimization utility time were screenied.The cell proliferation was detected on nmHMEC treated with E₂ with suitable concentration and an ER弓agonist(BAG) or BAG alone for 48 hours.After treatment on nmHMEC with E₂ cooperative with ICI182,780(an ERαblocking agent) or not for 48 hours,the expression of ERα,p85,PTEN and phospho-Akt(p-Akt) in the cells treated with E₂ were observed by Western blot analysis.The cultured primary gyHMEC and nmHMEC were exposed to E₂ for 48 hours,respectively.The expression of ERα,p-ERαand pAkt protein pre- and post-treatment was determined by Western blot.GyHMEC and nmHMEC were treated with E₂ additional with LY294002(blocking the PI3K pathway) respectively,and the expression of ERα,p-ERαand p-Akt protein were detected by Western blot.And the PIP3 protein expression in cells pre- and post-treatment was dectected by immunofluorescence.Results Treatment of nmHMEC cells with E₂ resulted in a marked increase in cell proliferation,and the increase presented significantly on 10^-10 M E₂.There was significant difference in the cell proliferation activity with different concentration of E₂ treatment(p<0.05).The promoting proliferation effects present concentration dependence.The cell proliferation activity presented markedly when E₂ treated for 48 hours and the difference was significantly in different group treated for different times. And,the promoting proliferation effects present time dependence.Exposure of nmHMEC to E₂ and BAG displayed higher cell proliferation than nmHMEC with BAG treatment alone.ERαexpression decreased in nmHMEC with E₂ treatment compared with control,but p85,p-Akt and PTEN expression increased(p<0.05).However,their expression difference was not observed in nmHMEC with E₂ and ICI182,780 treatment compared to control(p<0.05).The ERαexpression decreased in gyHMEC and nmHMEC after treatment with E₂.The decreasing degree of ERαin gyHMEC with E₂ treatment was more significantly than in nmHMEC.But,both p-Akt and p-ERαexpression increased markedly compared with control,and the increasing degree in gyHMEC was higher than in nmHMEC(p<0.05).After PI3K pathway was blocked by LY294002,the effects of E₂ on p-Akt and p-ERαexpression of gyHMEC was not markedly,but caused p-Akt and p-ERαexpression markedly decrease in nmHMEC(p< 0.05),and the immunofluorescence intensity of PIP3 in gyHMEC decreased markedly compared with control(p<0.05).Conclusions E₂ can promote the cell proliferation of nmHMEC cultured in vitro,and the promoting proliferation effects present time dependence and concentration dependence.The promoting proliferation effects may be mediated through ERαand the potential mechanism is to activate PI3K/Akt signal pathway.The results show that the down-regulated PTEN protein in gynecomastia mammary inactivates the inhibition for the down PIP3/Akt signal,promoting HMEC proliferation.The up-regulated expression of Akt induced by PTEN enhanced the phosphorylation of ERα,which raises responsiveness of the human mammary epithelial cells to E₂ resulting in the hyperplasia of male mammary.Both PTEN and E₂ can effect the breast proliferation of gynecomastia through of PI3K/PTEN/AKt signal pathway. PART 4 THE STUDY OF EFFECTS OF TAMOXIFEN ON MAMMARY EPITHELIAL CELLS AND FIBROBLASTS DERIVED FROM GYNECOMASTIAObjective Tamoxifen(TAM) is a drug used often to treat gynecomastia,but its certain curative effect and safety were not generally accepted.This present work is to explore the effects of tamoxifen on cultured in vitro nmHMEC,gyHMEC,normal male human mammary fibroblasts(nmFb) and gynecomastia fibroblasts(gyFb),providing a laboratory confirmation for its application on gynecomastia.Methods NmHMEC were exposed to different concentration tamoxifen or corresponding tamoxifen added with estrogen antagonist ICI182,780(1.0μM),and the cell growth inhibition rate(GIR) and cell proliferation labeling index(LI) were delected on 0 hour,12 hour,24 hour,48 hour and 72 hour.Then,gyHMEC and nmHMEC were exposed to tamoxifen(1.0μM) for 48 hours,and the effects of TAM on cell cycle distribution and apoptosis rate(AR) were assessed respectively.GyHMEC from florid type,intermediate type and fibrous type of gynecomastia were exposed to TAM(1.0μM) for 48 hours,and cell cycle distribution and AR were observed.The nmFb and gyFb were exposed to TAM(1.0μM) for 48 hours,and cell cycle distribution and AR were observed.GIR and LI were assessed by BrdU and MTT methods,the cell cycle distribution and apoptosis rate(AR) by flow cytometry,and the procollagen typeâ… mRNA synthesized by fibroblast by RT-PCR.Results 1.0μM TAM inhibited the nmHMEC activity,and suppressing effect of 5.0μM TAM significantly increased;the suppressing effect difference between different concentrations had pronouncedly statistical significance(p＜0.01).But,when the concentration was over 5.0μM,the increase in suppressing effect was not markedly, and the suppressing effect presented concentration dependence in a certain scope. Exposure to TAM for 48 hours,the increase of suppressing effect was markedly contrasted to other times,and the difference had statistical significance(p＜0.05).The extended exposure time did not increase the suppressing effect(p＞0.05),and the suppressing effect presented time dependence in a certain scope.The cell growth activity treated by TAM+ICI182,780 had no marked changes contrasted to TAM only. After exposure to TAM for 48 hour,the G₀/G₁ of nmHMEC and gyHMEC were 51.3% and 59.8%respectively,and increased markedly contrasted to control(p＜0.05);the AR of nmHMEC and gyHMEC were 10.1%and 18.1%respectively,and increased markedly contrasted to control(p＜0.01).The AR of florid type was 25.3%,which was markedly higher than AR(18.8%) of intermediate type and AR(10.3%) of fibrous type gynecomastia(p＜0.05).After exposure to TAM for 48 hours,the GIR of nmFb and gyFb were 36.81%and 56.32%respectively,and the difference had pronouncedly statistical significance(p＜0.05),the LI were 7.0%,and 5.2%respectively,which was at equal pace with GIR.The expression index of procollagen typeâ… mRNA in nmFb and gyFb pre- and post-treatment were 0.7,0.5 and 0.71,0.23 respectively,and the latter decreasing degree was more obviously;the difference between them had strikingly statistical significance(p＜0.05).Conclusions TAM had cell growth suppressing effects on nmHMEC, gyHMEC,nmFband and gyFb cultured in vitro,and the suppressing effects were mediated by ERa.The suppressing effects of TAM on gyHMEC from different histology gynecomastia had difference.The suppressing effect on gyHMEC from florid type of gynecomastia was most strong,which confirmed that TAM should be used in the early phrase of gynecomastia.The experiment using male HMEC cultured in vitro to study the medicine exposure effects provided an important method for the medicine intervention of gynecomastia.