The interaction between epidermal growth factor and matrix metalloproteinases induces the development of sweat glands in human fetal skin

BACKGROUND: The development of sweat glands is a very complicated biological process involving many factors. In this study, we explore the interrelationship among epidermal growth factor (EGF), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 7 (MMP-7), and the development of sweat glands in human embryos. Furthermore, we hope to elucidate the mechanism(s) underlying the induction of epidermal stem cells into sweat gland cells. MATERIALS AND METHODS: Skin biopsies of human embryos obtained from spontaneous abortions at different gestational ages from 11 to 31 weeks were used in this study. The dynamic expression of EGF, MMP-2, MMP-7, and keratin-7 (K7) in developing sweat gland cells or extracellular stroma surrounding the sweat gland cells was examined with SP immunohistochemical methods. The localization of the cellular sources of MMP-2 and MMP-7 was examined with in situ hybridization. RESULTS: At 14-20 weeks of gestation, a gradual increase in EGF immunoreactivity was observed not only in developing sweat gland buds but also in extracellular stroma surrounding the buds, and the expression intensity of EGF peaked at 20-22 weeks of gestational age. All mRNA-positive buds or cells in developing sweat glands contained corresponding immunoreactive proteins. Positive immunostaining for K7 appeared in early sweat gland buds at 14-16 weeks of gestation, and from then on, the positive signal of K7 was concentrated in developing sweat gland cords or cells. CONCLUSIONS: The morphogenesis of sweat glands in human fetal skin begins at 14-16 weeks of gestational age, and is essentially complete by 24 weeks. There is a close relationship among EGF, extracellular matrix remodeling, and morphogenesis of the sweat glands. EGF is one of the inducers in the development and maturity of sweat gland buds or cells.     

Epidermal stem cells are the source of sweat glands in human fetal skin: Evidence of synergetic development of stem cells, sweat glands, growth factors, and matrix metalloproteinases

The development of sweat glands is a complex biological process, and the extent of cellular trafficking between epidermal stem cells and the development of sweat glands is uncertain. Therefore, we studied the synergetic development effects of stem cells, sweat glands, growth factors, and matrix metalloproteinases (MMPs) in human skin. Human fetal skin was obtained from spontaneously aborted fetuses at 11-31 weeks of gestation. Paraffin sections were cut and stained with hematoxylin and eosin or immunostained with antibodies against beta(1) integrin, keratin (K)-19 and K7, MMP-2 and -7, and epidermal growth factor. In situ hybridization was used along with semiquantitative analysis of the positive expression of these proteins to analyze for mRNA expression of MMP-2 and -7. Histological studies revealed the fetal epidermis began to form a primary epidermal ridge at gestational age 13-14 weeks and these primordial basal cells became tightly packed to take the form of multiple hillocks between 14 and 16 weeks. Furthermore, these cells gave rise to chord-like columnar buds in the embryonic epidermis, and these buds gradually migrated downward into the dermis to form juvenile sweat glands at 18-20 weeks. Mature sweat glands were found in the fetal epidermis at the end of 24 weeks. beta(1) integrin and K19 immunoreactivities were first detected in those cells that gathered together to form primary epidermal ridges, including sweat gland cords, buds, and immature sweat gland cells. The positive immunostaining for K7 appeared in early sweat gland buds at 14-16 weeks, and from then on K7 was concentrated in developing sweat gland cords or cells. At 14-16 weeks, positive epidermal growth factor, MMP-2, and MMP-7 expression was first observed weakly in developing sweat gland buds. The immunoreactivity of these proteins was then gradually increased in the developing sweat gland buds and extracellular stroma from 14 to 20 weeks. The intensity of the positive signal peaked at 20-22 weeks of gestational age. After that, the intensity of immunostaining for MMP-2 and MMP-7 proteins was gradually weakened. However, the expression of epidermal growth factor did not show an apparent decrease. These results suggest that epidermal stem cells are the source of sweat glands. Epidermal growth factor is one of the main inducers in the development and maturity of sweat gland buds or cells and the local activated MMPs may play an important role in cleaving the major matrix components in the basement membrane.     

人胚胎期表皮干细胞与汗腺发生过程关系的研究

目的　观察表皮干细胞与胚胎期汗腺发生过程的关系 ,为诱导表皮干细胞向汗腺细胞定向分化奠定基础。　方法　分别取 13～ 3 1周胎龄人胎儿背部全层皮肤 ,采用常规组织学、免疫组织化学染色法 (SP法 ) ,动态观察汗腺原基细胞、汗腺胚芽细胞及汗腺细胞中 β1整合素与细胞角蛋白 19(K19)的表达特征。以细胞角蛋白 8(K8)免疫组化染色阳性为汗腺发生及成熟的鉴定标准。　结果　组织学观察显示 ,胎龄 16周的皮肤 ,初级表皮嵴基底层细胞呈灶性聚集 ,形成小丘状 ,继而形成圆柱状细胞索向深层切入 ;至胎龄第 2 4周时 ,细胞索末端部分形成蟠状 ,表现为成熟汗腺特征。不仅汗腺原基细胞、汗腺胚芽细胞表达 β1整合素与K19,成熟的汗腺细胞亦有表达 ,并持续存在于汗腺发生全过程。K8始于 14～ 16周 ,在汗腺胚芽细胞内表达并持续存在。　结论　汗腺于胎龄 14～ 16周开始发生 ,至第 2 4周基本成熟。胚胎期汗腺发生过程中 ,表皮干细胞是汗腺发生的源泉     

复合汗腺的组织工程三维皮肤模型的构建

目的：运用组织工程化方法在体外建立复合汗腺的三维皮肤模型,阐明汗腺体外再生的可行性,并为初步建立含有汗腺的仿生化功能性组织工程皮肤奠定实验基础。方法：分离培养人表皮细胞、成纤维细胞和汗腺细胞,将表皮细胞与汗腺细胞按1：1的比例共培养,在培养体系中分别加入含表皮细胞生长因子（EGF）的微球,噻唑蓝（MTT）法观察细胞增殖情况,并与培养时直接加入EGF及不加EGF的对照组进行比较。将共培养的细胞接种于复合成纤维细胞的胶原基质并通过三维培养方式构建组织工程皮肤模型,在模型中加入复合EGF的微球释放载体促进汗腺再生和上皮化进程,HE染色和免疫组织化学方法检测所构建组织工程皮肤以及体外再生汗腺的结构,并与模型中直接加入EGF和未加EGF的对照组进行比较。结果：MTT检测结果显示,与两对照组相比较,通过共培养方式获得的表皮与汗腺细胞团在复合EGF微球作用下生长良好,有明显增殖作用;组织学观察结果显示所构建组织工程皮肤模型具有和正常皮肤相似的结构,并在真皮浅层形成了类似汗腺结构的细胞密集区域,存在大量汗腺特异性标志——角蛋白19（CK19）和癌胚抗原（CEA）阳性的细胞,这种现象在单纯EGF组仅有微弱表现,在空白对照组则无此现象出现。结论：构建具有汗腺结构的体外皮肤模型具有可行性,这为汗腺再生和功能性组织工程皮肤的研究开创了新的途径。     

诱导表皮干细胞向汗腺上皮细胞分化的研究

目的通过探索体外诱导表皮干细胞向汗腺上皮分化可能性,为汗腺组织工程探索新的种子细胞来源。方法分别收集正常皮肤表皮细胞．利用流式细胞仪多参数分选表皮干细胞;于不同时间点连续观察,不同浓度EGF（50～1000ng／mL）作用于表皮干细胞后的细胞生长动力学变化,免疫荧光鉴定汗腺细胞表型（NHE-1,NKCC-1）流式细胞仪分析不同浓度EGF对于细胞的毒性。结果50ng／mL和100ng／mL EGF对表皮细胞促增殖能力明显,且二者无显著差异（P〉0．05）：500ng／mL和1000ng／mL EGF抑制细胞增殖。1000ng／mL EGF对细胞增殖的抑制作用更明显（P〈0．05）。表皮干细胞经过50ng／mLEGF诱导后表达汗腺上皮相对特异性标记NHE-1及NKCC-1。结论表皮干细胞可成为汗腺组织工程种子细胞的重要来源。50ng／ml EGF可作为相对优化的表皮干细胞诱导条件。     

The expression characteristics and biological significance of bFGF, EGF,TGF-β isoforms and their receptors in skins from fetus and adult

To observe the localization and expression characteristics of alpha-smooth muscle actin (AS-MA), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-β(TGF-β) isoforms, and their receptors in fetal and adult skins in order to explore their potential biological significance.Methods: The expression and the distribution of ASMA, bFGF, EGF, TGF-βisoforms, and their receptors were detected with immunohistochemistry and histopathology methods in 36 skin specimens. Among them, 30 specimens belonged to fetuses at different developmental stages and 6 were from adults. Results:Positive immunohistochemical signals of ASMA, bFGF, EGF, and TGF-βisoforms and their receptors could be found in fetal and postnatal skins.These factors were mainly distributed in the cytoplasm and extracellular matrix of epidermal cells, endothelial cells,hair follicle epithelial cells and some fibroblasts. Receptors of these factors were mostly located in the cellular membrane of the above mentioned cells, while protein particles of ASMA could be observed in myofibroblasts and sweat gland cells. Along with ascent in gestational age, the positive cellular rates of bFGF, EGF, TGF-βisoforms, their receptors, and ASMA in skin were elevated progressively. In skins specimens obtained from fetuses of late-trimester (29-31 week gestation) and adult, the positive rates of these proteins were significantly raised in comparison with skin of fetuses of early-trimester. Conclusion: The endogenous bFGF, EGF, three TGF-βisoforms and their receptors might be involved in the development of the skin in embryonic stage and in the cutaneous structure and function,and also wound healing in adult stage. The relative lack of these factors and their receptors might be one reason why the wound of fetus heal by regeneration rather than by scarring.     

采用基因芯片筛选与汗腺发育相关基因的初步研究

目的：探讨人胚胎皮肤汗腺在不同发育胎龄时的基因表达变化特征及其可能的生物学意义。方法：收集12周（H1）、16周（H2）、24周（H3）等3组不同胎龄胎儿皮肤,组织常规切片,显微镜下观察胚胎皮肤不同发育阶段的汗腺结构特征,提取不同胎龄的胚胎表皮组织总RNA,用逆转录-聚合酶链反应（RT-PCR）方法和基因芯片技术检测胚胎皮肤汗腺在不同发育阶段的基因表达变化规律。结果：光镜下可见不同发育阶段的胎儿皮肤具有典型的组织学结构,胎龄24周时汗腺结构初步形成,数量稳定。在95%的可信区间内,H2与H1样本相比较（H2/H1）,差异表达基因有113个,其中下调的基因有45个,上调的基因有68个;H3与H2样本相比较（H3/H2）,差异表达基因有91个,其中下调的基因有66个,上调的基因有25个;差异表达的基因包括生长因子、细胞外基质分子和外胚层发育因子等,与汗腺发育进程密切相关。结论：将基因芯片运用于汗腺发生机制的研究,可以较全面地反映汗腺发育及成熟过程中基因表达变化规律,寻找特异性调控基因。通过对汗腺不同发育阶段的基因表达及功能研究,可以筛选关键步骤调控基因,指导实现分子基因水平调控皮肤创面修复和汗腺组织再生。     

Regulation of matrix metalloprotease activity in malignant mesothelioma cell lines by growth factors

BACKGROUND: The extracellular matrix metalloproteases (MMPs) produced by tumour and stromal cells are believed to play a key role in tumour cell invasion and metastasis. Malignant mesothelioma is a highly aggressive tumour. Previous studies have shown that malignant mesothelioma cells express MMP-1, -2, -3, -7 and -9. However, the regulation of MMP expression in malignant mesothelioma cells has not been thoroughly investigated. METHODS: The effects of a number of growth factors on the secretion of MMP-2, -3 and -9 in malignant mesothelioma cells were studied by substrate zymography. The expression of relevant growth factor receptors in malignant mesothelioma cells was also examined using RT-PCR. RESULTS: The exposure of malignant mesothelioma cells to different growth factors including epidermal growth factor (EGF), transforming growth factor-alpha, amphiregulin, heparin binding EGF, beta-cellulin (BTC), stem cell factor, insulin-like growth factors I and II, acidic and basic fibroblast growth factors, and hepatocyte growth factor increased secretion of MMP-9 and/or MMP-3. EGF receptor ligands BTC and EGF had the most potent effect on MMP-9 and MMP-3 production, respectively. Production of MMP-2 was not affected by any growth factors used in this study. We have also demonstrated mRNA expression of different growth factor receptors in malignant mesothelioma cells, and found that the tyrosine kinase inhibitor genistein inhibited the increased production of MMPs resulting from stimulation with different growth factors. CONCLUSION: This study shows, for the first time, the broad extent of regulation of MMPs by various growth factors in malignant mesothelioma cells.     

表皮细胞生长因子通过诱导皮肤干细胞分化加速受创表皮再生的研究

目的：从皮肤干细胞增殖分化角度研究表皮细胞生长因子（EGF）加速受创皮肤再生的机制。方法：8例经EGF治疗创面于治疗后8天及14天对创面边缘部进行活检取材。用常规病理与SP免疫组织化学法研究β1整合素、角蛋白19（K19）、角蛋白14（K14）以及角蛋白10（K10）在不同修复阶段修复表皮的表达特征。另选研究β1整合素、角蛋白19（K19）、角蛋白14（K14）以及角蛋白10（K10）在不同修复阶段修复表皮的表达特征。另选7例同期未经EGF治疗创面为对照。结果：常规病理学检查与对照创面相比，EGF治疗创面后8天可见表皮层增厚，表皮脚增粗，治疗后14天以上特征更为明显。SP免疫组织化学对β1整合素与K19染色表明，EGF治疗8天的创面表皮干细胞与短暂扩充细胞不仅数量多，而且体积增大，治疗后14天创面面生表皮的棘细胞层中有干细胞岛出现。对照治疗后8天与14天创面除有一定数量的β1整合素与K19阳性染色细胞外，未见干细胞岛出现，在EGF治疗和对照治疗创面，治疗后8天及14天，K16、K10表达均见于再生上皮的表层，即那些已失去增殖能力与终末分化细胞。结论：EGF促进损伤皮肤再生的主要机制之一可能与它能诱导皮肤干细胞快速定向分化有关。     

Leptin受体在人胚胎皮肤附件发育过程中的表达特征

目的:探讨Leptin受体在人胚胎皮肤形成和附件发育过程中的表达特点及可能的生物学意义。方法:取8-38周龄人胚胎的背部全层皮肤,制成石蜡切片．进行常规组织学观察和免疫组织化学染色(SP法),动态观察皮肤形成过程和附件发育过程中Leptin受体的表达特点。结果:胚胎8周开始,胎儿周皮已有Leptin受体表达,随后逐渐增强。至胚胎11-16周表达最强,尤其在附件原基中呈局灶性表达;胚胎22周后,Leptin受体的表达逐渐减弱,至胚胎31周后仅在皮脂腺中表达。结论:Leptin可能通过其受体参与皮肤附件的发育过程。     

Neurogenic origin of human prostate endocrine cells

OBJECTIVES: To determine the histogenetic origin of prostate neuroendocrine cells in human embryos. METHODS: Prostatic tissue in human fetuses, ranging in gestational age from early week 10 to term, and infantile and pubertal glands were studied immunohistochemically. The distribution of neuroendocrine cells within the developing gland was semiquantitatively determined. Antibodies against the neuroendocrine markers chromogranin A (CgA) and protein gene product 9.5 (PGP), along with markers of prostatic secretion (prostate-specific antigen [PSA], prostatic acid phosphatase [PAP]), were used. They were applied either individually or in double-labeling experiments, as well as in experiments combining CgA immunohistochemical analysis with in situ hybridization or in situ end-labeling. RESULTS: In embryos of less than 65-mm crown-rump length (CRL) (ie, younger than 12 weeks of gestation), the epithelium of the urogenital sinus was free of endocrine cells. On either side of the future prostatic mesenchyme, paraganglia containing CgA-immunoreactive cells are present, which start to penetrate the urogenital mesenchyme. In the late 10th week, these CgA-immunoreactive cells are found dispersed in the urogenital mesenchyme. In embryos of 65-mm CRL, when prostatic anlagen start to sprout from the urogenital epithelium, very few (but typically shaped) neuroendocrine cells appear in the urogenital sinus epithelium. Later, after the 12th week, when solid prostatic ducts have started forming, CgA-immunoreactive neuroendocrine cells are also present in these buds. The number of neuroendocrine cells in the urethral epithelium is considerably increased, and with the continuous sprouting and lumen formation of prostatic anlagen, neuroendocrine cells are transported into the future gland. Neuroendocrine cells observed in stroma of prenatal and postnatal prostates may also contribute to the neuroendocrine cell population of the gland. CONCLUSIONS: Our results provide the first evidence that human prostate neuroendocrine cells represent a cell lineage of their own, being of neurogenic origin and therefore distinct from the urogenital sinus-derived prostate secretory and basal cells.     

人胎儿皮肤皮脂腺细胞和外泌汗腺细胞的分离培养及鉴定

目的建立人胎儿皮肤皮脂腺、外泌汗腺细胞的体外分离培养与鉴定方法。方法通过分离人胎儿皮肤皮脂腺腺体和外泌汗腺腺管,以DMEM/F12(1∶1)为基础培养基,分别添加不同浓度的胎牛血清、表皮生长因子、L-谷氨酰胺、氢化可的松、霍乱毒素、青霉素、链霉素、重组人表皮生长因子、三碘甲状腺氨酸、胰岛素、转铁蛋白、亚硒酸钠作为皮脂腺细胞培养基及外泌汗腺细胞培养基,置入37℃、体积分数5%CO2孵箱中进行原代及传代培养。倒置相差显微镜下观察人胎儿皮肤皮脂腺、外泌汗腺细胞的形态及变化,并进行细胞克隆形成率测定。采用油红染色和细胞角蛋白(CK)4.62、上皮膜抗原(EMA)免疫组织化学染色对传代培养的皮脂腺、外泌汗腺细胞进行鉴定。结果分离的人胎儿皮脂腺腺体和外泌汗腺腺管可以在体外贴壁生长繁殖;其皮脂腺细胞的克隆形成率为2.7%,明显低于人胎儿角质形成细胞(8.0%,P<0.01).人外泌汗腺细胞的克隆形成率为7.3%,与人胎儿角质形成细胞(7.7%)比较差异无统计学意义(P>0·05).油红染色显示,皮脂腺细胞含有少量脂质小滴,CK4.62、EMA免疫组织化学染色均为阳性;外泌汗腺细胞CK7、CK19免疫组织化学染色均为阳性。结论用酶消化法和显微分离法可体外分离人胎儿皮肤皮脂腺、外泌汗腺细胞,两者均具备上皮细胞的标志和生物学特点,但皮脂腺细胞增殖速度较为缓慢。     

The Role of NRG3 in Mammary Development

The Neuregulin gene family encodes EGF-containing ligands which mediate their effects by binding to the ERBB receptor tyrosine kinases, a signalling network with important roles in both mammary gland development and breast cancer. Neuregulin3 (NRG3), a ligand for ERBB4, promotes early mammary morphogenesis and acts during specification of the mammary placode, an aggregate of epithelial cells that forms during mid-embryogenesis. Recent studies have shown that NRG3 can alter the cell fate of other epidermal progenitor populations when NRG3 is mis-expressed throughout the basal layer of the developing epidermis with the K14 promoter. Here evidence for a key function for NRG3 in promoting early mammary morphogenesis and the implication for the role of NRG3 in breast cancer and establishment of the mammary lineage are discussed.     

汗腺样细胞经冻存复苏后再生汗腺功能的研究

目的:研究由人脐带间充质干细胞(UCMSCs)分化而来的汗腺样细胞在冻存与复苏后的生物学活性与促汗腺再生能力.方法:分离培养人UCMSCs 和人汗腺细胞,通过两种细胞共培养方式促进UCMSCs向汗腺细胞分化.分化后的干细胞经冻存、复苏处理后,局部移植于裸鼠烫伤脚掌,通过组织学观察和发汗试验观察汗腺组织再生修复情况.结果...     

阉割对大鼠颌下腺雄激素受体及表皮生长因子的影响

目的:观察阉割对大鼠颌下腺雄激素受体(AR)及表皮生长因子的影响。方法:60只30~60日龄的W istar雄性大鼠,随机分为阉割组、假手术组和正常对照组,每组20只。1周后摘除各组大鼠颌下腺。颌下腺匀浆后用于W estern印迹分析。结果:与其他两组相比,阉割组颌下腺中的AR含量明显降低(P<0.05),而且,颌下腺分泌的表皮生长因子亦低于其他两组(P<0.05)。结论:阉割影响了颌下腺中AR的产生,并进一步影响了颌下腺分泌表皮生长因子的功能。     

表皮细胞生长因子及其受体在胎儿和成人肠道组织中的表达特征

目的探讨表皮细胞生长因子(EGF)及其受体(EGFR)在不同发育阶段人小肠组织中的表达特征及其可能的生物学意义。方法24例被测标本中包括胎儿(胎龄13～31周)的小肠组织18例，成人(16～54岁)的小肠组织6例。用病理学技术和免疫组织化学方法确定EGF及EGFR两种蛋白在胎儿、成人小肠组织中的定位以及表达量的变化规律．结果EGF及EGFR在不同胎龄的胎儿和成人小肠组织中均有阳性表达。随着胎儿生长发育，小肠组织中EGF和EGFR的蛋白含量逐渐增加，这一变化趋势一直保持到成人小肠组织中。其中EGF主要存在于小肠黏膜上皮细胞、黏膜下层的血管内皮细胞内和浆膜上皮细胞的胞浆和胞外基质中，EGFR则分布于这些细胞的细胞膜上。结论EGF及EGFR在不同发育阶段的小肠组织中呈阳性表达，这种细胞因子可能以自分泌或旁分泌方式调节人肠道的生长发育、结构和功能的维持，并可能在小肠损伤后的修复中起重要作用。     

Regeneration of functional sweat gland-like structures by transplanted differentiated bone marrow mesenchymal stem cells

Regeneration of sweat glands after deep burns has been an unsolved problem. Owing to lack of perspiration, survivors of an extensive deep burn injury are leading a miserable life in sultry months. It was our contemplation to solve this problem by inducing bone marrow mesenchymal stem cells (MSCs) to acquire the phenotype of sweat gland cells in vitro. Then these cells were transplanted into fresh skin wounds resulting from excision of anhydrotic scars after healing of deep burn injury in five patients. Two to 12 months after the procedure, it was proved that there was recovery of perspiration function in all the MSCs' transplanted areas, as evidenced by positive iodine–starch perspiration test. Histological and biochemical observation confirmed the involvement of MSCs transformed sweat gland cells in the recovery of functional sweat glands, and the components of sweat collected from these areas were similar to that collected from normal skin. This is the first report of successful transplantation of MSCs in regenerating functional sweat glands, which may help solve the problem of depletion of sweat glands in patients surviving extensive deep burns in the future.     

脐带间充质干细胞向汗腺样细胞分化后的表型变化

目的：比较人脐带间充质干细胞（hUC-MSCs）向汗腺样细胞诱导前后表型特征的变化.方法：对hUC-MSCs进行分离、培养、鉴定,通过显微镜观察、免疫细胞化学、流式细胞术等方法比较hUC-MSCs经汗腺分化诱导培养液培养前后细胞形态变化及癌胚抗原（CEA）、角蛋白7（CK7）、CK8、CK14、CK18、CK19表达的差异.结果：hUC-MSCs向汗腺样细胞诱导前呈梭形,呈成纤维细胞样;流式细胞仪检测发现CD29、CD90表达阳性;而CD34、CEA、CK14、CK19表达阴性.经汗腺诱导培养基诱导后hUC-MSCs分化为外形肥大、不规则、类似铺路石样的细胞,聚集性增殖;免疫细胞化学检测显示CK7、CK8、CK18抗原表达阳性;流式细胞仪检测结果显示CEA、CK14、CK19的阳性表达率分别为77.98%,48.47%,20.85%.结论：hUC-MSCs经过汗腺分化诱导培养基培养后能够分化为表达汗腺细胞标记物抗原的汗腺样细胞.     

与人汗腺细胞共培养的人骨髓间充质干细胞表型转化中细胞外信号调节激酶通路的作用

目的观察人骨髓间充质干细胞(MSC)与热休克的人汗腺细胞(SGC)间接共培养后,其表型转化情况及细胞外信号调节激酶(ERK)通路所起的作用。方法体外分离和培养人MSC、SGC,采用二步免疫细胞化学法鉴定其均为纯化的SGC、MSC。将原代培养的SGC于47℃下行热休克处理后收集上清液。将第3代MSC作为实验对象并分组。对照组:行常规培养;SGC上清液组:采用含体积分数30％SGC上清液、体积分数1％胎牛血清、1×105U／L青霉素和0．1 g／L硫酸链霉素的DMEM／F12培养基培养;SGC上清液+表皮生长因子(EGF)组、SGC上清液+PD98059组同SGC上清液组处理后,分别添加50μg／L EGF、10μmol／L PD98059继续培养。培养7 d时用流式细胞术检测各组细胞中细胞角蛋白(CK)7、癌胚抗原(CEA)的阳性表达率,以蛋白质印迹法检测ERK和磷酸化ERK(pERK)的表达水平。结果SGC上清液组CK7、CEA阳性表达率分别为(5.76±0．10)％、(2．01±0．09)％;SGC上清液+EGF组分别为(7．31±0.21)％、(7．27+0.12)％;SGC上清液+ PD98059组分别为(1．63±0．11)％、(1．54±0．07)％。与对照组比较,前两组两项指标均明显升高(P<0.01),后一组却与之相近。各组细胞均表达ERK;但pERK水平以SGC上清液+EGF组最高,其次为SGC上清液组,SGC上清液+PD98059组和对照组几乎无表达。结论人MSC、SGC间接共培养可诱导MSC表型转化,ERK通路参与该过程并起着积极作用。     

Immunolocalization of estrogen receptor alpha and beta in human fetal prostate

PURPOSE: We examined the immunolocalization of estrogen receptor (ER)alpha and ERbeta in the human fetal prostate. MATERIALS AND METHODS: Tissue sections from human fetal prostates at 7 to 22 weeks of gestation were stained with antibodies to ERalpha, ERbeta, and cytokeratin 10 and 14. RESULTS: ERalpha expression was not detected until 15 weeks of gestation with sparse staining in the utricle. By 19 weeks increased ERalpha expression was seen in the luminal cells of the ventral urogenital epithelium (UGE), basal cells of the dorsal UGE, utricle, distal periurethral ducts, peripheral stroma and posterior prostatic duct. K14 was detected in basal cells of the UGE and in several posterior acini. At 22 weeks ERalpha expression was more intense in all of these areas. ERbeta was expressed throughout the UGE, ejaculatory ducts, müllerian ducts and entire stroma at 7 weeks. Intense ERbeta staining was observed in these areas and in the prostatic buds by 8 weeks with persistent intense staining through 22 weeks. CONCLUSIONS: To our knowledge we report the first immunolocalization of ERalpha in the human fetal prostate and the earliest demonstration of ERbeta expression in the prostate at 7 weeks of gestation. ERbeta expression is intense during ductal morphogenesis, suggesting a role in normal glandular growth and proliferation. The induction of squamous metaplasia in the UGE, distal periurethral ducts and utricle is associated with ERalpha expression in these areas, while the induction of squamous metaplasia in peripheral prostatic acini is associated with peripheral stromal ERalpha expression. This study suggests estrogen signaling pathways in the human fetal prostate via ERalpha that involve epithelial-epithelial and epithelial-stromal interactions.