男性乳房发育症发病机制及治疗干预的实验研究
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摘要
第一部分ERα,ERβ与PTEN在男性乳房发育症乳腺中的表达研究
目的男性乳房发育症(GYM)是最常见的男性乳腺疾病,其病理特征为乳腺腺体组织的增生。乳腺局部组织对激素的反应性改变是其重要的的发病机制之一。但目前还没有雌激素(ER)亚型及第10号染色体缺失的磷酸酶和张力蛋白同源物基因(PTEN)在GYM乳腺中表达研究的报道。本实验拟观察GYM乳腺中ERα、p-ERα、ERβ与PTEN的表达状况,以进一步深化对GYM的发病机制的认识。
方法以68例GYM乳腺组织为实验组,以24例正常男性乳腺为对照组。按病理学类型将GYM分旺炽型(13例)、中间型(18例)和纤维化型(37例);按年龄将GYM分为:青年组(21例)、中年组(27例)和老年组(20例)。采用免疫组织化学染色SP法检测ERα、p-ERα、ERβ与PTEN蛋白表达情况,表达情况根据染色强度及阳性细胞率计算表达指数来评估。
结果ERβ在GYM和正常男性乳腺组织的乳腺导管上皮细胞及间质成纤维细胞中均有表达,且表达水平GYM较正常男性乳腺组织明显上调;ERα、p-ERα和PTEN仅在GYM和正常男性乳腺组织的乳腺导管上皮细胞中表达,而间质细胞中不表达,且ERα、p-ERα蛋白在GYM中表达较正常男性乳腺升高,而PTEN蛋白在GYM中表达较正常男性乳腺下降;ERβ和ERα表达升高的程度及PTEN表达下降的程度在旺炽型、中间型和纤维化型GYM中依次递减,其间差异有统计学意义(p＜0.05);ERβ和ERα表达升高的程度及PTEN表达下降的程度在不同年龄组的表达也不同,在青年组、中年组和老年组GYM中依次递减,其之间差异也有统计学意义(p＜0.05)。
结论GYM和正常男性乳腺组织的乳腺导管上皮细胞及间质成纤维细胞中ERβ、ERα、p-ERα和PTEN表达不同,ERα和ERβ活性的增强和PTEN表达降低在GYM发病机制中具有重要作用;旺炽型、中间型和纤维化型GYM和青年组、中年组和老年组GYM发病机制存在差异。
第二部分正常男性乳腺及GYM乳腺上皮细胞和成纤维细胞的体外培养研究
目的目前还没有关于正常男性及GYM乳腺上皮细胞(HMEC)和成纤维细胞(fibroblast,Fb)体外培养的研究报道。本实验将探讨从正常男性乳腺及GYM乳腺组织中分离培养HMEC及Fb,以期建立男性HMEC和Fb体外培养细胞模型。
方法参照女性HMEC分离培养的方法,对新鲜的10例GYM乳腺组织标本和4例正常男性乳腺标本,用Ⅰ型胶原酶溶液(50 U/ml)于37℃水浴摇床振荡过夜消化乳腺组织碎块,用Percoll等密度梯度分离技术分离HMEC和Fb,分别以各自的培养液接种培养。相差显微镜动态观察细胞的生长形态,透视电镜观察细胞的超微结构;免疫细胞化学和免疫荧光法分别检测Cytokeratin 19和Vimentin在HMEC和Fb的特异性表达以鉴定其组织学来源。对分离培养的原代HMEC和Fb进行传代、冻存复苏,观察细胞生长活力。
结果所取的GYM和正常男性乳腺组织均能成功分离出HMEC和Fb,并能在各自培养液中良好生长。Cytokeratin 19和Vimentin免疫学标记均可分别在HMEC和Fb特异性表达,且表达率达到95%。HMEC在体外传代2次仍生长良好,且冻存复苏后可保持生长活力。Fb在体外生长活力较HMEC强。
结论参照女性HMEC分离培养的方法,从正常男性及GYM乳腺组织中分离培养HMEC和Fb是可行的,能成功建立男性HMEC和Fb体外培养模型。
第三部分E_2对体外培养的nmHMEC和gyHMEC的作用与PTEN在体外培养的nmHMEC和gyHMEC的作用研究
目的研究表明雌二醇(E_2)对女性HMEC作用的发挥是通过依赖ERα的PI3K/Akt信号途径实现的。正常女性乳腺腺泡上皮和导管上皮中存在PI3K/PTEN/AKt信号通路,ERα能结合PI3K的催化亚单位p85,激活PI3K。而GYM是否存在PI3K/PTEN/AKt信号通路,及其与E_2的关系尚未见文献报道。本实验拟通过观察E_2对体外培养的正常男性乳腺上皮细胞(nmHMEC)和GYM乳腺上皮细胞(gyHMEC)的作用及其对PI3K/PTEN/AKt信号通路的影响,PTEN在体外培养的nmHMEC和gyHMEC中的表达情况及其与ERα的关系,进一步探讨GYM的发病机制。
方法分别用10~(-12)、10~(-11)、10~(-10)、10~(-9)和10~(-8)M E_2处理处于对数生长期的原代nmHMEC,在0 h、12 h、24 h、48 h、72 h时间点,用BrdU掺入法和MTT法观察细胞生长和增殖情况,筛选出E_2合适浓度及最佳效用时间。用合适浓度的E_2处理乳腺上皮细胞,并在最佳作用时间用Western blot检测ERα,p85和Akt蛋白表达情况。用E_2、E_2+BAG(ERβ激动剂)、BAG分别处理nmHMEC 48 h,观察细胞增殖情况。E_2、E_2+ICI182,780(ERα特异性阻断剂)分别处理nmHMEC 48 h,观察细胞的ERα、p85、p-Akt及PTEN蛋白表达的变化。用E_2处理gyHMEC和nmHMEC 48h,检测细胞的ERα,p-ERα和p-Akt蛋白的表达变化。分别用E_2和LY294002(特异性阻断PI3K通路)处理gyHMEC和nmHMEC,用Western blot测定细胞的ERα,p-ERα和p-Akt蛋白表达的变化,免疫荧光检测细胞的PIP3表达的变化。
结果体外培养的nmHMEC在E_2刺激作用下生长明显增快,且在10~(-10) M浓度时表现明显,不同浓度之间细胞增殖活性存在显著性差异(p＜0.05);作用48 h时,细胞增殖活性显著,不同作用时间之间细胞增殖活性存在显著性差异(p＜0.05);E_2对nmHMEC的促增殖作用表现为浓度依赖性和时间依赖性。被E_2+BAG处理的nmHMEC增殖明显增强,但与E_2处理的细胞增殖程度差别没有统计学意义,仅用BAG处理的细胞生长变化不明显。E_2处理后的nmHMEC中ERα表达较对照组明显下降,但是p85、p-Akt和PTEN蛋白表达却较对照组显著升高(p＜0.05)。用E_2+ICI182,780处理后的nmHMEC中p85、p-Akt和PTEN蛋白表达与对照组相比,之间差异无统计学意义。E_2处理后的gyHMEC和nmHMEC中ERα蛋白表达较对照组明显降低(p＜0.05),而gyHMEC中下调的程度更显著,但相反的是,p-Akt与p-ERα蛋白表达却较对照组显著增加(p＜0.05),且在gyHMEC中的表达增加的程度要比nmHMEC高(p＜0.05)。LY294002特异性阻断PI3K通路后,E_2对gyHMEC中的p-Akt与p-ERα的表达影响不明显,但使nmHMEC中的p-Akt与p-ERα表达显著下降(p＜0.05),gyHMEC中PIP3表达的免疫荧光强度较对照组明显降低(p＜0.05)。
结论E_2能明显促体外培养的nmHMEC生长增殖,并有浓度依赖性和时间依赖性。E_2对nmHMEC的促增殖作用是通过ERα介导实现的,可能机制是激活了PI3K/Akt信号通路。GYM乳腺中表达下调的PTEN削弱了对下游PIP3/Akt信号通路的抑制作用,即促进Akt的相对增加,进而导致了乳腺上皮细胞的过度增殖。被PTEN诱导上调的Akt增加了对ERα的磷酸化,从而使ERα对E_2敏感性增加,更加促进了男性乳腺的过度增生。PTEN与E_2均可通过PI3K/PTEN/AKt信号通路影响GYM的增生。
第四部分他莫昔芬对体外培养的正常男性乳腺和GYM乳腺上皮细胞和成纤维细胞的作用研究
目的他莫昔芬(tamoxifen,TAM)是治疗GYM常用的药物,但其确切疗效和安全性尚未得到普遍公认。本实验拟观察TAM对体外培养的正常男性乳腺上皮细胞(nmHMEC)、GYM乳腺上皮细胞(gyHMEC)、正常男性乳腺成纤维细胞(nmFb)及GYM成纤维细胞(gyFb)生长的影响,以期为TAM治疗GYM提供
实验依据。
方法将nmHMEC分别暴露于不同浓度的TAM和相应浓度的TAM+ERα阻断剂ICI182,780(1.0μM)中,观察0、12、24、48及72 h的细胞抑制率(GIR)和细胞增殖指数(LI);将nmHMEC和gyHMEC分别暴露于TAM(1.0μM)48 h,观察对TAM对细胞周期分布和细胞凋亡率(AR)的影响。将旺炽型、中间型、纤维化型3种类型来源的gyHMEC分别暴露于TAM 48 h,观察TAM对细胞周期和细胞凋亡的影响。将正常男性乳腺成纤维细胞(nmFb)和gyFb分别暴露于TAM(1.0μM)48 h,观察细胞生长增殖的变化。GIR和LI用BrdU掺入法和MTT法分析;细胞周期分布和AR用流式细胞术测定;成纤维细胞合成前体胶原Ⅰ型mRNA的变化用RT-PCR评价。
结果TAM浓度在1.0μM时能明显抑制nmHMEC活性,5.0μM的抑制效应显著增加,不同浓度之间抑制效应差别具有显著的统计学意义(p＜0.01),但当浓度超过5.0μM时,抑制作用增加并不明显,TAM的抑制效应在一定范围内呈浓度依赖性。TAM暴露48 h对细胞的抑制作用增加最快,与其他时间点相比,其差异具有显著的统计学意义(p＜0.05),延长作用时间抑制作用增加并不显著(p＞0.05),表现为一定范围内的时间依赖性。TAM+ICI182,780处理组的细胞生长活性与单用TAM作用相比,没有明显变化。TAM暴露作用48 h后,nmHMEC和gyHMEC的G_0/G_1期分别为51.3%、59.8%,均较对照组明显增加(p＜0.05);nmHMEC和gyHMEC的AR分别为10.1%、18.1%,较对照组显著增加(p＜0.01)。旺炽型gyHMEC的AR为25.3%,较纤维化型(AR,10.3%)和中间型(AR,18.8%)明显增加(p＜0.05)。暴露于TAM 48h后,nmFb和gyFb的GIR分别为36.81%、56.32%,二者之间差别具有显著的统计学意义(p＜0.05);LI分别为7.0%、5.2%,与GIR变化一致;nmFb和gyFb中Ⅰ型胶原mRNA表达指数在暴露前后分别为0.7、0.5;0.71、0.23,后者降低程度更明显,二者之间差别具有显著的统计学意义(p＜0.05)。
结论TAM对体外培养的nmHMEC、gyHMEC、nmFb及gyFb具有明显的生长抑制效应,且TAM对nmHMEC抑制效应的发挥是由ERα介导的。不同组织学类型的gyHMEC对TAM抑制效应反应性的存在差异,TAM对旺炽型gyHMEC抑制效应最强,证实ATM的临床应用应选择在该病的早期阶段。
PART 1 THE STUDY OF ERα,ERβAND PTEN EXPRESSION IN GYNECOMASTIA
Objective 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 MAN
Objective 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(typeⅠ) 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 DEVELOPMENT
Objective Some researches disclosed that the effects of E_2 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 E_2.The purpose of this study is to determine the effects of E_2 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 E_2 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 E_2 with suitable concentration and an ER弓agonist(BAG) or BAG alone for 48 hours.After treatment on nmHMEC with E_2 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 E_2 were observed by Western blot analysis.The cultured primary gyHMEC and nmHMEC were exposed to E_2 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 E_2 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 E_2 resulted in a marked increase in cell proliferation,and the increase presented significantly on 10~(-10) M E_2.There was significant difference in the cell proliferation activity with different concentration of E_2 treatment(p<0.05).The promoting proliferation effects present concentration dependence.The cell proliferation activity presented markedly when E_2 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 E_2 and BAG displayed higher cell proliferation than nmHMEC with BAG treatment alone.ERαexpression decreased in nmHMEC with E_2 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 E_2 and ICI182,780 treatment compared to control(p<0.05).The ERαexpression decreased in gyHMEC and nmHMEC after treatment with E_2.The decreasing degree of ERαin gyHMEC with E_2 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 E_2 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 E_2 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 E_2 resulting in the hyperplasia of male mammary.Both PTEN and E_2 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 GYNECOMASTIA
Objective 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 typeⅠmRNA 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 G_0/G_1 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 typeⅠmRNA 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.
目的男性乳房发育症(GYM)是最常见的男性乳腺疾病,其病理特征为乳腺腺体组织的增生。乳腺局部组织对激素的反应性改变是其重要的的发病机制之一。但目前还没有雌激素(ER)亚型及第10号染色体缺失的磷酸酶和张力蛋白同源物基因(PTEN)在GYM乳腺中表达研究的报道。本实验拟观察GYM乳腺中ERα、p-ERα、ERβ与PTEN的表达状况,以进一步深化对GYM的发病机制的认识。
方法以68例GYM乳腺组织为实验组,以24例正常男性乳腺为对照组。按病理学类型将GYM分旺炽型(13例)、中间型(18例)和纤维化型(37例);按年龄将GYM分为:青年组(21例)、中年组(27例)和老年组(20例)。采用免疫组织化学染色SP法检测ERα、p-ERα、ERβ与PTEN蛋白表达情况,表达情况根据染色强度及阳性细胞率计算表达指数来评估。
结果ERβ在GYM和正常男性乳腺组织的乳腺导管上皮细胞及间质成纤维细胞中均有表达,且表达水平GYM较正常男性乳腺组织明显上调;ERα、p-ERα和PTEN仅在GYM和正常男性乳腺组织的乳腺导管上皮细胞中表达,而间质细胞中不表达,且ERα、p-ERα蛋白在GYM中表达较正常男性乳腺升高,而PTEN蛋白在GYM中表达较正常男性乳腺下降;ERβ和ERα表达升高的程度及PTEN表达下降的程度在旺炽型、中间型和纤维化型GYM中依次递减,其间差异有统计学意义(p＜0.05);ERβ和ERα表达升高的程度及PTEN表达下降的程度在不同年龄组的表达也不同,在青年组、中年组和老年组GYM中依次递减,其之间差异也有统计学意义(p＜0.05)。
结论GYM和正常男性乳腺组织的乳腺导管上皮细胞及间质成纤维细胞中ERβ、ERα、p-ERα和PTEN表达不同,ERα和ERβ活性的增强和PTEN表达降低在GYM发病机制中具有重要作用;旺炽型、中间型和纤维化型GYM和青年组、中年组和老年组GYM发病机制存在差异。
第二部分正常男性乳腺及GYM乳腺上皮细胞和成纤维细胞的体外培养研究
目的目前还没有关于正常男性及GYM乳腺上皮细胞(HMEC)和成纤维细胞(fibroblast,Fb)体外培养的研究报道。本实验将探讨从正常男性乳腺及GYM乳腺组织中分离培养HMEC及Fb,以期建立男性HMEC和Fb体外培养细胞模型。
方法参照女性HMEC分离培养的方法,对新鲜的10例GYM乳腺组织标本和4例正常男性乳腺标本,用Ⅰ型胶原酶溶液(50 U/ml)于37℃水浴摇床振荡过夜消化乳腺组织碎块,用Percoll等密度梯度分离技术分离HMEC和Fb,分别以各自的培养液接种培养。相差显微镜动态观察细胞的生长形态,透视电镜观察细胞的超微结构;免疫细胞化学和免疫荧光法分别检测Cytokeratin 19和Vimentin在HMEC和Fb的特异性表达以鉴定其组织学来源。对分离培养的原代HMEC和Fb进行传代、冻存复苏,观察细胞生长活力。
结果所取的GYM和正常男性乳腺组织均能成功分离出HMEC和Fb,并能在各自培养液中良好生长。Cytokeratin 19和Vimentin免疫学标记均可分别在HMEC和Fb特异性表达,且表达率达到95%。HMEC在体外传代2次仍生长良好,且冻存复苏后可保持生长活力。Fb在体外生长活力较HMEC强。
结论参照女性HMEC分离培养的方法,从正常男性及GYM乳腺组织中分离培养HMEC和Fb是可行的,能成功建立男性HMEC和Fb体外培养模型。
第三部分E_2对体外培养的nmHMEC和gyHMEC的作用与PTEN在体外培养的nmHMEC和gyHMEC的作用研究
目的研究表明雌二醇(E_2)对女性HMEC作用的发挥是通过依赖ERα的PI3K/Akt信号途径实现的。正常女性乳腺腺泡上皮和导管上皮中存在PI3K/PTEN/AKt信号通路,ERα能结合PI3K的催化亚单位p85,激活PI3K。而GYM是否存在PI3K/PTEN/AKt信号通路,及其与E_2的关系尚未见文献报道。本实验拟通过观察E_2对体外培养的正常男性乳腺上皮细胞(nmHMEC)和GYM乳腺上皮细胞(gyHMEC)的作用及其对PI3K/PTEN/AKt信号通路的影响,PTEN在体外培养的nmHMEC和gyHMEC中的表达情况及其与ERα的关系,进一步探讨GYM的发病机制。
方法分别用10~(-12)、10~(-11)、10~(-10)、10~(-9)和10~(-8)M E_2处理处于对数生长期的原代nmHMEC,在0 h、12 h、24 h、48 h、72 h时间点,用BrdU掺入法和MTT法观察细胞生长和增殖情况,筛选出E_2合适浓度及最佳效用时间。用合适浓度的E_2处理乳腺上皮细胞,并在最佳作用时间用Western blot检测ERα,p85和Akt蛋白表达情况。用E_2、E_2+BAG(ERβ激动剂)、BAG分别处理nmHMEC 48 h,观察细胞增殖情况。E_2、E_2+ICI182,780(ERα特异性阻断剂)分别处理nmHMEC 48 h,观察细胞的ERα、p85、p-Akt及PTEN蛋白表达的变化。用E_2处理gyHMEC和nmHMEC 48h,检测细胞的ERα,p-ERα和p-Akt蛋白的表达变化。分别用E_2和LY294002(特异性阻断PI3K通路)处理gyHMEC和nmHMEC,用Western blot测定细胞的ERα,p-ERα和p-Akt蛋白表达的变化,免疫荧光检测细胞的PIP3表达的变化。
结果体外培养的nmHMEC在E_2刺激作用下生长明显增快,且在10~(-10) M浓度时表现明显,不同浓度之间细胞增殖活性存在显著性差异(p＜0.05);作用48 h时,细胞增殖活性显著,不同作用时间之间细胞增殖活性存在显著性差异(p＜0.05);E_2对nmHMEC的促增殖作用表现为浓度依赖性和时间依赖性。被E_2+BAG处理的nmHMEC增殖明显增强,但与E_2处理的细胞增殖程度差别没有统计学意义,仅用BAG处理的细胞生长变化不明显。E_2处理后的nmHMEC中ERα表达较对照组明显下降,但是p85、p-Akt和PTEN蛋白表达却较对照组显著升高(p＜0.05)。用E_2+ICI182,780处理后的nmHMEC中p85、p-Akt和PTEN蛋白表达与对照组相比,之间差异无统计学意义。E_2处理后的gyHMEC和nmHMEC中ERα蛋白表达较对照组明显降低(p＜0.05),而gyHMEC中下调的程度更显著,但相反的是,p-Akt与p-ERα蛋白表达却较对照组显著增加(p＜0.05),且在gyHMEC中的表达增加的程度要比nmHMEC高(p＜0.05)。LY294002特异性阻断PI3K通路后,E_2对gyHMEC中的p-Akt与p-ERα的表达影响不明显,但使nmHMEC中的p-Akt与p-ERα表达显著下降(p＜0.05),gyHMEC中PIP3表达的免疫荧光强度较对照组明显降低(p＜0.05)。
结论E_2能明显促体外培养的nmHMEC生长增殖,并有浓度依赖性和时间依赖性。E_2对nmHMEC的促增殖作用是通过ERα介导实现的,可能机制是激活了PI3K/Akt信号通路。GYM乳腺中表达下调的PTEN削弱了对下游PIP3/Akt信号通路的抑制作用,即促进Akt的相对增加,进而导致了乳腺上皮细胞的过度增殖。被PTEN诱导上调的Akt增加了对ERα的磷酸化,从而使ERα对E_2敏感性增加,更加促进了男性乳腺的过度增生。PTEN与E_2均可通过PI3K/PTEN/AKt信号通路影响GYM的增生。
第四部分他莫昔芬对体外培养的正常男性乳腺和GYM乳腺上皮细胞和成纤维细胞的作用研究
目的他莫昔芬(tamoxifen,TAM)是治疗GYM常用的药物,但其确切疗效和安全性尚未得到普遍公认。本实验拟观察TAM对体外培养的正常男性乳腺上皮细胞(nmHMEC)、GYM乳腺上皮细胞(gyHMEC)、正常男性乳腺成纤维细胞(nmFb)及GYM成纤维细胞(gyFb)生长的影响,以期为TAM治疗GYM提供
实验依据。
方法将nmHMEC分别暴露于不同浓度的TAM和相应浓度的TAM+ERα阻断剂ICI182,780(1.0μM)中,观察0、12、24、48及72 h的细胞抑制率(GIR)和细胞增殖指数(LI);将nmHMEC和gyHMEC分别暴露于TAM(1.0μM)48 h,观察对TAM对细胞周期分布和细胞凋亡率(AR)的影响。将旺炽型、中间型、纤维化型3种类型来源的gyHMEC分别暴露于TAM 48 h,观察TAM对细胞周期和细胞凋亡的影响。将正常男性乳腺成纤维细胞(nmFb)和gyFb分别暴露于TAM(1.0μM)48 h,观察细胞生长增殖的变化。GIR和LI用BrdU掺入法和MTT法分析;细胞周期分布和AR用流式细胞术测定;成纤维细胞合成前体胶原Ⅰ型mRNA的变化用RT-PCR评价。
结果TAM浓度在1.0μM时能明显抑制nmHMEC活性,5.0μM的抑制效应显著增加,不同浓度之间抑制效应差别具有显著的统计学意义(p＜0.01),但当浓度超过5.0μM时,抑制作用增加并不明显,TAM的抑制效应在一定范围内呈浓度依赖性。TAM暴露48 h对细胞的抑制作用增加最快,与其他时间点相比,其差异具有显著的统计学意义(p＜0.05),延长作用时间抑制作用增加并不显著(p＞0.05),表现为一定范围内的时间依赖性。TAM+ICI182,780处理组的细胞生长活性与单用TAM作用相比,没有明显变化。TAM暴露作用48 h后,nmHMEC和gyHMEC的G_0/G_1期分别为51.3%、59.8%,均较对照组明显增加(p＜0.05);nmHMEC和gyHMEC的AR分别为10.1%、18.1%,较对照组显著增加(p＜0.01)。旺炽型gyHMEC的AR为25.3%,较纤维化型(AR,10.3%)和中间型(AR,18.8%)明显增加(p＜0.05)。暴露于TAM 48h后,nmFb和gyFb的GIR分别为36.81%、56.32%,二者之间差别具有显著的统计学意义(p＜0.05);LI分别为7.0%、5.2%,与GIR变化一致;nmFb和gyFb中Ⅰ型胶原mRNA表达指数在暴露前后分别为0.7、0.5;0.71、0.23,后者降低程度更明显,二者之间差别具有显著的统计学意义(p＜0.05)。
结论TAM对体外培养的nmHMEC、gyHMEC、nmFb及gyFb具有明显的生长抑制效应,且TAM对nmHMEC抑制效应的发挥是由ERα介导的。不同组织学类型的gyHMEC对TAM抑制效应反应性的存在差异,TAM对旺炽型gyHMEC抑制效应最强,证实ATM的临床应用应选择在该病的早期阶段。
PART 1 THE STUDY OF ERα,ERβAND PTEN EXPRESSION IN GYNECOMASTIA
Objective 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 MAN
Objective 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(typeⅠ) 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 DEVELOPMENT
Objective Some researches disclosed that the effects of E_2 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 E_2.The purpose of this study is to determine the effects of E_2 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 E_2 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 E_2 with suitable concentration and an ER弓agonist(BAG) or BAG alone for 48 hours.After treatment on nmHMEC with E_2 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 E_2 were observed by Western blot analysis.The cultured primary gyHMEC and nmHMEC were exposed to E_2 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 E_2 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 E_2 resulted in a marked increase in cell proliferation,and the increase presented significantly on 10~(-10) M E_2.There was significant difference in the cell proliferation activity with different concentration of E_2 treatment(p<0.05).The promoting proliferation effects present concentration dependence.The cell proliferation activity presented markedly when E_2 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 E_2 and BAG displayed higher cell proliferation than nmHMEC with BAG treatment alone.ERαexpression decreased in nmHMEC with E_2 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 E_2 and ICI182,780 treatment compared to control(p<0.05).The ERαexpression decreased in gyHMEC and nmHMEC after treatment with E_2.The decreasing degree of ERαin gyHMEC with E_2 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 E_2 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 E_2 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 E_2 resulting in the hyperplasia of male mammary.Both PTEN and E_2 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 GYNECOMASTIA
Objective 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 typeⅠmRNA 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 G_0/G_1 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 typeⅠmRNA 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.
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